University of Nevada, Reno

Development of Small Molecule Therapies Targeting Regeneration for the Treatment of Duchenne Muscular Dystrophy

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Cellular and Molecular Pharmacology and Physiology

by

Tatiana M. Fontelonga

Dr. Dean Burkin/Dissertation Advisor

December, 2018

© by Tatiana M. Fontelonga 2018 All Rights Reserved

THE GRADUATE SCHOOL

We recommend that the dissertation prepared under our supervision by

TATIANA M. FONTELONGA

Entitled

Development Of Small Molecule Therapies Targeting Regeneration For The Treatment Of Duchenne Muscular Dystrophy

be accepted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Dean Burkin, Advisor

Patricia Berninsone, Committee Member

Normand LeBlanc, Committee Member

Thomas Gould, Committee Member

Thomas Kidd, Graduate School Representative

David W. Zeh, Ph. D., Dean, Graduate School December, 2018

i

Abstract

Duchenne muscular dystrophy (DMD) is a devastating, X-linked, neuromuscular disease that affects 1 in 5,000 male children worldwide. DMD causes severe muscle wasting that confines individuals to wheelchairs and ventilators early on in life. Disease progression leads to pulmonary infections and cardiac failure, ultimately leading to the untimely death of patients. DMD is characterized by mutations in the Dmd gene, resulting in a loss of functional dystrophin protein.

The lack of dystrophin causes an associated reduction in proteins of the dystrophin glycoprotein complex (DGC). In the absence of the DGC, the muscle is subject to contraction-induced sarcolemmal weakening, muscle tearing, fibrotic and inflammatory infiltration, calcium dysregulation and rounds of degeneration and regeneration affecting satellite cell populations. Currently, there is no cure for

DMD and treatments are scarce. The α7β1 integrin has been implicated in increasing myogenic capacity of satellite cells therefore restoring muscle viability, increasing muscle force and preserving muscle function in dystrophic model mice. Our studies have identified two small molecule therapies capable of increasing α7β1 integrin and halting DMD disease progression. SU9516 was identified using a novel cell-based screen developed in our lab and Sunitinib was discovered as an FDA-approved, structural analog of SU9516. Both small molecules are hereafter characterized as α7β1 integrin enhancers capable of promoting myogenic regeneration. Specifically, Sunitinib stimulates satellite cell activation and increased myofiber fusion via transient inhibition of SHP-2/ERK1/2 ii and activation of the STAT3 pathway. Treatment with Sunitinib in mdx mice demonstrated decreased sarcolemmal damage via myofiber regeneration and enhanced structural support. Additionally, treatment with Sunitinib decreases fibrotic accumulation in the heart of dystrophic mice, making it an appealing therapeutic for DMD. This study identifies two small molecule compounds capable of halting skeletal muscle disease progression in the mdx mouse model of DMD, Sunitinib also showing potential as a treatment for dystrophic, dilated cardiomyopathy. iii

Dedication

To my mother Maria Jose, for her kind soul, endless love and constant support

To my father Antonio, for always being there and helping me become the scientist I am today

Thank you both for being my parents. iv

Acknowledgements

I would first like to thank Dr. Dean Burkin for accepting me into his lab and allowing me to work on such an amazing project, in a most rewarding field. Thank you for your mentorship and for allowing my independence in the lab, you helped me become the scientist I am today.

I would also like to extend a large thank you to my committee members Dr.

Patricia Berninsone for initially helping me get into the program, Dr. Normand LeBlanc for all his helpful questioning, Dr. Thomas Kidd for his mentorship throughout the years

I’ve been at UNR and Dr. Thomas Gould for allowing me my first rotation in the CMPP program. Thank you all for taking time out of your hectic lives to help me become a better scientist.

A big thank you to Dr. Mick Hitchcock for his support throughout my Ph.D.

Thank you to Dr. Andreia Nunes, my Portuguese friend for all her help and for always fixing my computer problems! Thanks to Dr.’s Apurva Sarathy and Ryan Wuebbles for your scientific input. Thank you to Brennan Jordan, my faithful undergrad for spending hours helping me with my project. Thank you to Nicholas Bolden for his assistance on my project as well.

A huge thank you to all my friends in the CMM building for making this experience a little less painful, I am lucky to have shared it with all of you. Suzanne

Duan, thank you for making my days brighter with your sweet demeanor. Miguel

Hernandez you will always make me laugh and I’ll be waiting for you on the East Coast.

Jon Evasovic (aka Fallulah) thank you for always bringing me goodies and making my v days a little brighter. Dr. Mariam Ba thank you for your constant encouragement and friendship, I hope to soon see you running your own lab. Dante, thanks for always bailing me out of my car troubles! Vivian Cruz, thank you for your help in the lab but mostly for becoming one of my good friends. To my friends Samantha Lee, Kristina Sumauskaite,

Cheryl Lee and Ariel Frey, thank you for the support and laughs. Thank you Marisela

Dagda and the pharmacology admins, Annette, Lorraine and Alex for keeping things running. To Monica Rice, I wish I could’ve celebrated the day you graduated, you were truly a wonderful person, rest in peace my friend.

Pamela Barraza-Flores, thank you for your disturbing sense of humor, for being there when science wasn’t working, for making me cry laughing, for taking the absolute worse candid photos of myself and for reviewing my dissertation. Thank you for all the trips we took together and for keeping me sane during this journey; but most of all, thank you for becoming one of my closest friends. I expect amazing things from you, GBC!

A huge thank you to my mother, you are truly my biggest fan, you are the one person I don’t ever doubt loves me beyond all bounds, thank you for being my rock and for always picking up the phone when I need you but also for celebrating all my victories.

Lady, you deserve a medal!

To my father, thank you for making me question everything, for making me listen to classical music, for telling me tales about Albert Einstein, Wolfgang Pauli, Max

Planck, etc., even when I didn’t want to listen (trust me, there were SO many tales). You made me the tough woman I am today, medal for you too!

There is no obtaining a Ph.D. without the help, love and support of all these people and for that, I am truly thankful to you all. vi

Table of Contents

Abstract...... i

Dedication...... iii

Acknowledgements...... iv

Chapter 1 Introduction: Duchenne Muscular Dystrophy – The Path to Discovery of Small Molecule Treatments...... 1

Chapter 2 SU9516 Increases α7β1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy...... 50

Abstract...... 51 Introduction...... 52 Materials and Methods...... 54 Results...... 62 Discussion...... 84

Chapter 3 Sunitinib promotes myogenic regeneration via transient SHP-2 inhibition/STAT3 activation and prevents Duchenne muscular dystrophy disease progression...... 96

Abstract...... 97 Introduction...... 98 Materials and Methods...... 100 Results...... 105 Discussion...... 123

Chapter 4 Cardiac Dystrophy and the Role of α7 Integrin...... 134

Abstract...... 135 Introduction...... 136 Materials and Methods...... 147 Results...... 150 Discussion...... 164

Chapter 5 Conclusions and Future Directions in the Development of Small Molecule Therapies for Duchenne Muscular Dystrophy...... 173

Appendix A Additional Publications...... 190 vii

Appendix B Provisional Patent: SHP2 Inhibitors for the Treatment of Muscular Dystrophy...... 217

Appendix C Copyright releases for use of published work...... 272

Bibliography...... 28 viii

List of Tables

Chapter 2

Table S1. Dose response curves for eight CDK inhibitors tested in α7+/lacZ myotubes...... 92

Table S2. Kinase targets of SU9516 in human DMD myotubes...... 95

Chapter 4

Table 1. β-adrenergic challenge in mdx mice...... 165 ix

List of Figures

Chapter 1

Figure 1. The dystrophin glycoprotein complex (DGC) and the integrin α7β1 complex...... 9

Figure 2. Collapse of the DGC at the sarcolemma of DMD patients...... 11

Figure 3. High-throughput, α7+/LacZ cell-based screen...... 48

Chapter 2

Figure 1. SU9516 increases α7 integrin in myogenic cell lines through the inhibition of the SPAK/OSR1 pathway...... 66

Figure 2. SU9516 expedites differentiation in C2C12 myoblasts...... 69

Figure 3. SU9516 increases α7β1 integrin levels in the skeletal muscle of mdx mice...... 73

Figure 4. SU9516 improves in vivo outcome measures and diaphragm muscle function in the mdx mice...... 76

Figure 5. SU9516 promotes myofiber regeneration through the inhibition of the p65-NF-κB pathway in mdx skeletal muscle...... 79

Figure 6. SU9516 ameliorates pathology in mdx skeletal muscle...... 82

Supplemental Figure 1. Linear dose response curves for SU9516 and STOCK1S- 50699 in myogenic cell lines...... 87

Supplemental Figure 2. Pharmacokinetics of SU9516...... 89

Supplemental Figure 3. Contraction of diaphragm to phrenic nerve stimulation in wild type, vehicle and SU9516-treated mice...... 90

Chapter 3

Figure 1. Sunitinib treatment increases α7B integrin via activation of Myod1 and Myog transcription factors...... 107 x

Figure 2. Sunitinib improves specific muscle contractile function and overall muscle strength...... 109

Figure 3. Sunitinib promotes muscle repair and improves markers of DMD disease progression...... 112

Figure 4. Sunitinib treatment promotes satellite cell proliferation and myoblast fusion...... 117

Figure 5. Sunitinib inhibits SHP-2 - ERK1/2 and activates the STAT3 pathway in C2C12 cell line...... 120

Figure 6. Proposed mechanism of SHP2 - ERK1/2 inhibition and STAT3 activation by Sunitinib...... 122

Supplemental Figure 1. Determination of Sunitinib dosing schedule...... 131

Supplemental Figure 2. Increased expression of titin protein with Sunitinib treatment...... 132

Supplemental Figure 3. SU9516 does not activate STAT3 nor inhibit ERK activation in C2C12 myoblasts...... 133

Chapter 4

Figure 1. Electrocardiogram assessed conduction changes in Itga7-/- mice...... 152

Figure 2. Levels of fibrosis remain unchanged in Itga7-/- mice...... 155

Figure 3. Histological analysis of WT and Itga7 null cardiac muscle...... 158

Figure 4. Sunitinib treatment decreases fibrosis in mdx mouse hearts...... 161

Figure 5. Sunitinib treatment increases α7β1 integrin in the heart...... 162

Figure 6. β-adrenergic challenge increases mdx-utrn+/- heart size...... 165

Figure 7. Echocardiogram assessment on mdx-utrn+/- isoproterenol treated mice...... 166

Figure 8. Fibrosis is elevated in mdx-utrn+/- isoproterenol treated hearts...... 167 1

Chapter 1

Introduction

Duchenne Muscular Dystrophy – The Path to Discovery of Small Molecule

Treatments

2

History of Duchenne Muscular Dystrophy:

Considering the human body as a whole, one can easily agree upon the great importance of skeletal muscle tissue. Comprising about 40% of whole body weight, skeletal muscle is essential for everyday activities; be it walking, running, holding a pencil or tasks as simple as standing up and maintaining posture. Home to circa 50-75% of all body proteins it is apparent how this essential structural and ever dynamic machinery can break down 1.

The muscular dystrophies are a set of diseases that cause progressive weakness leading to skeletal muscle wasting. Muscular dystrophy was first believed to have been described in the beginning of the 19th century by English physician Charles Bell, as well as Italian physicians Gaetano Conte and L. Gioja.

Definitive, clinical examination came later in the century by physicians Richard

Partridge, Edward Meryon and William Little 2. These cases all described young male patients ages 8-17 developing proximal muscle weakness, increased muscle mass of the calf muscles progressing into difficulty standing and maintaining posture. Disease descriptions also included severe contractures, tendon rigidity and significant adipose replacement. Even with all these past descriptions of what would, today, undoubtedly be classified as Duchenne muscular dystrophy, the physician awarded with its discovery/classical description was the French physician Guillaume Duchenne in the year 1858.

Identification of the missing protein product responsible for Duchenne muscular dystrophy (DMD) disease pathology only came in 1987. Dr.’s Kunkel 3

Hoffman and Brown used positional cloning to identify the absence of this protein in both DMD patient and mouse model skeletal muscle tissue 3. They named this protein dystrophin and determined its molecular weight to be about 400kD with an expression profile to include skeletal muscle, cardiac muscle, smooth muscle and minute amounts in brain tissue.

Nearly two centuries have passed since the description of the first case of

DMD and three decades since the discovery of the protein culprit. To this date, this destructive disease has no cure and limited treatment options.

Duchenne Muscular Dystrophy: Prognosis

Duchenne muscular dystrophy (DMD) is a common X-linked neuromuscular disease with an incidence of 1 in 5000 males worldwide, leading to the premature death of affected individuals 4. Clinical features of DMD present in children as early as 3-5 years of age, these include delays in motor capacity, weakness in the limbs and pseudohypertrophy of the calf muscles 5. From this point on, progressive muscle wasting occurs leading to significant motor delays and development of positive Gower’s sign, defined as substantial use of forearms to assist in rising from the floor 6. If left untreated most children between 11-14 years of age will become wheelchair bound requiring substantial intervention from caregivers. In addition to muscle wasting, DMD is also characterized by severe pulmonary failure owing to weakened diaphragm and intercostal muscles. Starting at around age 11, patients lose 4-8% of their vital capacity per year 4 placing them at risk for 4 pulmonary infections. Ultimately, if left untreated, most patients will die prematurely in the second decade of life, most commonly from respiratory infections, but also from dilated cardiomyopathy manifested as cardiac arrhythmias and heart failure.

Fortunately, in the last decades, significant advances have been made toward the clinical care of DMD patients. Early diagnosis of DMD is a crucial aspect of care and while the symptoms used to diagnose DMD have not changed significantly over the years, female carrier and newborn screening have made early disease detection and prevention more attainable 6. One major advancement in DMD disease management came with the use of nocturnal ventilators, reported to increase survival to age 25 by 53% 7,8. Additionally, standard of care has significantly improved with patients being followed by a team of clinical specialists and treatment plans beginning earlier in life 9.

In addition to DMD, there exists a milder form of the disease called Becker’s muscular dystrophy (BMD). BMD is characterized by in-frame mutations in the dystrophin gene (Dmd) giving rise to mutated but semi-functional dystrophin protein which translates into a milder form of the disease where patients exhibit normal survival rates 10. Depending on the type of mutated dystrophin expressed, patients can present as asymptomatic and range toward a loss of ambulation by age 16 11.

Currently, there are limited treatment options available for DMD patients due to the nature of the disease. The difficulty in finding treatment options lies in the fact that this is a rapidly progressing, degenerative disease affecting all 5 skeletal, cardiac and smooth muscle. Developed treatments would require reaching all affected tissues to prevent their detriment.

The Extracellular Matrix

All tissues present in organisms are equipped with a dynamic, non-cellular structure called the extracellular matrix (ECM). The ECM is composed of a variety of glycoproteins and polysaccharides that form a connective network, allowing cells to communicate with their substrates 12. There are two types of ECM that differ based on their location; the interstitial connective tissue matrix (pericellular), which surrounds cells and the basement membrane which separates the epithelium from the stroma. The ECM is not a static structure required only for support and elasticity but is in fact constantly undergoing remodeling to maintain tissue homeostasis. For this reason, the ECM is essential for tissue survival as it regulates proliferation, differentiation, migration, apoptosis and adhesion, all in response to outside signals. Some of the signals incorporated by the ECM are growth factors such as fibroblast growth factor (FGF), epidermal growth factor

(EGF) and transforming growth factor-β (TGF-β) as well as signaling molecules such as wingless/integrated (WNTs) and amphiregulin 13,14.

The skeletal muscle ECM is composed of the basal lamina, directly linked to the sarcolemma, and the reticular lamina. Muscle fiber ECM is composed of collagens, laminins, fibronectin and proteoglycans bound to a protein core. The main accepted function of the ECM is to provide structural and mechanical support 6 during myofiber contractions 15. New insights into the molecular structure of the

ECM now show that it is also responsible for significant signaling transmission, mainly via integrins (discussed in Laminin Binding Partners-Integrins). In addition, the ECM was shown to regulate the satellite cell niche, allowing proper the communication required for muscle regeneration 16,17. In order to perform all its functions, the ECM relies on the complex network of protein structures embedded within the basal lamina that will now further be discussed.

Dystrophin Glycoprotein Complex: Dystrophin

DMD is caused by mutations in the gene encoding the dystrophin protein.

The human gene encoding dystrophin is found on X chromosome at position 21 and is the largest known gene in the human body. The dystrophin gene encompasses 2.4 megabases of genomic DNA 18, with an open reading frame of

11 kilobases, encoding a protein sequence 3865 amino acids long; making it also one of the largest proteins in the human body at 427kDa 19. Due to dystrophin’s large genomic span, circa 7000 mutations have been described that are capable of disrupting the reading frame and producing dysfunctional, truncated dystrophin protein. These mutations encompass everything from large deletions/insertions, being the most common, to smaller deletions/insertions and even point mutations.

Of these 7000 mutations circa 70% are inherited from the mother and the remaining 30% are spontaneous mutations 20. In addition to the lack-of-function mutations in the dystrophin gene that cause DMD, there are also in-frame 7 mutations that give rise to mutated albeit functional dystrophin protein causing

BMD, a milder form of DMD 11.

Dystrophin is found at the sarcolemma of skeletal, cardiac and vascular smooth muscle and concentrated at neuromuscular junctions 3,21. Dystrophin is part of a complex of proteins routinely named the dystrophin glycoprotein complex

(DGC) or dystrophin associated protein complex (DAPC) (Fig. 1). Structurally, dystrophin is essential to anchor the intracellular actin filaments to sarcolemmal proteins (α, β)-dystroglycan, α-dystrobrevin, (α, β, γ,δ)-sarcoglycans, (α1, β1)- syntrophins and sarcospan 22–24. The sarcoglycans bind α-dystrobrevin, which associates with α-syntrophin and together bind the signaling molecule nNOS.

Sarcospan is a tetraspanin-like protein that tightly binds the sarcoglycans forming a sub-complex that affects α-dystroglycan glycosylation and was shown to interact with integrins and laminins to regulate signaling 25. Intracellular dystrophin anchors the complex by binding to the transmembrane protein β-dystroglycan and intracellular actin, while α-dystroglycan binds laminin in the basal lamina 26.

Together this complex of proteins anchors the sarcolemma providing membrane stability, elasticity and allowing force transduction to the extracellular matrix (ECM) during muscle contractions.

It has been proposed that lack of dystrophin prevents association of the

DGC with laminin, thus disrupting DGC associated intracellular signaling, affecting myofiber survival 27,28. This proposal was developed from the many studies that, to date, have shown an interaction of the DGC with all the following signaling proteins: stress-activated protein kinase (SAPK) 3, nitric oxide synthase, 8

phosphatidylinositol 4,5-biphosphate, calmodulin kinase II, integrins, growth factor

receptor-bound protein 2. In addition, it was suggested that transduction of

signaling via these proteins is dependent on laminin binding to the DGC 29. In the absence of dystrophin the DGC becomes unstable and protein elements of the

DGC become significantly reduced at the membrane (Fig. 2). This leaves the sarcolemma susceptible to contraction induced tearing thus causing muscle weakness 30,31. The constant sarcolemmal damage translates into rounds of

muscle fiber degeneration and improper regeneration. Fibrotic/fatty and

inflammatory infiltration exacerbate regenerative impairment and add to the

cascade of muscle necrosis 32,33. Membrane tearing causes a dysregulation of

basal intracellular calcium, leading to leaky calcium channels that ultimately

activate apoptotic pathways initiated by caspases 34,35. In fact, lack-of-function

mutations in any of the proteins of the DGC can cause several types of muscular

dystrophy. The disrupted mechanical and signaling potential of the DGC leads to

a cascade of events, all leading to severe muscle necrosis that explain the rapid

progression of DMD disease pathology.

Dystrophin Glycoprotein Complex: nNOS

Maintaining the DGC complex is extremely important, not only structurally

but as a binding receptor complex to non-structural signaling molecules such

neuronal nitric oxide synthase (nNOS) 36. nNOS is expressed in the brain and at

higher levels in skeletal muscle, where it is essential for the localized production 9

Figure 1. The dystrophin glycoprotein complex (DGC) and the integrin α7β1 complex. The DGC and integrin α7β1 complexes are found at the sarcolemma of myofibers. Both complexes are laminin-211/221 binding partners. As depicted laminin-211/221 binds directly to integrin α7β1, which in turn binds actin filaments.

Laminin-211/221 also binds α-dystroglycan which in turn binds both β-dystroglycan and dystrophin which can then bind intracellular actin filaments. The sarcoglycans and sarcospan span the extracellular membrane adding support the DGC and communication between complexes.

10 of nitric oxide (NO). NO is a short-lived free radical that mediates developmental and physiological processes at the tissue biosynthesis level. It does so by transducing signaling events in a calcium-dependent manner 37. In adult skeletal muscle nNOS is expressed at higher levels in fast-twitch fibers where it opposes contraction and has been shown to promote precocious myoblast fusion 38,39.

Therefore it is unsurprising that in the absence of dystrophin there is a related decrease in nNOS activity, leading to decreased production of NO that causes oxidative stress in the muscle 40–42. The loss of nNOS in DMD was shown to exacerbate the damage produced by eccentric contractions and cause increased inflammation 43. Forced expression of nNOS was shown to revert the effects by decreasing inflammation and reducing DMD muscle pathology 44. Decreased nNOS activity is also responsible for the increased fatigue experienced by patients with neuromuscular disorders such as DMD. This is attributed to a lack of vasodilation of the vessels responsible for supplying blood to muscle fibers post- exercise 45,46. Loss of nNOS activity in DMD pathophysiology is one more drop in the ocean of events that cause severe muscle damage and progression of the muscle disease.

Dystroglycan (DG) is an extracellular matrix receptor encoded by the DAG1 gene and highly expressed in skeletal muscle. DG undergoes posttranslational cleavage into α and β-DG subunits, the α-DG subunit undergoes additional, significant posttranslational glycosylation, in part by the glycoprotein LARGE, required for binding to its extracellular partners containing laminin globular domains such as laminin-211, agrin, neurexin, pickagurin and perlecan 47–49. 11

Figure 2. Collapse of the DGC at the sarcolemma of DMD patients. DMD pathology is characterized by a severe loss of the DGC in the absence of dystrophin protein production. This loss of protein leads to large breaks in the sarcolemmal membrane that lead to severe and rapidly progressing disease pathology. Collapsed DGC is accompanied by excessive bouts of degeneration and regeneration, fibrotic and fatty tissue replacement, intracellular calcium leaking, leading to apoptosis and inflammatory responses. As depicted, the α7β1 integrin complex remains intact at the sarcolemma and could, if overexpressed, provide the structural support that has gone missing in the absence of dystrophin.

12

Dystrophin Glycoprotein Complex: Dystroglycans

Proper binding of glycosylated α-DG to its partners is essential for structural purposes but has also been implicated in satellite cell mediated muscle regeneration 50. The β-DG subunit is the transmembrane portion of DG that binds the C-terminal domain of dystrophin providing the anchor between the ECM and the intracellular actin, bound to the N-terminus of dystrophin 51.

Dystroglycanopathies are a group of congenital muscular dystrophies linked to abnormalities in the dystroglycan protein. Primary dystroglycanopathies are caused by abnormal DG, while secondary dystroglycanopathies involve mutations in the genes encoding glycosyltransferases responsible for glycosylation of DG.

The secondary group of dystroglycanopathies includes congenital muscular dystrophy type 1D produced by mutations in the Large gene encoding a glycosyltransferase, Fukuyama congenital muscular dystrophy produced by mutations in the FCMD gene encoding Fukutin, also a glycosyltransferase, muscle eye brain disease a type of congenital muscular dystrophy produced by mutations in the gene encoding POMGnT1 a protein similar to glycosyltransferases, Walker

Warburg syndrome caused by mutations in the gene encoding POMT1 and congenital muscular dystrophy type 1C caused by mutations in the FKRP gene encoding fukutin-related protein 52. Mutations not only in α-DG itself and the various proteins involved in glycosylation affect the binding of α-DG to laminin, demonstrating the importance of DGs in binding the basal lamina to intracellular dystrophin and providing structural stability to the sarcolemma. 13

Dystrophin Glycoprotein Complex: Laminins

The laminins are heterotrimeric, basal lamina glycoproteins made up of one

of five α chains, one of three β chains and one of three γ chains. Laminins consist

of one α-helical, coiled long arm and three short arms and composed one of each

chain. The α-chain at the distal end of the long arm adds five LG domains required

for laminin binding specificities, while the homologous short arms contain the N-

terminal laminin (LN) and laminin-type epidermal growth factor (LE) domains 53.

Binding specificities lie in the LG motifs of the α chains with LG motifs 1-2 having

a high affinity for integrins α6β1, α6β4, α3β1, α7X1β1, and α7X2β1 and LG motifs

4-5 binding a-dystroglycan as part of the DGC 54.

Laminin-111 (α1, β1, γ1) is the predominantly embryonic form of laminin

expressed in both developing skeletal and cardiac muscle tissue. Laminin-111 is

required for myoblast proliferation, mobility and myogenesis via initial recruitment

to α-dystroglycan and further assembly support via α7β1 integrin 55,56.Recent studies have shown that Laminin-111 regulates myogenesis via the sonic hedgehog pathway (SHH) which is also associated with the transcription of the myogenic regulatory factor (MRF) Myf5, one of the early myogenic markers.

Laminin-111 is also determined to be the first required in the assembly of the myotomal basement membrane 57,58.

Later in development laminin-111 replaced by laminin-211 (α2, β1, γ1) and

laminin-221 (α2, β2, γ1), the adult forms of laminin expressed in skeletal muscle,

where they bind predominately to α-dystroglycan in the DGC, but also nidogen, 14 providing a link between laminin and type IV collagen, to form the bond between the basal lamina and sarcolemma 59. Laminin-211 is therefore also involved in force transmission across the membrane and eccentric contraction force absorption. In addition to providing mechanical support and stabilization to the sarcolemma via its association with the dystroglycans, laminin-211 is involved in cross-membrane signaling by its association to α7β1 integrin and regulating downstream pathways such as the PI3K/Akt kinase cascade, Bcl-2 and Bcl-XL

(apoptotic suppressors) 48,60. Additionally, mutations in the laminin α2 chain cause a severe type of congenital muscular dystrophy previously known as congenital muscular dystrophy type 1A (MDC1A), currently known as LAMA2- related muscular dystrophy. This muscular dystrophy causes sever joint contractures, muscle wasting, weakness and hypotonia with many patients never reaching full ambulation and succumbing to premature death 61–63. This information suggests that the laminins are essential for developmental myogenesis and adult structural stability of skeletal muscle sarcolemma.

Laminin Binding Partners: Utrophin Glycoprotein Complex

The structural and signaling capacity of the DGC is contingent upon its capacity to structurally connect intracellular actin to laminin in the basal lamina.

Without the structural support of intracellular dystrophin, dystroglycan is no longer capable of binding laminin. Due to the large size of dystrophin, replacement therapy is currently not an option, although micro-dystrophin is emerging as a 15 potential treatment. For this reason it was important to identify other laminin binding partners that could potentially compensate for the lack of dystrophin in

DMD.

Utrophin is a smaller analog of dystrophin, it is expressed during development at the sarcolemma pre-dystrophin expression, later found localized to neuromuscular junctions where it co-localizes with acetylcholine receptors 64.

With greatly shared homology to dystrophin, utrophin binds actin at its N-terminal domain with similar affinity to dystrophin as well as the DGC at its C-terminal 65,66.

Utrophin was shown to be slightly upregulated in dystrophin deficient mice, possibly as a compensatory mechanism allowing laminin binding to strengthen the sarcolemma 67. Additionally, mice that lack both dystrophin and utrophin have their otherwise near-normal lifespan (dystrophin null, mdx mouse model) cut down to

20 weeks of age 25. Transgenic expression of full length utrophin in mdx skeletal muscle rescues dystrophic phenotype 68. Global, transgenic expression of full length utrophin is safe with regards to other tissues making it a plausible treatment option in humans 69. Studies also determined that a truncated form of utrophin, transgenically expressed in the skeletal muscle of dystrophin/utrophin null mice

(Dmdmdx;Utm-/-), can prolong survival and prevent dystrophic muscle phenotype 70.

These studies suggest that utrophin is a plausible compensatory, laminin-binding protein capable of providing sarcolemmal stability in the absence of dystrophin and ameliorating dystrophic disease pathology. Currently, small molecule therapies for utrophin up-regulation are being tested and will be discussed later in Current DMD

Therapies. 16

Laminin Binding Partners: α7β1 Integrin

Integrins are transmembrane, alpha-beta (αβ) heterodimers having 24 heterodimers described in humans as a result of 18 α and 8 β available subunits.

The vast combination of α and β subunits in conjunction with post-transcriptional splicing and post-translational processing add much versatility to their ligand specificities and their consequential functional roles. Integrins are unique, transmembrane cell adhesion receptors involved in transducing signals into the cell interior (outside-in signaling) as well as receiving intracellular signals to regulate their ligand binding affinities (inside-out signaling) 71–73. Integrins expressing the β1 subunit are the ones involved in ECM signaling 74,75. Signaling pathways activated by integrin receptor binding have been implicated in a series of cellular functions such as survival, proliferation, differentiation, polarity and migration 76. Regulation of cellular functions occurs via activation of downstream integrin signaling pathways, involving kinases such as phosphoinositide 3-kinase

(PI3K), src kinase and focal adhesion kinase (FAK). Activation of these kinases signals downstream via Akt, ERK, Jnk, RhoA, Rac1 and Cdc42 to regulate the various cellular functions mentioned above 77.

The α7β1 integrin is the predominant and most abundant laminin binding integrin expressed in adult skeletal muscle. Differential splicing of α7 integrin mRNA gives rise to three cytoplasmic domain variants: α7A, α7B and α7C, all with different potential signaling transductions 78,79. Additional variations in α7 integrin signaling arise from alternative splicing of the extracellular domain, giving rise to 17

mRNA splice variants X1 and X2. Both variants are expressed in myoblasts and

cardiac muscle, while only the X2 variant is expressed in adult skeletal muscle

tissue 80. Studies aimed at determining differences between the X1 and X2 variants

have demonstrated differential receptor binding affinities of α7 integrin, with those

expressing the X2 variant being constitutively active while those that express the

X1 domain being regulated in a cell specific manner 81. Additionally, the X2 isoform

is more potent at mediating laminin-induced acetylcholine receptor clustering 82.

Together, cytoplasmic and extracellular domain alternative splicing of integrin α7 subunit can give rise to six different integrin isoforms with different binding affinities and downstream signaling pathway activation. The α7 integrin subunit pairs strictly with the β1D integrin subunit in skeletal and cardiac muscle and together α7β1 integrin is the receptor for laminin-1 and laminin-2/4 in the basal lamina 83. While smaller in size, the α7β1 complex anchors similar members to the DGC and could be a plausible replacement for the loss of dystrophin in DMD (Fig.1).

The α7β1 integrin is expressed peripherally in cardiac and skeletal muscle fibers and concentrated at neuromuscular and myotendinous junctions. Like utrophin, α7β1 integrin is a major laminin binding partner involved in transmembrane signaling, mechanosensing and sarcolemmal anchoring 84. The

α7β1 integrin expression is moderately increased in both mouse and human

skeletal muscle suggesting it is endogenously up-regulated as a compensatory

laminin binding receptor, in a similar manner to utrophin 84,85. Mutations causing

the lack of α7 integrin reportedly result in congenital myopathies in humans 86 and

loss of α7 integrin exacerbates muscle disease progression in mdx and sarcospan- 18 null mice 87–90. Additionally, studies demonstrated that transgenic increases of rat

α7 integrin in mdx/utrn-/- mice can restore viability, increase muscle force, increase regeneration and preserve muscle function 91,92. More recent studies show that

AAV delivery of human α7 integrin in mdx and mdx/utrn-/- mice can protect against eccentric force contraction injury 93,94. Together these findings suggest that therapies that target an increase in α7β1 integrin expression can ameliorate dystrophic disease pathophysiology.

Current Therapies for the Treatment of Duchenne Muscular Dystrophy:

DMD is a multi-faceted disease consisting of primary disease pathology, directly related to the lack of dystrophin protein. This leads to membrane instability and significant declines in muscle force, presenting as severe skeletal muscle weakness. Secondary disease pathology is related to the membrane tearing caused by the primary lack of dystrophin protein. It includes everything from inflammatory infiltration 95–98, fibrotic and fatty tissue deposition/replacement 99–101, bouts of degeneration and failed regeneration of muscle fibers 33, dysregulation of intracellular calcium causing activation of apoptotic caspases 34,102,103 and decreased activity and activation of satellite cell populations 104,105. Novel therapeutic strategies targeting several different pathological aspects of the disease are presently under development.

Corticosteroids 19

Current available therapies for DMD are limited. Aside from the previously mentioned improvements in the standard of patient care, children suffering from

DMD are limited to treatment with corticosteroids or with the novel exon-skipping drug, Exondys50. The first described use of steroids for the treatment of DMD came in 1974 where a cohort of fourteen patients were treated with prednisone, a synthetic glucocorticoid, thirteen of them showed improvements in muscle force

106. Since then, several double-blinded studies performed on significantly larger cohorts have described the benefits of daily prednisone treatment to include increased motor function and delayed loss of ambulation 107. Albeit significant improvements are presented with prednisone use, one drawback of treatment is the intense side effects described by patients to include increased weight gain, cushinoid facies and behavioral changes 108. For these reasons a new corticosteroid, Deflazacort, was developed and shown to provide similar benefits with respect to ambulatory extension and muscular strength when compared to prednisone, with milder side effects and benefits of treatment observed into the second decade of life 109–111. The extension in ambulatory decline provided by steroid use is undoubtedly significant for the patient population. Unfortunately the severe side effects of daily steroid use consistently force the discontinuation of this therapy. Significant efforts have been made to determine the effectiveness of lower doses of corticosteroids, intermittent dosing and research on the anti-inflammatory pathways involved in the production of muscle force.

Several studies have now shown that reducing corticosteroid dosing from daily to bi-weekly or weekly has the same beneficial effect on muscle force with 20 lower observed side effects and therefore increased tolerability 108,112,113. One study described chronic corticosteroid use as promoting the atrophy of muscle, therefore shedding light on the mechanism of action of steroids 114. This study went on to show that weekly dosing with either prednisone or its derivative Deflazacort, increases muscle force by promoting sarcolemmal repair. This improvement in membrane repair is in accordance with an earlier study describing corticosteroids as protective against exercise-induced damage 115. Additionally, suppression of inflammatory responses has recently been correlated with improvements in skeletal muscle regeneration. This was demonstrated by increases in mRNA levels of myogenic regulatory factors such as Pax7, Myf5, Myf6 and MyoD 116. Together these studies shed light into the repair and regeneration capabilities of corticosteroids and the importance of dosing schedule in decreasing detrimental side effects. Unfortunately, while steroid treatments do both improve quality of life and extend ambulation in patients by 2-3 years they do eventually succumb to the disease in their second to third decade of life.

Exon-skipping therapies

A brand new line of therapies has recently emerged with significant potential to treat DMD. While the technology itself was discovered 20 years ago only recently was it put to use for a number of disorders, with a significant emphasis on

DMD. These are exon-skipping “drugs”, more specifically morpholinos, used to target specific mutations in the pre-mRNA stage causing premature end codons to be skipped and reading frames to be restored, therefore giving rise to truncated 21 yet functional proteins 117. Two different exon-skipping drugs were being tested, a

2’O-methyl-phosphorothioate-dinucleoside (2’O-Me(PS)) named drisapersen and a phosphorodiamidate morpholino oligomer (PMO) named eteplirsen or

Exondys51. In 2016, eteplirsen was given accelerated approval by the US Food and Drug Administration (FDA) and is currently under use. Eteplirsen functions as an exon-skipping drug capable of restoring the dystrophin reading frame by skipping exon 51 of the dystrophin gene, where the mutations cause premature end codons to be encoded. Eteplirsen is applicable to about 14% of the DMD population that harbors the specific mutation in exon 51 118. However, by restoring the reading frame patients will produce a truncated, albeit functional dystrophin variant giving them a Becker’s-like phenotype with milder skeletal muscle pathology and significantly extended survival 119. Drisapersen did not receive FDA approval due to unexpected adverse effects when dosed intravenously as well as not producing significant clinical benefit to patients 120. While providing a very significant improvement in DMD treatment, eteplirsen is only applicable to 14% of the patient population, leaving the other 86% still searching for new treatments.

Novel Therapies for the Treatment of Duchenne Muscular Dystrophy

Protein Therapies (Adapted from: ECM-Related Myopathies and Muscular

Dystrophies: Pros and Cons of Protein Therapies, 2017121):

22

Currently there are over 130 proteins or peptides approved by the FDA for clinical use, with several others in the process of development. One of the advantages of protein therapies over pharmacological therapies is that proteins are highly specific and have a defined set of functions, therefore producing less off-target effects. Second, most proteins are already produced physiologically and immune responses are less likely to occur 122. Third, research has shown that clinical development and FDA approval time for protein therapeutics is faster compared to small molecule development 123. Lastly, the specific and defined nature of proteins as replacement therapeutics makes them ideal for development through commercialization. Advancements in the production of recombinant proteins have provided a means to manufacture large quantities of inexpensive purified biologics 122. In 2006 Myozyme© human recombinant acid α-glucosidase was approved by the FDA as a protein enzyme replacement therapy for infantile onset Pompe disease 124. The pathway paved by Myozyme from bench to the bedside is a model for protein therapies being developed for muscle diseases.

Laminin-111 In 1979 Timpl et al. discovered and isolated an ECM molecule secreted by

Engelbreth-Holm-Swarm (EHS) tumors 125 later identified as laminin-111. Laminin-

111 is predominantly expressed embryonically in epithelium 126, it is the embryonic form of adult skeletal muscle laminin-211/221 and can also be found in the placenta, kidney, liver, testis ovaries and blood vessels 127. Initial in vivo experiments were performed using intramuscular injections with 100µL of 100 nM 23 commercially available EHS laminin-111 into the tibialis anterior (TA) muscles of

10 day-old mdx muscle, the contralateral leg was injected with equal volume of

PBS. The TA muscle was harvested and used to assess changes in disease pathology. This study provided evidence that laminin-111 treatment increased α7 integrin and utrophin, both known to slow down disease progression in the mdx mouse 128. These experiments also provided proof of principle that laminin-111 can be delivered throughout skeletal muscle and can re-establish muscle repair and regeneration (Fig. 2b) 129. Building on this result, the possibility of systemic delivery was investigated in the mdx mouse model of DMD, and the dyW-/- mouse model of

LAMA2-CMD 130,131. Mdx mice received intraperitoneal (IP) injections of 1 mg/kg at 10 days of age with an endpoint assessment at 5 weeks of age. This study demonstrated that laminin-111 protein could be systemically delivered to skeletal muscle to reduce disease progression through increased α7 integrin and utrophin.

Furthermore, this study showed improvements in neuromuscular junctions and serum creatine kinase levels.

In laminin-α2 deficient dyW-/- mice the dose of laminin-111 had to be increased 10-fold to 10mg/kg/week to produce therapeutic benefits131. The difference in treatment regimen was attributed to increased rate of ECM turnover in the dyW-/- mice. These observations suggest patients may need a loading dose of laminin-111 and then a maintenance dose to restore the laminin-rich ECM.

Studies showed that laminin-111 treatment in the dyW-/- mouse model of LAMA2-

CMD decreased disease pathology and increased function and longevity 128,131.

Several mechanisms of action may be responsible for the success of laminin- 24 receptors such as the α7β1 integrin and other components of the ECM niche that aid muscle regenerative capacity 130–132. Additional benefits include a lack of immune response to the therapy and ability to provide benefits after disease onset

132. The lack of immune response can be attributed to the presence of endogenous laminin-111 found embryonically and in several adult tissues 126,128,131. For these reasons laminin-111 has the potential to be a successful protein therapy.

There are several issues that need to be addressed before laminin-111 can be used as a therapeutic in patients. Laminin-111 is a large 900 kDa glycosylated heterotrimetric protein which is a significant barrier to producing sufficient quantities of recombinant protein 128. Preclinical studies have used IM

(intramuscular) and IP (intraperitoneal) injections to deliver laminin-111 protein in mouse models however; studies need to be conducted using IV delivery as this would be the most appropriate for the treatment of LAMA2-CMD in patients. If similar results are obtained with this mode of delivery and production obstacles are overcome, then laminin-111 could have potential therapeutic value for several

ECM-related muscle diseases.

Insulin-like Growth Factor Growth factor therapeutics such as insulin-like growth factor-1 (IGF-1) could effectively counter the secondary hallmarks of DMD progressive muscle disease.

IGF-1 promotes muscle regeneration by regulating the proliferation, differentiation and fusion of satellite cells to existing muscle fibers 133. Increased muscle specific

IGF-1 expression effectively eliminated age-related fibrosis in the diaphragm of the 25 mdx mouse 134. Additional effects of enhanced IGF-1 expression in mdx dystrophic muscle showed muscle hypertrophy associated with an increase in muscle strength and a reduction in early myofiber necrosis 134,135. Exogenous administration of IGF-1 resulted in an increase in specific force output of the diaphragm of mdx mice, as well as an improved resistance to fatigue 136.

Combination therapies utilizing stem cells along with exogenous growth factors are currently being explored for treating muscular dystrophy. A recent study showed IGF-1 injected systemically with mesenchymal stromal cells significantly reduced muscle inflammation fibrosis, and improved muscle strength in LAMA2 dy2J dystrophic mice 137.

Other molecular targets for growth factor therapy include members of the transforming growth factor-β (TGF-β) superfamily. TGF-β is a pro-fibrotic cytokine, which is elevated in DMD patients and mouse models. Therapies that target the inhibition of TGF-β in diseased muscle have been shown to be effective in ameliorating the pathology and improving function in the muscle of the mdx mouse

138. Losartan is an angiotensin II receptor type 1 (AT1) antagonist associated with attenuating the levels of TGF-β.

The drawback of growth factor therapy is that these treatments do not target the primary defect on muscle sarcolemmal integrity which remains compromised.

While they may ameliorate secondary symptoms of the disease, growth factor therapies may be more suitable to combinatorial treatment approaches. 26

TAT-Utrophin Utrophin has been shown to be a major modifier of disease progression in

DMD 69,139,148,140–147. Replacement of dystrophin has been shown to induce humoral or cytotoxic immune responses in mdx mice and in DMD patients 149.

Owing to its sequence similarity to dystrophin, utrophin is an excellent therapeutic target in dystrophin deficient dystrophies. The homology of utrophin to dystrophin allows binding to complementary proteins with nearly identical results to dystrophin

150–152. IM injections of AAV mediated gene transfer of mini-utrophin in mdx mice and in the golden retriever dog model for muscular dystrophy (GRMD) show a significant reduction of dystrophic phenotype with sustained expression of mini- utrophin for approximately 60 days 153,154. Two drawbacks of this treatment are that animals receive immunosuppressive drugs like cyclosporine to mitigate immune response to both the virus and transgene; and second, IM injections limit the treatment area.

The production of murine recombinant TAT-utrophin (TAT-Utr) and TAT- micro-utrophin (TAT-µUtr) addresses both of these problems 155. By using the protein transduction domain of the HIV-1 TAT protein instead of the AAV, the need for immunosuppression in mdx mice was alleviated. The TAT protein allows the recombinant protein to travel through the cell membrane into the cytoplasm 156,157.

Several research groups have shown that the TAT fusion protein delivery is a viable option in all tissue types. When injected systemically, flag tagged TAT-Utr and TAT-µUtr migrated to the skeletal muscle, kidney, brain and liver. Although both recombinant proteins were able to rescue dystrophic histology, TAT-µUtr 27

provided greater improvements in contractile force. Treatment of mdx:utr-/- mice with TAT-μUtr, mirrored results observed in the mdx mouse 158. The results using

TAT-µUtr protein suggest a promising therapeutic approach for the treatment of

DMD.

Galectin-1 Galectin-1 is a small 14.5 kDa, non-glycosylated protein encoded by the Lectin

family of genes, Galactoside-binding, Soluable-1 (LGALS1) gene. Galectin-1 is

found in the cell nuclei and cytosol and is secreted into the extracellular matrix 159–

165. Galectin-1 is expressed in many tissues with concentration dependent,

monomeric and non-covalent homodimeric structures determining glycoside-

binding affinities. Although Galectin-1 binding dynamics are complex, the native

dimeric form has been shown to preferentially bind immobilized extended glycans

166. This binding induces diverse physiological activities including: cell migration,

cell growth, angiogenesis, and immune tolerance 167. Galectin-1 is known to have

several impactful roles in the initiation, magnification and termination of

inflammatory responses 168.

The role of Galectin-1 as a pro- or anti-inflammatory factor is tissue, dose and

disease dependent 168. Through inhibitory T-cell interactions, in vivo administration

of Galectin-1 prevents chronic inflammation and thus disease progression in the

following experimental models: arthritis 169 , colitis 170, hepatitis 171, and chronic

pancreatitis 172. The muscular dystrophies are inflammatory diseases with

heightened T-cell response. Galectin-1 reduces immune response, apoptosis 28

and/or cell turnover 173. Binding of dimeric Galectin-1 to activated T helper (Th)

cells promotes regulatory type 1 (Tr1) cell differentiation. Tr1 mediated secretion

of interleukin-27 and interleukin-10 efficiently dampens inflammation in mouse

models of autoimmunity, and immune evasion or tolerance in murine cancer

models. Several studies show a correlation in the reduction of T-cells in DMD

patients treated with prednisone with a reduction in muscle necrosis and fibrosis

174,175; thus, the supposition that Galectin-1 interactions may modify disease in

dystrophic deficient mice and patients.

In skeletal muscle, Galectin-1 plays a role in the conversion of dermal

fibroblasts to myogenic cells during muscle repair and regeneration, 176–179. It also directly interacts with both laminin and the α7β1 integrin to modulate myoblast fusion during muscle repair 180,181. Together, the activities of Galectin-1 in skeletal

muscle make it an intriguing protein therapeutic candidate for the treatment of

DMD patients.

Recently published findings have characterized Galectin-1 as a novel protein

therapeutic for DMD. Recombinant mouse Galectin-1 (rMsGal-1) protein was

delivered systemically and treatment improved activity and muscle strength in mdx

mice 182. Mice treated with rMsGal-1 exhibited reduced skeletal muscle disease

pathology demonstrated by a reduction in centrally located nuclei and Evans blue

dye uptake. rMsGal-1 treatments led to elevated levels of two integral sarcolemmal

complexes in skeletal muscle: the UGC and α7β1 integrin. Since utrophin and

α7β1 integrin have been identified as major modifiers of DMD disease progression,

as previously mentioned, increased levels of these complexes result in 29

sarcolemmal stabilization 183. The improvements in muscle pathology observed in

this study may also be attributed to Galectin-1’s role in the immunomodulation of

leukocytes179. Modulating immune response to muscle damage in mdx mice limits

muscle damage and leads to elevated strength 184. These results are similar to

those reported with Galectin-1 treatment. Thus the immunomodulatory actions of

Galectin-1 could be responsible for many of the observed improvements in the

study. Although many aspects of Galectin-1 treatment still need to be elucidated,

preclinical studies indicate Galectin-1 protein has exciting therapeutic potential for

the treatment of DMD.

Biglycan Biglycan is a protein endogenously found in the ECM and is currently in

development as a potential therapeutic for muscle diseases. Biglycan belongs to

the class 1 small leucine-rich proteoglycans (SLRPs) and the gene encoding

biglycan is located on the X-chromosome 185,186. Some of the known receptors for

biglycan in skeletal muscle include key members of the DGC: α-DG 187, α- and γ-

sarcoglycan, dystrobrevin, syntrophin and nNOS 186. Biglycan has two binding

sites for α-DG through its carboxy-terminal; this activity does not require the

glycosylated form of biglycan 187. Unlike some proteins, non-glycosylated biglycan

is able to functionally attach through the N-terminus of its core protein as a ligand

188. Non-glycosylated biglycan is found in cartilage and intervertebral discs 189. The functionality of both forms of biglycan has caused researchers to refer to it as a

“part time” proteoglycan 186. 30

Biglycan is involved in muscle, tendon, bone development and signaling 190.

It plays a structural role in the ECM in bone formation, muscle integrity and synapse stability at the neuromuscular junctions 186. Research has implicated biglycan in the mechanism of innate immune response and as an activator of the inflammasome 186. Although biglycan is not essential for embryogenesis, it is highly developmentally regulated during tendon and skeletal muscle development

190.

Studies in mdx mice reveal that loss of dystrophin results in an upregulation of biglycan in skeletal muscle 187. Results from experiments in biglycan knockout mice show that it regulates expression and localization of α- and γ-SG 191, dystrobrevin, syntrophin, and nNOS. Treatment with core recombinant biglycan rescued the mild histopathology of biglycan null mice 192. In vitro and in vivo treatments with recombinant human biglycan (rhBGN) increase utrophin expression in dystrophic muscle resulting in less pathological markers for disease, an increase in sarcolemmal stabilizing proteins, less susceptibility to contraction- induced injury and an increase in muscle strength 193. Additionally, treatment with rhBGN was shown to stabilize synapses through the extracellular binding of biglycan to the receptor tyrosine kinase MuSK 194. Non-glycosylated rhBGN is commercially available and has been shown to be active for up to 3 weeks after delivery. Studies have shown that rhBGN does not produce an immune response

192–194. These studies suggest biglycan is a disease modifying protein therapy for

DMD and potentially other muscle diseases. 31

MG53 and Sarcolemma Repair Mitsugumin 53 (MG53) is a tripartite interaction motif (TRIM) protein, also known as TRIM72 195. MG53 is preferentially expressed in cardiac and skeletal muscle 195,196. Protein therapy using recombinant MG53 facilitates cell membrane repair prevalent in many forms of muscular dystrophy. In vitro studies using myogenic and non-myogenic cells treated with green fluorescent protein fused to

MG53 and recombinant human MG53 (rhMG53) prior to damage, provided proof- of-concept that MG53 treatment can both protect and aid in repair of skeletal muscle. Subcutaneous or intravenous treatments with rhMG53 reduced muscle damage, increased repair and provided protection in mdx mice 197. Treatment of

δ-sarcoglycan-deficient TO-2 hamsters with rhMG53 produced similar effects to those observed in the mdx mouse with additional improvements in heart functions

198.

The mechanism by which rhMG53 operates is only beginning to be elucidated 195,197,199. Results thus far show that when membrane damage occurs, endogenous cytoplasmic MG53 attaches to phosphatidylserine on the interior plasma membrane and on intracellular vesicles. The oligomerization of MG53 mediated repair occurs in response to exposure of the interior of the cell to the oxidative extracellular environment 200. Members of the repair complex include: annexin V, caveolin-3, polymerase 1 transcription release factor (PTRF), and dysferlin 197,201,202. Recent studies have also shown that non-muscle myosin 2a is involved in MG53 mediated recruitment of vesicles during membrane repair 203.

The increase in Ca2+ at the point of damage aids in the fusion of recruited 32 intracellular vesicles to repair damaged membrane 199. Suppositions as to the mechanism of action for rhMG53 include a positive feedback loop initiated with the release of MG53 into the oxidative extracellular space 195. This mechanism explains the MG53-phosphatidylserine interaction initiating repair of damaged cells and the repair of neighboring cell surfaces. Additionally, this proposed action would account for the protection of neighboring cells and reduction in pro-inflammatory response. This hypothesized mechanism still needs to be confirmed but thus far been proven correct.

Other studies provided evidence that MG53 protection in δ-sarcoglycan- deficient TO-2 hamsters is mediated by the activation of the Akt-ERK survival pathway and by inhibition of the pro-apoptotic protein Bax 198. In a recent paper,

MG53 was shown to be an E3 ligase that targets the insulin receptor and insulin receptor substrate 1 (IRS1) leading to a negatively regulated mechanism for increased myogenesis 204. This research provides another mechanism for MG53 mediated repair in muscle and may broaden its possible use as a therapeutic in metabolic syndromes involving insulin resistance 204,205. Building on this research, it was recently shown that focal adhesion kinase (FAK) is a second target of MG53 during skeletal myogenesis 205. The potential of MG53 to repair the sarcolemma makes it an intriguing new protein therapeutic for the muscular dystrophies.

Wnt7a The loss of muscle regenerative capacity is a hallmark of many ECM-related muscular dystrophies. Recently, the non-canonical Wnt (Wingless-Type MMTV 33

Integration Site Family) pathway, specifically Wnt7a protein was shown to induce satellite cell expansion and generate larger, stronger muscle fibers in mdx mice

206. These effects are produced via Frizzled family-1 (Fzl1) receptors found exclusively in satellite cells. Indeed, Wnt7a recombinant protein therapy was shown to increase the force produced by muscles in mdx mice, as well as wild- type mice. This increase in force was attributed to a shift in muscle type fibers from fast to slow twitch fibers in response to Wnt7a therapy 207. More recently it was found that Wnt7a/Fzl1 signaling is capable of enhancing the directional migration of satellite stem cells allowing for better engraftment of myogenic cells post-cell transplantation into dystrophic muscle. Enhancing the ability of muscle to repair, especially after contraction-induced muscle injury is a promising and exciting area for therapeutic development.

Although there are many advantages to using biologics for the treatment of muscle disease, there are also potential problems. These include large scale production and purification which can be difficult for large proteins or proteins that are post-translationally modified. Proteins may also contain contaminants (e.g.

LPS using bacterial systems) that can neutralize the beneficial effects of the therapy. Immune response to non-native or chimeric protein therapies may act to reduce the potency of the biologic and off-target effects are possible, although likely reduced compared to small molecule therapeutics. Given these potential shortcomings, biologics hold significant promise for the current, near and/or long- term treatment for the muscular dystrophies. The ideal protein therapy will aid in strengthening the ECM and restoring transmembrane and/or intracellular 34 components which are absent in the respective muscular dystrophies. Many existing and newly discovered ECM muscular dystrophies and myopathies may be amenable to biologics that replace the missing protein. Combination therapies using protein therapeutics or other treatment modalities that modify disease progression may be an effective approach in treating muscular dystrophy.

Dystrophin Replacement Therapies:

Micro-dystrophin gene therapy

The most efficient therapy for DMD would be the replacement of the mutated dystrophin gene with a healthy copy of the gene that could be translated into intact, functional dystrophin protein. Conceptually, fixing the primary cause of the disease would be the ideal treatment, unfortunately the incredibly large size of the DMD gene (2.6 megabytes) as well as the broad distribution throughout the body makes this task a bit more complicated than presumed. In 1990, a study revealed that patients expressing a dystrophin protein about half the size of normal dystrophin experienced very mild dystrophic symptoms. The mini-dystrophin ∆17-

48 discovered in these patients was highly functional and provided excellent muscle protection 208. Use of adenoviral and retroviral vectors to transfer mini- dystrophin to muscle was evaluated in transgenic mice but never relayed onto

DMD patients due to the high immunotoxicity of the vectors. Since the discovery of the adeno-associated virus (AAV) in 1965, recombinant forms have been 35 produced for use with gene therapy. However, the AAV vectors allow packaging of only 5kb of DNA whereas the previously described mini-dystrophin was 6.4kb, alterations had to be made. By 2009, more than 30 different configurations of micro

(µ)-dystrophin genes (no longer named mini-dystrophin) had been published, all with deletions of different dystrophin genomic areas including the very important addition of the nNOS binding domain 209. Many of these µ-dystrophins have since been tested in dystrophic animals showing improvement in dystrophic pathophysiology 210–215. Additionally, AAV delivery of µ-dystrophin has been tested in non-human primates 216,217.

These studies were all gearing up for clinical trials in DMD patients, three of which began in the U.S. in 2017 with one more in Europe. The trials differ in regards to the AAV production, serotype, purification, promotor used, µ-dystrophin configuration, patient age and gene mutation 209. Innate immunity to the AAV vector is the major concern in these studies, as high percentages of people have been exposed to AAVs in their lifetime and could have adverse reactions. If immunity is overcome, µ-dystrophin gene replacement would become the go-to therapy for DMD patients.

CRISPR-Cas9 dystrophin gene correction

In 2012, Jennifer Doudna along with Emmanuelle Charpentier described the discovery of a new biotechnological system that would change the scientific world. Clustered regularly interspaced short palindromic repeat/CRISPR- associated 9 (CRISPR-Cas9) is a biological tool discovered in bacterial and 36 archaea genomes, capable of targeting specific DNA sequences for editing. Cas9 is the endonuclease that is assembled, together with two guide RNA sequences, to target a specific DNA sequence to be edited. Cas9 then introduces double stranded breaks in the DNA that are repaired by the organisms’ own repair mechanisms of homologous recombination or non-homologous end-joining 218.

The scope of this technology with regards to DMD becomes immediately apparent.

The most common genetic modification encountered in DMD patients is the deletion of one or more exons found in the “hotspot” genomic area between exons

45 and 55. Whole exon deletion results in the disruption of the reading frame and production of truncated dystrophin protein that is targeted for degradation. Several groups are concentrating on using CRISPR-Cas9 technology to insert specific double strand breaks into the DNA before and after the deleted exon. This would cause the deletion of the selected area, now restoring the reading frame and giving a shorter, albeit functional dystrophin protein. Proof of concept studies were performed in vitro in 293T and human patient DMD skeletal muscle cells, showing that CRISPR-Cas9 corrected myotubes went on to produce internally deleted dystrophin protein in vitro as well as in vivo post-transplantation of corrected myoblasts into immunodeficient mice 219,220. Studies have since been performed on induced pluripotent stem cells (iPSCs) and postnatal as well as adult murine models of DMD, namely several different strains of dystrophin null mice. All studies described significant expression of dystrophin protein in vivo (endogenous or transplanted iPSC derived) in skeletal and cardiac muscle, leading to the prevention of DMD disease progression 221–226. This year, as is the norm after 37 successful murine trials, CRISPR-Cas9 editing was successfully performed in a canine model of DMD showing high levels of dystrophin expression in skeletal and cardiac muscles and proving that with further development of the technology clinical trials are in the foreseeable future 227.

Like µ-dystrophin therapy, CRISPR-Cas9 is currently packaged and delivered in AAV vectors. As aforementioned, innate immunity to the vector is the single most important challenge that needs to be overcome before either therapy can move forward. Additionally, studies have shown that AAV is not successful at infecting skeletal muscle stem cells (satellite cells), meaning that treatment would need to be ongoing throughout the patients’ lives as newly formed muscle fibers would not be expressing dystrophin 228. Taking into consideration the predicted price of therapy, this would be a significant financial burden on patient families, therefore, researching how to infect satellite cells with the CRISP-Cas9 complex would put this technology at the forefront of DMD treatments.

Stem Cell Therapies for Duchenne Muscular Dystrophy:

Adult skeletal muscle is developed from precursor stem cells called satellite cells, given their name due to their sub-laminar location around the myofiber.

Satellite cells are present in adult muscle in a quiescent state that can be primed for proliferation and differentiation in response to growth signals 229. Satellite cells are merely activated in the case of exercise or injury where they begin the process of differentiation into mature muscle fibers commonly referred to as asymmetric 38 division. Additionally, upon activation, satellite cells can undergo a process known as symmetric division where they self-replicate and expand the satellite cell pool, giving them the ability to support several rounds of muscle regeneration 230–233.

Several myogenic factors are involved in maintaining the proliferative state of satellite cells and/or promoting their activation. The paired box transcription factor (Pax) 3 is expressed embryonically and Pax7 is expressed postnatally in all satellite cell populations and is involved in maintaining their quiescent state by restricting differentiation programs and regulating cell-cycle entry 234–236. In addition to Pax7, murine satellite cell quiescence is determined by the expression of the cell surface markers M-cadherin, CD34, α7 integrin and Vcam1 while proliferating satellite cells also express lamin A/C and emerin 237,238. Once activated, the family of myogenic basic helix-loop-helix (bHLH) transcription factors, MyoD, Myf5, MRF4 and myogenin all play essential roles in the specification and differentiation of satellite cells 104,239. Interestingly, it was discovered that Myf5 is transcribed in a large population of Pax7 positive cells that do not retain the same self-renewing capacity as the ~10% of cells which do not transcribe Myf5 240. This suggests that even within the population of Pax7 expressing satellite cells, some are already primed for differentiation, whilst only

10% retain their proliferative potential, making them more successful in the event of transplantation. MyoD is commonly referred to as the master regulator of muscle specification, its expression can convert many non-skeletal muscle cell types into muscle 241. While there is significant overlap amongst Myf5, MyoD and MRF4 expression during satellite cell fate specification, no overlap exists between those 39

three bHLH factors and myogenin, suggesting their involvement in the downstream

expression of myogenin. Once expressing myogenin, cells are fully committed to

differentiate into skeletal muscle 242. The capacity of these myogenic precursors to

develop into adult skeletal muscle makes them an appealing target for

transplantation studies and a viable treatment option for DMD patients.

Several different methods have been explored in the stem cell field with

regards to DMD. In the early 1990’s, several clinical trials were performed in both

BMD and DMD patients using intramuscular myoblast injections. Early studies

hinted at successful myoblast engraftment, but ultimately myoblast transfer was

deemed unsuccessful in improving strength in patients. Additionally, they

produced very low levels of myoblast implantation, proving the lack of proliferative

capacity of myoblasts provides no regenerative benefit to patients 243–247. Most myoblasts have already entered the commitment phase of differentiation, they already express most, it not all, bHLH transcription factors and their proliferative capability is not that of satellite cells. In 2005, Montarras first described a method for the isolation of healthy satellite cells using a Pax3GFP/+ expressing mouse with

subsequent transplantation into an mdx mouse model. Results from the study

suggest that the healthy cells significantly contributed to the repair of muscle fibers

and addition to the satellite cell pool 248. Since then, the purity of satellite cell

isolation has been perfected by using different markers to select the most “stem

like” cells out of the satellite cell pool and transplant them with high efficacy into

dystrophic muscles 249–251. 40

Differences exist between muscle stem cell membrane surface markers expressed in murine myogenic stem cells and human myogenic stem cells that must be taken into consideration when developing studies 252,253. One cell surface marker common to most stem cells, found initially in hematopoietic CD34+ cells, is CD133, shown to be on the surface of undifferentiated cells and used as a marker to isolate stem cells from human blood and muscle 254. Phase I clinical trials conducted to test the safety of transplanting autologous, muscle derived

CD133+ cells into DMD patients showed no adverse effects, suggesting their safety for clinical applications 255. However, more current studies found that

CD133+ cells derived from DMD patients are compromised in terms of their regenerative capabilities, making autologous transplantation of CD133+ cells safe, albeit non-effective in providing benefit to patients 256. To overcome this problem several studies are currently developing methods for the isolation of human fetal skeletal muscle myogenic cells. Microarray studies determined the cell surface marker, melanoma cell adhesion molecule (MCAM) to be an excellent marker of a highly myogenic cell population found in human fetal skeletal muscle 252,257.

However, MCAM is expressed in both Pax7 positive and Pax7 negative cells as well as mature myofibers, making it too broad of a marker for the isolation of truly myogenic cells. Recently, KAI/CD82 was identified as a potential cell surface marker that can further refine the MCAM population of cells to select for the subset of cells with true myogenic potential 253. Isolating stem cells using these cell surface markers will therefore increase the probability of implantation and expansion within the host. 41

While satellite cells are the holy grail of pluripotent skeletal muscle stem cells, in addition to embryonic stem cells (ESCs), other types of cell populations exist with the capacity of giving rise to mature skeletal muscle. These cell types include mesenchymal stem cells (MSCs), muscle side population cells (SPs), mesoangioblasts, pericytes, muscle-derived stem cells (MDSCs) and induced- pluripotent stem cells (iPSCs) 258. MSCs, mesoangioblasts and pericytes are particularly appealing as they have been shown to infiltrate dystrophic muscle from the circulation and can therefore be administered intra-arterially 259. In fact, intra- arterial delivery of wildtype, canine, mesoangioblast stem cells recovered dystrophin expression and ameliorated muscle function in the GRMD canine model of DMD 260. Phase I-IIa clinical trials in humans using HLA-matched donor mesoangioblasts proved their safety and showed moderate levels of donor derived dystrophin expression 261. Additionally, a phase I clinical trial using umbilical cord

MSCs presented skeletal muscle stabilization and was proven safe for clinical administration 262. More recently, cardiosphere derived cells (CDCs) have emerged as a new modality of stem cell capable of improving ambulatory and cardiac function in the mdx mouse model 263 and are currently gearing up for Phase

I clinical trials. The main obstacles for the stem cell field include the expansion of cells without loss of myogenic capability, the potential immune responses to transplanted cells and intravenous delivery of the cells to reach all skeletal and cardiac muscle. While the field of stem cell therapy for DMD is quickly advancing and many different types of stem cells are in the early phases of clinical trials, currently there is no viable stem cell treatment option for patients. 42

Pharmaceutical Therapies for Duchenne Muscular Dystrophy:

Utrophin enhancing drug therapeutics

As previously mentioned, the UGC is a major laminin binding complex with utrophin sharing 80% homology with the dystrophin protein and capable of binding many of the same proteins found in the DGC (see Laminin binding partners-

Utrophin Glycoprotein Complex). Utrophin forms its own complex at the sarcolelmmal membrane named the utrophin glycoprotein complex (UGC). Due to analogous binding partners to dystrophin, several studies are aimed at the up- regulation of utrophin protein with the purpose of compensating for the loss of the

DGC by replacement with the UGC, recovering membrane stability. Transgenic overexpression of utrophin at the sarcolemma of dystrophin deficient mice is proof of concept that up-regulation of utrophin can decrease degenerative markers and improve muscle force 68. Therefore, pharmaceutical up-regulation of utrophin is a viable treatment option for DMD patients.

Ezutromid (SMT C1100, Summit Therapeutics) was one of the first compounds obtained from an extensive cell-based screen using mdx myoblasts.

The luciferase reporter sequence was placed under control of the utrophin promoter with which a series of compounds were tested for their ability to increase luciferase activity and later confirmed in both mouse and human cell lines to up- regulate endogenous UTRN transcription. Pre-clinical studies using SMT C1100 in mdx mice showed a two-fold up-regulation of the UTRN transcript and increased protein levels in skeletal and cardiac muscles. This increase in utrophin translated 43

into decreased DMD muscle pathology in mdx mice and increased muscle force

production 264. Ezutromid passed Phase I safety trials and was deemed tolerable

for pediatric patients 265. Unfortunately, in June 2018 Summit Therapeutics

announced they would discontinue Phase II trials due to lack of treatment efficacy

with patients not showing benefits after a month long treatment with Ezutromid.

Utrophin up-regulation can also be achieved by promoting the slow,

oxidative myogenic program by triggering the switch from fast to slow fiber types.

This can be achieved by treatment with AICAR (5-aminoimidazole-4-carboxamide-

1-β-D-ribofuranoside) an AMP-activated protein kinase (AMPK) activator.

Treatment of mdx mice with AICAR was shown to elicit the shift in fiber type to

slow fibers therefore increasing levels of utrophin and its partner β-dystroglycan

and promoting myofiber stability and prevention of contraction induced muscle

damage 266. Proof of concept of the benefits of increased AMPK activity was shown

in DMD patients treated with L-arginine (an NO precursor) and metformin, an

AMPK activator which in patients showed improved clinical scores 267. In addition,

nabumetone, heregulin and resveratrol all were previously described to modulate

the utrophin promotor and improve disease pathology in mdx mice 268. While studies are underway, no clinical trial is yet reported for these utrophin modulating drugs.

Anti-inflammatory and anti-fibrotic therapeutic agents

The current standard of care relies solely on the anti-inflammatory glucocorticoids, prednisone and Deflazacort, both acting via inhibition of the Nf-κB 44 pathway. Due to the severe secondary effects of glucocorticoids, new drugs are currently under development that provide Nf-κB inhibition without severe secondary effects. One of these drugs is called Vamorolone (VBP15), it has been developed as a specific Nf-κB inhibitor capable of providing membrane stability without any growth, hormonal or immunosuppressive side effects 269. Vamorolone passed Phase I clinical trials with tolerability observed at all doses and no side effects observed 270 and is currently in Phase IIa trials. Edasalonexent (CAT-1004) is another oral, NF-κB modulator developed by Catabasis showing good tolerability in Phase I clinical trials 271 but ultimately failing to demonstrate benefit in DMD patients after 12 weeks of treatment. Nonetheless, these studies pave the way to the development of specific NF-κB inhibitors with potential to alleviate DMD disease pathology with no side effects, proving a very promising therapeutic avenue.

Another secondary aspect of DMD is the excessive accumulation of fibrotic and fatty tissue within muscle that replaces necrotic skeletal muscle fibers and accelerates disease progression. Transforming growth factor-β (TGF-β) is the pathway responsible for fibrotic deposition, making it an attractive target for therapies. Halofuginone is an anti-fibrotic agent targeting the TGF-β pathway that showed promising anti-fibrotic, anti-inflammatory and muscle regenerative potential 272. Unfortunately, Phase II clinical studies resulted in the death of one patient and further studies were discontinued. However, new anti-fibrotic agents such as losartan and tamoxifen are currently being studied.

45

Therapeutics targeting the dysregulation of intracellular calcium

DMD is characterized by a dysregulation of basal intracellular calcium and

leaky calcium channels leading to activation of apoptotic pathways 34,35. Drugs aimed at controlling levels of intracellular calcium could prove beneficial to DMD pathology. Blocking stretch-activated calcium channels is one way to prevent the influx of calcium into the muscle fibers. One such drug, AT-300 has shown modest benefits in mdx mice and is currently underway for clinical trials 273. Due to dissociation of the calcium calstabin-ryanodine receptor channel in dystrophic muscle, a stabilizer of this complex, ARM210/S48168, shows functional

improvement in mice.

Secondary to the influx of calcium is the increase in reactive oxygen species

(ROS) that lead to increased oxidative stress of muscle cells. Raxone/idebenone

are antioxidants and synthetic derivatives of Coenzyme Q10 that entered Phase

III clinical trials showing respiratory improvements in patients 274. The efficacy of

this antioxidant paved the way for the new antioxidant therapies such as melatonin,

N-acetylcysteine and simvastatin, all currently under investigation. Increased

calcium influx also causes mitochondrial dysfunction leading to increased ROS.

Alisporovir/Debio-25 (an analog of cyclosporine) is a cyclophilin D inhibitor shown

to decrease inflammation in mdx mice with better efficacy than prednisone 275. All

of these therapies are currently being investigated.

nNOS dysregulation 46

nNOS is downregulated in dystrophic muscle due to the lack DGC binding partners responsible for anchoring nNOS to provide local synthesis of nitric oxide

(NO). Dysregulated nNOS prevents the NO-dependent S-nitrosylation required for inhibition of Histone deacetylase 2 (HDAC2), therefore leading to constitutively activated HDAC2. Givinostat is an HDAC inhibitor that has shown promising effects in Phase II clinical trials for DMD 276. HDAC inhibitors are, however, quite likely to be toxic due to their global epigenetic effects. Caution should be used when studying these inhibitors aiming to make them as muscle specific as possible.

nNOS dysregulation is associated with a lack of NO production, required to promote vasodilation and oxygen dispersion throughout the muscle. Therefore, sympathetic vasoconstriction occurs via cyclic guanosine monophosphate (cGMP) causing muscles to not be properly oxygenated during exercise 277. To promote vasodilation, phosphodiesterase-5 inhibitors (PDE5) such as Tadalafil have been studied and shown to decrease muscle ischemia due to extended vasoconstriction

278. Unfortunately, Phase III trials showed no functional improvements, possibly due to the lack of exercise of patients which is required to increase cGMP levels.

Myostatin inhibitors

Myostatin is a transforming growth factor-beta (TGF-β) superfamily member. Myostatin was shown via genetic, antibody and propeptide-mediated 47 inhibition to be involved in negatively regulating muscle hypertrophy, suggesting its inhibition as a potential therapeutic target. Indeed several inhibitors have been carried into clinical trials including ACE-031 (failing due to side effects) and PF-

06252616 (ongoing Phase II trials) 279. Additionally, newer and more specific inhibitors conferring fewer side effects are being studied such as ACE-083, a locally acting myostatin trap administered via intramuscular injections 280. While not yet perfected, pharmaceutical inhibition of myostatin is still very much a viable treatment option for DMD.

Small Molecule Drug Screen for α7 Integrin Enhancement:

The main purpose of this dissertation is the identification of small molecules capable of increasing α7 integrin expression and slowing DMD disease progression. As previously mentioned, several studies have determined that α7 integrin is a positive modifier of disease progression in DMD via transgenic and, more recently, AAV delivery of mouse and human ITGA7 84,87,283,88,90–94,281,282. For this reason we embarked on a journey aimed at developing small molecules capable of enhancing α7 integrin using a high-throughput muscle cell based screen.

For this screen our lab collaborated with the National Chemical and

Genomics Center (NCGC) and the National Institutes of Health (NIH) to screen

350,000 compounds in the Molecular Library and Small Molecule Repository

(MLSMR). To screen the compounds, a novel knockout cell line was produced by placing LacZ in the place of exon 1, downstream of the α7 integrin promotor (Fig.3). 48

Figure 3. High-throughput, α7+/LacZ cell-based screen. Schematic representation of the development of a novel cell based screen used to identify small molecule compounds capable of promoting increased α7 integrin transcription.

Levels of β-galactosidase were quantified in both myoblasts and myotubes treated with the various drugs using Fluorescein di-β-D-galactopyranoside (FDG) cleavage and fluorescein detection. Several drugs were identified and are currently under development in our lab. The identification of the most robust compound from the screen, SU9516, is the topic of chapter 2 in this dissertation. Chapter 3 is focused on the discovery of an already FDA-approved small molecule compound,

Sunitinib (SUTENT®) with structural similarities to SU9516, shown to be a more potent enhancer of α7 integrin as well as disclosing new regenerative capabilities.

The fourth chapter of this dissertation will follow the implications of α7 integrin in the cardiac profile of DMD patients as well as patients with new found cardiac 49 arrhythmias. As most patients will succumb to cardiopulmonary failure it is important to determine the effects of any therapeutic treatment on the heart, do they aid in diminishing the accumulation of fibrosis in the heart? Is cardiomyopathy severity changed post treatment? Do these therapeutics have adverse side effects on the heart, not previously shown in skeletal muscle? These are questions essential to any pre-clinical study performed on a disease affecting not only skeletal as well as cardiac and smooth muscle tissues. Additionally, some aspects of the potential benefits of Sunitinib treatment on cardiomyopathies and arrhythmias will be discussed. Finally, the future of the therapeutics mentioned in the dissertation will be addressed as well as the potential to cover other muscular dystrophies and the potential for near future clinical trials.

50

Chapter 2

SU9516 increases α7β1 integrin and ameliorates disease progression in the

mdx mouse model of Duchenne Muscular Dystrophy

Published 7 June, 2017, in Molecular Therapy

http://dx.doi.org/10.1016/j.ymthe.2017.03.022

(Permission to reprint in Appendix C)

51

ABSTRACT

Duchenne Muscular Dystrophy (DMD) is a fatal muscle disease caused by mutations in the dystrophin gene resulting in a complete loss of the dystrophin protein. Dystrophin is a critical component of the dystrophin glycoprotein complex

(DGC) that links laminin in the extracellular matrix to the actin cytoskeleton within myofibers and provides resistance to shear stresses during muscle activity. Loss of dystrophin in DMD patients results in a fragile sarcolemma prone to contraction- induced muscle damage. The α7β1 integrin is a laminin receptor protein complex in skeletal and cardiac muscle and a major modifier of disease progression in

DMD. In a muscle cell-based screen for α7 integrin transcriptional enhancers, we identified a small molecule, SU9516, which promoted increased α7β1 integrin expression. Here, we show that SU9516 leads to increased α7B integrin protein in murine C2C12 and human DMD patient myogenic cell lines. Oral administration of SU9516 in the mdx mouse model of DMD increased α7β1 integrin in skeletal muscle, ameliorated pathology and improved muscle function. We show that these improvements are mediated through SU9516 inhibitory actions on the p65-NF-κB pro-inflammatory and SPAK/OSR1 signaling pathways. This study identifies a first in-class α7 integrin enhancing small molecule compound with potential for the treatment of DMD.

Contribution to Chapter 2: Author performed histology, immunofluorescence, aided in contraction studies and phrenic muscle stimulation studies. 52

INTRODUCTION

Duchenne muscular dystrophy (DMD) is a common form of muscular

dystrophy affecting 1 in every 5000 males 284. It is caused by mutations in the Dmd

gene, resulting in complete loss of the dystrophin protein 285–287. In healthy muscle,

dystrophin stabilizes the dystrophin glycoprotein complex (DGC) which links

laminin in the extracellular matrix (ECM) to the actin cytoskeleton 22,288. The absence of dystrophin in skeletal muscle leads to significant sarcolemmal tearing and myofiber damage, as levels of compensating structural proteins are inadequate to withstand normal contractile forces 289. The progressive muscle

damage and subsequent rounds of degeneration/regeneration, are accompanied

by elevated levels of inflammation, necrosis and fibrosis. Until recently

glucocorticoids and corticosteroids, prednisone and deflazacort were the only

palliative treatments available for DMD 290–292. Although current clinical practice

guidelines recommend corticosteroid therapy for all DMD patients starting at an

early ambulatory stage 293,294, there are several side effects associated with

sustained use. These include stunted growth, obesity, cataracts and propensity to

skeletal fractures 295,296. In September 2016, an exon skipping

phosphorodiamidate morpholino oligomer (PMO) Exondys 51 (eteplirsen), was

approved by the Food and Drug Administration (FDA) for the treatment of patients

with amenable mutations in exon 51 of the dystrophin gene 297. Exondys 51 skips

its target exon 51 thereby restoring the dystrophin reading frame in these patients.

One of the limitations of this drug is that it is applicable to only 13% of DMD patients 53 as it is mutation specific 297. Current therapeutic approaches under investigation such as gene replacement and stem cell therapies seek to treat all patients irrespective of their dystrophin mutation 298–300. Another strategy that has near term therapeutic promise is the enhancement of structural proteins like utrophin and α7β1 integrin that compensate for the loss of dystrophin. Transgenic enhancement of these proteins has been demonstrated to significantly rescue disease phenotype in mouse models of DMD 91,92,301.

The α7β1 integrin protein is the predominant laminin-binding integrin in skeletal, cardiac and vascular smooth muscle 302. It is normally distributed along the sarcolemma at costameres and is elevated at neuromuscular and myotendinous junctions in skeletal muscle 303,304. α7β1 integrin has structural and signaling functions that contribute to muscle development and physiology and was originally identified as a marker for muscle differentiation 79. Loss of the α7 integrin in dystrophin deficient mdx mice leads to a severe dystrophic phenotype and mice do not survive past 4 weeks of age 88. Conversely, transgenic over-expression of the α7β1 integrin ameliorates disease pathology and extends longevity of severely dystrophic mice 91. Mechanisms that contribute to α7 integrin mediated rescue of dystrophin deficient muscle include maintenance of myotendinous and neuromuscular junctions, enhanced muscle hypertrophy and regeneration, decreased apoptosis and cardiomyopathy 92,305–307. Recent evidence suggests that prednisone may maintain function in the GRMD dog model by stabilizing α7 integrin protein levels 308. Together, these observations support the idea that the 54

α7β1 integrin is a major disease modifier in DMD and a target for drug-based therapeutics.

Here we report the discovery and preclinical assessment of a first in-class

α7 integrin enhancing small molecule called SU9516. We show that SU9516 treatment in human patient cell lines and mdx mice increases levels of the α7β1 integrin protein complex. This effect possibly occurs through inhibition of the

SPAK/OSR1 pathway and downregulation of p65 NF-κB pathway activation. Most importantly, treatment with SU9516 led to improved muscle function and reduced dystrophic pathology in the mdx mouse model of DMD. Therefore, we believe

SU9516 represents a novel small molecule that has translational potential for the treatment of DMD.

MATERIALS AND METHODS

Study design

To assess the benefits of SU9516 as a therapeutic for DMD, we conducted in vitro experiments to compare α7 integrin levels in murine C2C12 and human DMD myogenic cell lines. These experiments were followed by a preclinical assessment of the drug in mdx mice treated with a dose of 5mg/kg/day SU9516 for 7 weeks.

Assessment of different measurements such as body weights and forelimb grip- strength, ex vivo muscle physiology and quantifications of disease markers such 55 as centrally nucleated fiber counts, Sirius Red and Feret’s diameter of myofibers in diaphragm muscle of mdx mice were performed in a blinded fashion.

Cell Culture

C2C12 myoblasts were originally purchased from ATCC and grown and maintained in growth media comprising DMEM (Sigma) containing 20% FBS

(Atlanta Biologicals), 1% Penicillin/Streptomycin (P/S) (GIBCO) + 1% L-Glutamine

(GIBCO). Myoblasts were maintained below 70% confluence until use in assay.

Myoblasts were differentiated into myotubes in DMEM, 2% horse-serum (Atlanta

Biologicals), and 1% P/S + L-Glutamine. All cells were incubated at 37ºC with 5%

CO2. Assays were performed on myoblasts and myotubes between passages 8 and 14. Human DMD myoblasts were a generous gift from Dr. Kathryn North (The

Royal Children’s Hospital, Victoria, Australia) and used under an approved IRB from the University of Nevada, Reno. DMD myogenic cells were cultured as described previously 308. α7+/LacZ myoblasts were originally isolated and maintained as described 309. A total of 5000 α7+/LacZ myoblasts (25,000 for myotube assays) were dispensed in 100µL growth media (DMEM without phenol red) using a 12-well multi-pipette (Rainin) onto Nunc black-sided TC coated 96- well plate. For myotube formation, differentiation media was changed daily between 72 and 120hrs. Dose response curves were generated for SU9516

(TOCRIS Biosciences) and STOCK1S-50699 (Asinex).

Drug screen 56

In collaboration with NCGC, the Burkin lab identified SU9516 as an enhancer of

α7 integrin expression from the LOPAC library. The screen used to identify the

compound utilized a myogenic cultured cell line derived from Itga7+/LacZ strain of mice developed in the Burkin lab. The cells were derived from heterozygous mice

in order to maintain the α7 integrin protein in these myogenic cells, as its loss

significantly alters many signaling pathways 309 . On the opposing allele, exon 1 of

the Itga7 gene was replaced with the LacZ gene. The β-galactosidase expressed

from this allele acts as a reporter of Itga7 promoter activity and has been previously

shown to mimic normal α7 integrin protein levels during muscle differentiation 309.

The enzymatic activity of β-galactosidase levels were then translated into a

readable fluorescent signal through the addition and cleavage of Fluorescein-Di-

β-D-Galactopyranoside (FDG) to fluorescein.

C2C12 Myotube Analysis

The measurements for myotube width and fusion index were performed according

to a protocol modified from Wang et al. 310. To analyze myotube diameter, fifteen

fields were selected randomly, and three myotubes were measured per field. The

diameter per myotube was computed as the maximum width taken along the long

axis of the myotube. Myotube nuclei were counted in approximately 100 randomly

chosen MyHC-positive myotubes containing two or more nuclei. Myotubes were

categorized into five groups (2 to 5, 6 to 10, 11 to 15, 16 to 20 and 21 to 25 nuclei

per myotube) and were expressed as a percentage of the total myotube number. 57

The fusion index was calculated as the ratio of nuclei in MyHC-positive myotubes to the total number of nuclei in the field for fifteen random fields.

KiNativ Assay

A probe based chemoproteomics (KiNativ) assay was used to identify muscle specific kinase(s) regulated by SU9516 311. DMD myotubes were cultured as previously described 308 and treated with the 0.1, 0.5 and 1µM SU9516 for 48 hours. Proteins from control and treated DMD myoblasts and myotubes were harvested and incubated with the KiNativ probe (biotinylated acyl phosphates of

ATP and ADP) following the manufacturer’s protocol (ActivX Biosciences, Inc.,

Torrey Pines, CA). Proteins were sent to ActiveX Biosciences and subjected to

LC-MS/MS to identify the molecular signature of biotin labeled proteins in the samples and control vs treated proteins compared and kinases in which activation was inhibited by SU9516 were identified.

Immunoblotting

Protein was extracted from cell pellets or tissue powdered in liquid nitrogen. RIPA was used as lysis buffer with a 1:100 dilution of NaF and Na3VO4 and a 1:500 dilution of protease inhibitor cocktail. Protein was quantified using a Bradford assay and separated using SDS polyacrylamide gel electrophoresis. α7B and β1D integrin were detected as previously described 281. P-p65 and p65-NF-κB antibodies (1:1000) blotting was followed by rabbit anti-HRP (1:2000, Cell

Signaling) and WesternSureTM PREMIUM Chemiluminescent Substrate (LI-COR). 58

Protein loading was normalized to either α-tubulin (1:1000 mouse-monoclonal,

Abcam) or GapDH (1:1000, rabbit-monoclonal, Cell Signaling). Quantitation was performed using Image J.

Histology and Immunofluorescence

C2C12 cells were grown in chamber slides, fixed with 4% paraformaldehyde and blocked in PBS containing 2% bovine serum albumin (Sigma), 0.1% Tween 20, and 0.05% Triton X-100 (American Bioanalytical) for 1h at room temperature. The cells were then incubated with the MF20 monoclonal antibody against Myosin

Heavy Chain (MHC) (1:100; DSHB) for 2.5h and subsequently with an Alexa Fluor

488-conjugated secondary antibody (1:200; Invitrogen) for 1h at room temperature. For in vivo immunofluorescence analysis, the hemi-diaphragm was embedded in a mixture of 2:3 (v/v) OCT and 30% sucrose. 10μm sections of diaphragm and gastrocnemius muscles from mice were immunostained using antibodies against α7B integrin and β1D as previously described 79 followed by

FITC-donkey-anti-rabbit (1:1000, Jackson ImmunoResearch, Baltimore, MD) incubation. Embryonic MHC (Mab 1:50, DSHB) overnight incubation at 4°C was performed on cryosections followed by 1hr incubation with Alexa Fluor 488- conjugated secondary antibody (1:200; Invitrogen). Wheat germ agglutinin

(1:1000) and Sirius Red stained cryosections were used for minimum Feret’s diameter measurements and percent fibrosis respectively using ImageJ. Cell preparations and cryosections were mounted using Vectashield containing DAPI 59

(4′, 6-diamidino-2-phenylindole) (H-1500, Vector Laboratories). Images were taken using an Olympus Fluoview FV1000 Laser Confocal Microscope.

Animals

It was determined that SU9516 was soluble in 10% hydroxypropyl-β-cyclo-dextrin

(HPβCD) and 90% saline (Vehicle) and could be delivered by oral gavage. Three- week old female mdx mice were administered a daily dose of 5mg/kg SU9516 until ten weeks of age. Female mdx mice were selected owing to availability for the study. All female mdx mice used in the study were homozygous for the DMDmdx allele. All animals were treated according to rules and regulations specified in the

University of Nevada Reno IACUC. At the end of the study, the mice were euthanized by cervical dislocation under anesthesia and diaphragms harvested for either contractile measurements 312 or phrenic nerve stimulation studies.

Pharmacokinetic and Toxicity studies with SU9516

Initially, it was determined that SU9516 was soluble in 10% hydroxypropyl-β-cyclo- dextrin (HPBCD) and 90% saline (vehicle) and could be delivered by oral gavage.

For initial PK studies a single 10mg/kg SU9516 dose in CD1 mice and examining serum, intestine, and muscle concentrations of SU9516 were determined by mass spectrometry over a 24 hour period.

Forelimb grip-strength measurement 60

Forelimb grip-strength was measured with a computerized grip-strength meter

(Columbus Instruments, Columbus, OH) according to guidelines published by the

Treat-NMD neuromuscular network. The single best recorded value for each mouse is represented in the data analysis.

Intracellular microelectrode recording post phrenic nerve stimulation

The diaphragm was dissected and pinned flat in a Sylguard lined 6 cm petridish continuously perfused with a modified Krebs-Ringer solution (NaCl, 121.0mM; KCl,

5.0mM; NaHCO3, 24.0mM; NaH2PO4, 0.4mM; MgCl2, 0.5mM; CaCl2, 1.8mM; glucose, 5.5mM (continuously gassed with 5% CO2–95% O2, pH 7.3–7.4) maintained at 23°C. The phrenic nerve was drawn into a suction electrode and stimulated with suprathreshold square pulses (0.1ms) using a Grass S48 stimulator (Quincy, MA, USA) coupled to a stimulus isolation unit (Grass, SIU5).

Endplate potentials (EPPs) were recorded in the presence of 2.3µM µ-conotoxin

(Peptides International, Louisville, KY, USA) with borosilicate micro-electrodes

(resistance = 30 MΩ) filled with 3M KCl. The endplate region containing neuromuscular junctions was identified by the recording of miniature EPPs with rise-to-peak times less than 2ms and was confirmed by post-hoc fluorescent α- bungarotoxin labeling. EPPs were only collected from muscle fibers with resting membrane potentials more negative than -65mV. EPPs were amplified using an

Axoclamp 900A amplifier, digitized at 2 KHz using a Digidata 1550 and recorded using Axoscope software before being analyzed with the Clampfit data analysis module within pCLAMP 10 software (Molecular Devices, Sunnyvale, CA, USA). 61

For synaptic rundown experiments, the phrenic nerve was continuously stimulated for 60 seconds and half-maximal EPP amplitudes were measured in relation to the initial EPP. A minimum of 3 trains of EPPs from each diaphragm was recorded

(n=3). Differences in EPP amplitude as well as time to half-maximal EPP were assessed by unpaired Student’s t-tests assuming equal variance.

Functional imaging was performed on a Nikon Eclipse FN1 upright fluorescence microscope using Nikon Plan Fluor 4X lens (Nikon, USA). Image sequences were captured using an Andor Neo (Andor Technology, Belfast, UK) sCMOS camera using Nikon NIS Elements 4.1 (Nikon, USA). Image sequences were recorded at

25 frames/s, processed as 8-bit intensity units and analyzed using in-house custom-written software (Volumetry G7; G.W.H.).

Statistical Analysis

Graphpad prism software was used to fit dose response curves using nonlinear regression analysis with log (agonist) vs. response with a variable slope. A constraint equal to 1 was placed on the bottom of the curve and either 2 or 2.5 at the top (when needed) in order to produce appropriate IC50 values. Analysis of

C2C12 fiber size, central nuclei, hydroxyproline content, eMHC positive fibers were compared using Student’s t-test. Specific force measurements and neuromuscular parameters were compared using one-way and two-way ANOVA followed by Tukey’s post hoc test. Averaged data are reported as the mean ± the standard error of the mean. P<0.05 was considered statistically significant.

62

RESULTS

SU9516 enhances α7 integrin levels through the inhibition of the SPAK/OSR1 kinase pathway

Using a muscle cell-based assay previously described 309 we screened the

LOPAC library and identified SU9516, a compound described in literature as a selective CDK2 inhibitor 313. SU9516 dose-response curves for α7+/lacZ myoblasts

(Fig. 1A) and α7+/lacZ myotubes (Fig. 1B) were generated for treatments ranging from 0.5-40µM with maximal calculated increase (~3-fold increase) within the established effective therapeutic range 92. The dose-response curves have also been represented in a linear scale in Supplemental Figure S1A and S1B. The maximum effective concentration varied between myoblasts (~5µM) and myotubes

(~12µM), likely due to a reduction in proliferation observed in myoblasts (data not shown). The α7B integrin protein enhancing effects of SU9516 were initially verified in C2C12 myotubes (Fig. 1C) and subsequently in human DMD patient myotubes over a range of concentrations (Fig. 1D). The maximum effective concentration was ~1µM in human DMD myotubes with statistically significant elevation of α7B integrin protein levels at a concentration of 20nM. Together, these data demonstrate that SU9516 treatment of human and mouse myogenic cell lineages leads to increased α7 integrin protein.

We previously tested eight CDK inhibitors (Supplemental Table S1) with different selectivity and did not see an increase in α7 integrin in myotubes.

Therefore, we believe that the increase in α7 integrin mediated by SU9516 is not 63 related to CDK activity. To better determine the specific kinase inhibitory profile of

SU9516 in human DMD patient myotubes, we performed a KiNativ profile assay.

Human DMD patient myotubes were treated for 48 hours with, 0.1, 0.5 and 1µM

SU9516, after which, cell lysates were subjected to a KiNativ in situ profiling assay to identify kinase targets (Supplemental Table S2). The purpose of the KiNativ assay was to elucidate the mechanism of action of SU9516, namely a specific kinase target that potentially regulates α7 integrin. Based on results depicted in

Fig.1D, we observed that in human DMD myotubes, significant increases in α7B integrin protein levels were seen at concentrations above 20 nM. The maximum effective concentration (maximum increase in α7B integrin) was ~1µM in human

DMD myotubes. In order to narrow down the kinases that were inhibited by

SU9516 within the effective dose range of 100 nM to 1 µM in human DMD myotubes, we normalized the three concentrations against the ‘non-effective’ dose of 0.01 uM (10nM). All kinases that were inhibited at this concentration or lower were hence eliminated as possible candidates for mechanism of action of SU9516.

The KiNativ data confirmed that SU9516 inhibited CDK2, 4 and 5 by >60% inhibition at 1µM. In addition, two kinases were inhibited across all concentrations of SU9516, namely the STE20/SPS1-related proline-alanine-rich protein kinase

(STK39/STLK3/SPAK) and the SPAK homolog oxidative stress response -1

(OSR1) kinase. These kinases were inhibited ~80% at the lowest concentration of 0.1µM SU9516 (Table 1). To further test whether inhibition of SPAK/OSR1 kinases increased α7 integrin, utilizing α7+/lacZ myotubes we generated a dose response curve for a potent suppressor of SPAK/OSR1 activity designated 64

STOCK1S-50699 (Fig. 1E). The linear dose response curve for STOCK1S-50699 is depicted in Supplemental Figure S1C. STOCK1S-50699 is a conserved carboxy terminal (CCT) domain inhibitor, a domain that is present on SPAK/OSR1 kinases.

Analysis of the dose curve for STOCK1S-50699 in myogenic cells showed an increase in α7 integrin expression levels nearing the maximum fold increase attained by SU9516, although STOCK1S-50699 exhibited toxicity in myotubes at higher concentrations (Fig. 1E). Additionally, western blot analysis utilizing

STOCK1S-50699 at a concentration as low as 25nM in human DMD myotubes showed an increase in levels of α7B integrin (Fig. 1F). Together, these data suggest that SU9516 inhibits the SPAK/OSR1 kinase activity in myogenic cells, which leads to elevated α7 integrin in skeletal muscle.

SU9516 promotes myogenic differentiation

Myoblasts treated with SU9516 exhibited significant morphological changes and differentiation of myogenic cells was promoted irrespective of serum concentrations. In order to determine if SU9516 treatment promoted myogenic fusion/differentiation rates, C2C12 cells were allowed to differentiate in the presence of 12µM SU9516 or DMSO alone. At 72 hours post differentiation,

SU9516 treated myotubes were larger and contained more nuclei than DMSO treated controls. Myofiber size was quantified by measuring the average myofiber width, which increased ~3-fold in SU9516 treated cells over DMSO (Fig. 2 A and

B). The fusion index was also increased ~3-fold in SU9516 treated myoblasts (Fig.

2C) with a shift toward myotubes containing more nuclei per myotube relative to 65

DMSO treated controls (Fig. 2D). We evaluated α7B integrin as a marker of muscle differentiation through the course of differentiation of C2C12 myoblasts.

Western blot assays showed that SU9516 treatment upregulated α7B integrin by

~2.5-fold over DMSO as early as 48 hours post initiation of differentiation (Fig. 2E).

Together, these results support SU9516 as a mediator of myogenic fusion and differentiation, which make it a candidate drug for the treatment of dystrophies with impaired myogenic regeneration programs.

Pharmacokinetic profile of SU9516 in dystrophic mice

The uptake and metabolism of SU9516 delivered by oral gavage was investigated in order to better define the optimal dose selection for initiating preclinical studies in mdx mice. For pharmacokinetic (PK) studies, CD1 mice were treated via oral gavage with a single dose of 10mg/kg. The intestines, muscle and serum (n=3) were assayed at various time points over the next 24-hours for SU9516. The serum half-life- t1/2 was determined to be 1.03 hours (Supplemental Figure S2A).

The 10 mg/kg SU9516 dose appears to be near the maximum tolerated dose as the CD1 mice used in the study showed torpidity up to 24 hours post-treatment.

Elevated levels of SU9516 were observed in the intestine and plasma

(Supplemental Figure S2B) while only low transient levels were observed in muscle (Supplemental Figure S2C). The poor distribution in muscle might be attributed to a low volume of distribution or enhanced secretion. A second dosing study was then performed with SU9516 in C57BL10 mice in order to determine the long-term tolerated dose and test α7 integrin enhancing activity. Daily treatments 66 67

Figure 1. SU9516 increases α7 integrin in myogenic cell lines through the inhibition of the SPAK/OSR1 pathway. SU9516 shows an increase in β-

Galactosidase activity in (A) α7+/lacZ myoblasts and (B) α7+/lacZ myotubes over a wide range of concentrations. Western blot analysis confirmed that treatment with

12µM SU9516 increased the levels of α7B integrin post 48hrs in (C) C2C12 myotubes (n=3) and (D) telomerized human DMD patient myotubes over a wide range of concentrations (n=3/conc.). (E) STOCK1S-50699 showed an increase in

β-galactosidase activity and obtained maximum fold fluorescence levels, nearly as high as SU9516 in α7+/lacZ treated myotubes (n=10/conc.). (F) Western Blot analysis in patient human DMD myotubes treated with STOCK1S-50699, showed a 1.8-fold increase in α7B integrin levels (n=3). ***P<0.001, **P<0.01, *P<0.05

68 of vehicle, 2.5mg/kg, 5mg/kg and 10mg/kg doses of SU9516 were administered via oral gavage for 4 days. After 3 days, dosing of the 10mg/kg/day group was stopped due to increasingly obvious mouse distress. As the 5mg/kg/day SU9516 dose appeared to be well tolerated, with no obvious changes in mouse behavior, it was chosen for preclinical pharmacodynamic studies in the mdx mouse model.

SU9516 promotes an increase in α7B and β1D integrin in mdx skeletal muscle.

In order to determine if SU9516 exhibited on target in vivo activity, female mdx mice were treated from 3 to 10 weeks of age with either 5mg/kg/day of

SU9516 or vehicle (2-hydroxypropyl-β-cyclo-dextrin). During the course of treatment, none of the experimental mice showed adverse reactions to this dose of SU9516. At 10 weeks of age, diaphragm and gastrocnemius tissues used to quantify α7B and β1D integrin protein levels. A 2.0- and 2.9-fold increase was observed for α7B and β1D integrin respectively, in the diaphragm muscle of

SU9516 treated mdx mice relative to vehicle treated controls (Fig. 3 A, B and C).

The gastrocnemius showed a similar 2-fold increase in α7B integrin but a lower

1.7-fold increase in β1D integrin levels with SU9516 treatments compared to vehicle treated mice (Fig. 3 D, E and F). Immunofluorescence analysis displayed normal sarcolemmal localization of both α7B and β1D integrin in both the diaphragm and the gastrocnemius muscles of all mice (Fig. 3 G and H). Together, these data show that daily oral treatments of SU9516 increased the α7β1 integrin to therapeutic levels in the skeletal muscle of mdx mice. 69

70

Figure 2. SU9516 expedites differentiation in C2C12 myoblasts. C2C12 myoblasts were differentiated with 12µM SU9516 or DMSO. Post 72hrs

(n=3/group) were fixed and immunostained for MHC. (A) Myofibers stained at

72hrs (green-MHC; blue-DAPI) Magnification 40X, Scale bar=100µm. (B) At 72 hrs, myotube diameters were increased with SU9516 treatment versus DMSO (C)

SU9516 treated myogenic cells showed a greater fusion index defined as the ratio of the number of nuclei in MHC-positive myotubes to the total number of nuclei in one field for five random microscopic fields. (D) SU9516 treatment increased the fraction of total myotubes with higher numbers of nuclei. Results represent means and SEM for three independent experiments. (E) SU9516 treated C2C12 cells showed an early increase in α7B integrin with differentiation versus DMSO *P<0.05

**P<0.01 *** P < 0.001

71

SU9516 treatment improves in vivo outcome measures and muscle function in mdx mice

In order to assess SU9516 safety and efficacy, several parameters including body mass and forelimb grip-strength were measured. After ~5-weeks of preclinical treatments, the SU9516 treated mdx mice displayed decreased body mass gain with a progressive shift towards WT body weight compared to vehicle treated controls and were significantly different from vehicle treated animals at 9 and 10 weeks of age (Fig. 4A). Similarly, normalized average forelimb grip- strength indicated that SU9516 treated mdx mice had progressive improvements in muscle function compared to vehicle treated animals, with significant improvements at 8 and 9-weeks of age (Fig. 4B). We asked whether treatment with integrin enhancing drug, SU9516 restores the force deficit seen in the diaphragm of mdx mice 312 by measuring the maximum isometric force produced by isolated diaphragm strips (2-4 mm in width). Measurements of in vitro active force developed by diaphragm muscle strips from WT mice, vehicle treated mdx and SU9516 treated mdx mice were performed in independent experiments.

Results revealed that diaphragm muscles from SU9516 treated mice had higher developed tetanic force (118.1 ± 7.914mN/mm2) compared to vehicle treated mice

(87.5 ± 6.106mN/mm2) (Fig. 4C). The diaphragm muscle of SU9516 treated mdx mice produced higher specific force amplitudes (~26-29% increase) compared to vehicle treated counterparts at peak tension (150Hz) as well as stimulation frequencies within the range of 100-150Hz (Fig. 4D). Diaphragm fatigue was also evaluated, defined as percent decline in amplitude relative to the initial amplitude, 72 after a bout of serial stimulations. No improvements in resistance to fatigue were evident in SU9516 treated mdx (Fig. 4E), however, after five and ten minutes of recovery post the fatigue protocol, the SU9516 treated mdx diaphragms recovered to a greater extent (~8%) compared to the vehicle treated diaphragms (Fig. 4F).

Taken together, these results indicate that treatment with the small molecule compound SU9516 restored mdx body mass and strength towards WT levels.

Studies have shown that the neuromuscular junction (NMJ) is structurally and functionally altered in mdx mice 314. To determine if SU9516 improve NMJ function, we examined muscle force in response to nerve stimulation using a novel optical method. Bright-field videos of phrenic nerve stimulation-induced diaphragm contraction were recorded and post-process analyzed using in-house designed custom software 315 (Volumetry G7, G.W.Hennig). Representative traces of the contractile response, measured as the displacement of an identified region of muscle fiber in response to a train of continuous phrenic nerve stimulations, are depicted in Supplemental Figure S3A. In response to 10 and 20Hz nerve stimulation, no differences in peak amplitudes were observed between vehicle and

SU9516 treated animals. However, at 40Hz frequency, the average peak amplitude of displacement attained by the SU9516 treated diaphragms was ~2.6- fold higher than the vehicle group (Supplemental Figure S3B). Analysis of the integrated area under the curve (AUC) from the contractile responses showed trends toward (not significant) greater cumulative displacement over time in

SU9516 treated diaphragms (P=0.07 for SU9516 vs Vehicle at

40Hz) (Supplemental Figure S3C). These results suggest that mdx mice exhibit 73

Figure 3: SU9516 increases α7β1 integrin levels in the skeletal muscle of mdx mice. Western blot analysis performed in the diapoahragm and the gastrocnemius muscle of 10-week old mdx mice showed (A) an increase in 74 levels of α7B and β1D integrin in the diaphragm of the SU9516 treated mdx mice. This increase is quantified in (B) where an ~2-fold increase in α7B and an

(C) ~3.4-fold increase in β1D was observed in the diaphragm of SU9516 treated mdx mice. (D) Western blot analysis in the gastrocnemius showed an increase in levels of α7B and β1D integrin in SU9516 treated mdx mice. These increases were quantified in (E) where an ~2-fold increase in α7B and an (F) ~1.7-fold increase was observed in SU9516 treated mdx mice. Immunofluoresence performed on 10 µm cryosections for α7B and β1D integrin in (G) the diaphragm and (H) the gastrocnemius muscle of mdx mice showed sarcolemmal localization. (N=4 diaphragms/group, N=4-6 gastrocnemius/group, *P<0.05,

***P<0.001)

75 impaired neuromuscular function, and that SU516 partially rescues this impairment. In order to directly test this hypothesis, we examined the effects of sustained phrenic nerve stimulation on acetylcholine (ACh) release by measuring end plate potentials (EPP) in WT and mdx mice.

The time to 50% EPP amplitude from peak EPP amplitude during 10Hz stimulation did not vary between WT, vehicle or SU9516 treated mdx mice; and did not reach 50% of peak amplitude within the first 60 seconds of stimulation

(Supplemental Figure S3D). However, at 20Hz stimulation frequencies, the vehicle-treated mdx EPP reached half-maximal EPP amplitude significantly faster

(Supplemental Figure S3D and E), compared to SU9516-treated mdx mice.

Together, these studies show that mdx mice exhibit enhanced synaptic rundown of neurotransmitter release in response to high-frequency nerve stimulation, and that treatment with SU9516 partially rescues this deficit.

SU9516 improves regeneration in the mdx diaphragm through the inhibition of the p65-NF-κB signaling pathway

In order to assess the physiologic effects of SU9516 treatment in mdx mice, we performed muscle histological analyses. Histopathological analysis of the diaphragm showed a 5.5% increase in the percentage of centrally nucleated myofibers (a marker of damage and/or regeneration) in SU9516 treated mdx diaphragms relative to vehicle treatment alone (Fig. 5A). Furthermore, immunofluorescence for embryonic myosin heavy chain (eMHC) positive fibers in mdx mouse diaphragms treated with SU9516 relative to vehicle treated controls 76

77

Figure 4. SU9516 improves in vivo outcome measures and diaphragm muscle function in the mdx mice. All comparisons were made across WT,

Vehicle treated mdx and SU9516 treated mdx mice. A) SU9516 treated mice showed a smaller gain in body mass compared to vehicle treated controls in weeks

9 and 10. WT animals showed the least increase in body weight over time. Weekly forelimb grip-strength B) SU9516 treated mdx mice showed greater muscle strength compared to vehicle treated mdx mice at weeks 8 and 9. C) At 10 weeks of age, mouse diaphragm muscle function was assessed. At a 100Hz tetanic stimulus, SU9516 treated mdx mice showed greater isometric tetanic tension compared to vehicle treated controls. D) A force-frequency protocol to measure tetanic tension generated by the diaphragm showed that SU9516 treated diaphragms produced higher tension compared to vehicle treated diaphragms when stimulated between 100-150Hz frequencies. E) Mdx diaphragms in both

SU9516 and vehicle treated groups fatigued to ~80% of the initial force. F)

SU9516 diaphragms recovered by ~8% compared to vehicle, 10 minutes post recovery from fatigue. Tukey post-hoc test annotations:*P<0.05, **P<0.01,

***P<0.01 for SU9516-mdx vs Vehicle-mdx treated mdx mice. #P<0.05, ##P<0.01,

### P<0.001. #### P<0.0001 Vehicle-mdx vs WT. +P<0.05, ++P<0.01 for WT vs

SU9516-mdx mice.

78

(Fig. 5B and C). It has previously been shown that pharmacological inhibition of p65-NF-κB pathway in 3 week old mdx mice increased the number of eMHC fibers by 47% 96. We hypothesized that elevated levels of regeneration seen with

SU9516 might be, at least partially attributed to, the inhibition of the p65-NF-κB pathway. Therefore, lysates from the diaphragms of 10-week-old WT, vehicle and

5mg/kg/day SU9516 treated mdx mice were analyzed via western blot for p-p65

NF-κB and normalized to total p65. We found that the SU9516 treated mdx mouse diaphragms had a ~2.83-fold decrease in the levels of phospho-p65 over total p65 compared to vehicle treated controls (Fig. 5D). In vitro, treatment of human DMD patient myotubes with 1µM SU9516 showed a similar 3-fold inhibition of p-p65-NF-

κB compared to vehicle treated myotubes (Fig. 5E). These results indicate that

SU9516 treatment attenuated the activation of the p65- NF-κB pathway, thereby promoting myofiber regeneration in vivo.

SU9516 improves fiber size, muscle regeneration and reduces fibrosis in the mdx diaphragm

Two common histological changes in mdx muscle are changes to fiber diameter and fibrosis. To assess whether SU9516 altered these outcome measures, we performed minimum Feret’s diameter and Sirius red staining for evaluation of collagen content in the diaphragms of experimental mice. We observed a fiber of size shift toward larger fibers in the SU9516 treated mdx muscles relative to the vehicle treated mdx group (Fig. 6A). Sirius red staining showed decreased area fibrosis expressed as a percentage of total area (Fig. 6B). Together these results 79

Figure 5: SU9516 promotes myofiber regeneration through the inhibition of the p65-NF-κB pathway in mdx skeletal muscle. A) SU9516 treated mdx diaphragms showed a 5.5% increase in percent of centrally nucleated fibers over vehicle treated diaphragms. B) The percentage of embryonic myosin heavy chain

(eMHC) positive fibers in the diaphragm of SU9516 treated mdx was higher than vehicle treated mdx by 3.3%. C) eMHC staining in immunofluorescence images of diaphragms from vehicle and SU9516 treated mdx mice. SU9516 treated 80 diaphragms show an increase in eMHC positive fibers. D) SU9516 treatment decreases level of p-p65-NF-κB by ~2.83-fold in lysates of mdx diaphragm compared to vehicle treated controls E) Western blot analysis for total phosphorylated p65-NF-κB showed that SU9516 treatment decreases the level of p-p65-NF-κB by > 3-fold in human DMD patient myotubes, compared to the vehicle treated counterparts. (N=3/mdx treatment group,*P<0.05, **P<0.01)

81 indicate SU9516 improved muscle regeneration and reduced pathology in mdx muscle by enhancing sarcolemmal stabilization through elevated α7β1 integrin and also improved regeneration through p65-NF-κB inhibition (Fig. 6C).

SU9516 improves fiber size, muscle regeneration and reduces fibrosis in the mdx diaphragm

Two common histological changes in mdx muscle are changes to fiber diameter and fibrosis. To assess whether SU9516 altered these outcome measures, we performed minimum Feret’s diameter and Sirius red staining for evaluation of collagen content in the diaphragms of experimental mice. We observed a fiber size shift toward larger fibers in the SU9516 treated mdx muscles relative to the vehicle treated mdx group (Fig. 6A). Sirius red staining showed decreased area of fibrosis expressed as a percentage of total area (Fig. 6B).

Together these results indicate SU9516 improved muscle regeneration and reduced pathology in mdx muscle by enhancing sarcolemmal stabilization through elevated α7β1 integrin and also improved regeneration through p65-NF-κB inhibition (Fig. 6C).

82

Figure 6: SU9516 ameliorates pathology in mdx skeletal muscle. A) Myofiber size distribution in diaphragms of WT, vehicle and SU9516 treated mice was 83 assessed utilizing 10 µm cryosections stained with wheat germ agglutinin followed by minimum Ferret’s diameter measurements. The distribution of fiber size in

SU9516 treated diaphragms shifted towards WT fiber size distribution i.e. larger myofibers. E) The percent of fibrotic area as quantified by Sirius Red staining showed a decrease in collagen positive areas within the SU9516 treated mdx diaphragm cross-sections, compared to vehicle treated mdx mice. (N=3 WT,

N=4/mdx treatment group,*P<0.05, **P<0.01) C) Proposed model for mechanism of action by which SU9516 ameliorates dystrophic pathology and enhances α7β1 integrin in dystrophic muscle fibers.

84

DISCUSSION

This study identifies SU9516 as a novel α7 integrin enhancing compound in muscle and demonstrates the benefits of using this therapeutic to modify disease progression in the mdx mouse model of DMD. SU9516 is an indolinone compound, which has been shown to be a potent inhibitor of CDK2 along with a host of other kinases 316. In vitro experiments in this study showed that SU9516 increased the protein levels of α7B integrin in human DMD patient and C2C12 myogenic cells. Additionally, a seven-week treatment of 5mg/kg/day SU9516 increased the protein levels of α7B and β1D integrin in the skeletal muscle of dystrophin-deficient mdx mice, thereby demonstrating in vivo on-target activity. In

DMD patients, the skeletal muscles progressively weaken, pathology is severe and patients lose their ability to walk by 13 years of age 317. In mdx mice, however, skeletal muscle pathology is comparatively mild and appears to plateau after 3 months of age. In contrast, the mdx diaphragm is more severely and progressively affected in mdx mice and thus, more representative of muscle pathology in DMD patients 318. The ex vivo muscle contraction experiments performed in diaphragms of mdx mice showed that SU9516 increased the specific force developed by the mdx diaphragm. Additionally, phrenic nerve stimulation and intracellular recordings of myofibers in the diaphragm showed that SU9516 treated mdx muscles demonstrated higher peak amplitudes of displacement and slowed synaptic fatigue. It is likely that these improvements are partially due to elevated levels of α7β1 integrin in muscle, with SU9516 treatment. 85

Our study also identified SU9516 as an inhibitor of p65-NF-κB signaling activation in skeletal muscle. In mdx mice, increased NF-κB activity exacerbates

DMD pathology through increased immune cell activity and the inhibition of myogenic differentiation of muscle precursors319. Inhibiting NF-κB signaling either genetically or by pharmacological means promoted formation of new myofibers in response to degeneration 96,320. Hence, we conclude that the increase in centrally nucleated myofibers and eMHC-positive myofibers seen in the diaphragm of

SU9516 treated mdx mice could be attributed to SU9516 inhibition of p65-NF-κB activation. Recently it was shown that β1 integrin was the sensor of the satellite cell (SC) niche in skeletal muscle and the activation of β1 integrin signaling in the mdx mouse promoted expansion of the SC population giving rise to robust myofiber regeneration as well as improved function 321. Hence, it is also possible that SU9516 promotes myofiber regeneration through enhanced expression and activity of β1 integrin.

In addition to blocking CDK2 activity, we determined that in DMD muscle cells, SU9516 is a potent inhibitor of SPAK/OSR1 kinases (SPAK). Functionally,

SPAK kinases are stress sensors that activate the p38 MAP kinase pathway, having a cell-type-dependent and a stimuli-dependent subcellular distribution322.

Importantly, specific stress stimuli such as hyperosmotic conditions significantly increased their expression levels and recruitment to membranes of cells323.

Moreover, previous authors have shown the capacity of p38α to modulate NF-κB transcriptional activation and TNF-α production through phosphorylation of p65-

NF-κB, mediated by mitogen- and stress-activated protein kinase 1 (MSK1) 324. 86

Aligning with these observations, it seems that inhibition of SPAK/OSR1 kinases

in DMD muscle cells by SU9516 can downregulate the stress related downstream

p65-NF-κB activation. Further pharmacological experiments utilizing STOCK1S-

50699, a known inhibitor of SPAK/OSR1, showed that α7 integrin levels increase

with suppression of SPAK/OSR1 activity. STOCK1S-50699 is highly hydrophobic,

exhibits poor solubility and cannot be used in animal models, but the data obtained

in our experiments provides evidence that development of SPAK/OSR1 inhibitors

is feasible for targeting α7 integrin in muscle. While further experiments are

warranted to evaluate the relevance of this pathway in DMD, our results shed light

on a novel mechanism of action for the regulation of integrin 7. In our study, we

demonstrate for the first time that a small molecule α7β1 integrin enhancing

compound can act to prevent muscle disease progression in the mdx mouse model

of DMD. Previous studies have investigated the benefits of utilizing SU9516 as an

apoptotic drug for the treatment of leukemia 325. It was observed that at

concentrations of >=5 µM SU9516, apoptotic pathways were triggered in U937 and

other leukemia cell lines. It has been shown that apoptosis is a response to the

downregulation of the antiapoptotic protein Mcl-1 with SU9516 treatment 325. This is also the likely explanation for the narrow therapeutic range of SU9516, with toxicity observed at higher doses in the mdx murine model. Hence, derivatives of

SU9516 with reduced toxicity are warranted for clinical trials. This study leads the way for further development of small molecule therapeutics targeting the α7β1 integrin complex in DMD.

87

88

Figure S1: Linear dose response curves for SU9516 and STOCK1S-50699 in myogenic cell lines. This experiment shows that both drugs increased α7 integrin promoter activity. A) SU9516 shows an ~2.3-fold increase in β-galactosidase activity in α7+/lacZ myoblasts over DMSO treated cells. B) SU9516 shows an ~2- fold increase in β-galactosidase activity in α7+/lacZ myotubes over DMSO treated cells. C). STOCK1S-50699 treatment shows an ~1.5-fold increase of β- galactosidase activity over DMSO treated α7+/lacZ myotubes.

89

Figure S2: Pharmacokinetics of SU9516. A) SU9516 has a serum half-life of

30 minutes. B) A large portion of the drug is cleared in the intestine which indicates minimal absorption. C) The concentration of the drug in the target tissue namely muscle, after a single dose is very low. 90

91

Figure S3: Contraction of diaphragm to phrenic nerve stimulation in wild type, vehicle and SU9516-treated mice. A) Representative traces of the contractile response of diaphragms from a WT, vehicle-mdx and SU9516-mdx mouse to repeated stimulations at 10, 20 & 40Hz for ~40s. B) WT mice showed greater peak amplitude of displacement in response to 10, 20 and 40Hz nerve stimulation, compared to mdx mice in both treatment groups. SU9516-treated mdx mice showed a higher peak amplitude in response to 40Hz stimulation, compared to vehicle treated mdx mice (N=5, *P<0.01) C) The composite integration of the area under the curve showed a trend toward increased total displacement in the SU9516 treatment group compared to the vehicle treated group in response to 20 and 40Hz nerve stimulation (N=4, P=0.07). D) Synaptic rundown of neurotransmitter is enhanced in mdx mice relative to WT mice, and significantly improved in SU9516-treated mdx mice, relative to vehicle-treated mdx mice, in response to trains of continuous 20Hz nerve stimulation, as measured by the time to half-maximal EPP. E) Representative images of EPP trains in response to continuous 20Hz nerve stimulation in WT and both treatment groups of mdx mice. (N=3,*P<0.01).

92

Table S1. Dose response curves for eight CDK inhibitors tested in α7+/lacZ myotubes. These experiments were done to eliminate the possibility that the known CDK inhibitor- SU9516 increases a7 integrin promoter activity through the inhibition of CDKs. For each CDK inhibitor, a list of CDKs inhibited has been summarized in the table above. 93

SU9516 SU9516 SU9516 Kinase Reference Sequence Labeling Site 1 µM 0.5 µM 0.1 µM IC50 (µM) ABL, ARG UniRef100_P005 LMTGDTYTAHAGAKFPIK Activation Loop ‐4.4 33.1 32.0 >1 ABL, ARG UniRef100_P005 YSLTVAVKTLKEDTMEVEEFLK Lys1 ‐92.4 ‐33.6 15.8 >1 AKT1 UniRef100_P3174GTFGKVILVK ATP Loop Anchor G2 ‐10.8 ‐22.6 ‐9.0 >1 AKT2, AKT3 UniRef100_Q9Y2GTFGKVILVR ATP Loop Anchor G2 ‐19.3 ‐33.7 ‐15.0 >1 AMPKa1, AMPKa2 UniRef100_P5464DLKPENVLLDAHMNAK Lys2 ‐50.9 ‐1.5 ‐3.8 >1 ANPb UniRef100_P2059GMAFLHNSIISSHGSLKSSNCVLys2 4.8 ‐73.2 ‐48.8 >1 ARAF UniRef100_P1039DLKSNNIFLHEGLTVK Lys2 ‐11.9 ‐13.6 ‐2.8 >1 AurA UniRef100_O149DIKPENLLLGSAGELK Lys2 30.8 14.4 18.9 >1 AurA UniRef100_O149FILALKVLFK Lys1 18.2 31.6 29.9 >1 BRAF UniRef100_P1505DLKSNNIFLHEDLTVK Lys2 11.6 18.0 13.6 >1 CaMK1a UniRef100_Q140LVAIKCIAK Lys1 ‐21.5 ‐17.7 ‐14.0 >1 CaMK1d UniRef100_Q8IU LFAVKCIPK Lys1 ‐16.4 ‐17.0 ‐14.3 >1 CaMK2b UniRef100_Q53HLCTGHEYAAKIINTK Lys1 20.0 22.5 18.3 >1 CaMK2d UniRef100_Q135IPTGQEYAAKIINTKK Lys1 21.0 27.6 7.6 >1 CaMK2g UniRef100_Q135TSTQEYAAKIINTK Lys1 27.2 21.4 12.3 >1 CaMKK2 UniRef100_Q96RDIKPSNLLVGEDGHIK Lys2 ‐34.8 12.2 26.4 >1 CASK UniRef100_O149ETGQQFAVKIVDVAK Lys1 10.3 16.0 ‐1.4 >1 CCRK UniRef100_Q8IZLDLKPANLLISASGQLK Lys2 ‐2.9 ‐25.3 ‐19.2 >1 CDC2 UniRef100_Q5H9DLKPQNLLIDDKGTIK Lys2 9.8 13.5 32.4 >1 CDK2 UniRef100_P2494DLKPQNLLINTEGAIK Lys2 60.3 61.8 45.0 <0.1 CDK4 UniRef100_P1180DLKPENILVTSGGTVK Lys2 64.0 45.1 26.9 0.59 CDK5 UniRef100_Q005DLKPQNLLINR Lys2 76.9 70.3 31.4 0.26 CDK7 UniRef100_P506 DLKPNNLLLDENGVLK Lys2 ‐20.8 ‐87.4 ‐92.6 >1 CHK2 UniRef100_O960DLKPENVLLSSQEEDCLIK Lys2 10.6 ‐0.4 1.9 >1 CSK UniRef100_P4124VSDFGLTKEASSTQDTGKLPVKDFG Motif 4.8 ‐1.1 ‐2.1 >1 DCAMKL1 UniRef100_O150DIKPENLLVYEHQDGSK Lys2 17.3 20.9 26.2 >1 DGKA UniRef100_P2374IDPVPNTHPLLVFVNPKSGGK ATP ‐49.5 ‐58.4 ‐37.6 >1 DGKQ UniRef100_P5282GRLLTALVLPDLLHAKLPPDSC ATP ‐26.5 ‐85.8 ‐27.6 >1 DNAPK UniRef100_P7852KGGSWIQEINVAEK ATP ‐42.2 ‐15.3 ‐39.1 >1 eEF2K UniRef100_O004YIKYNSNSGFVR ATP ‐1.1 ‐36.8 ‐24.8 >1 EGFR UniRef100_P0053LLGAEEKEYHAEGGKVPIK Activation Loop 17.8 ‐18.1 ‐48.5 >1 EGFR UniRef100_P0053IPVAIKELR Lys1 ‐14.4 ‐24.3 ‐43.8 >1 Erk1 UniRef100_P2736DLKPSNLLINTTCDLK Lys2 22.1 13.1 23.6 >1 Erk2 UniRef100_P2848DLKPSNLLLNTTCDLK Lys2 21.0 8.9 16.5 >1 FER UniRef100_P1659TSVAVKTCKEDLPQELK Lys1 ‐4.2 8.1 ‐1.4 >1 FES UniRef100_P0733LRADNTLVAVKSCR Lys1 10.6 ‐0.4 12.4 >1 FRAP UniRef100_P4234IQSIAPSLQVITSKQRPR ATP ‐16.9 ‐8.8 ‐1.2 >1 FYN, SRC, YES UniRef100_P1293QGAKFPIKWTAPEAALYGR Activation Loop 12.0 26.1 33.5 >1 GCK UniRef100_Q128DIKGANLLLTLQGDVK Lys2 48.1 34.6 31.8 >1 GCN2 UniRef100_Q9P2DLKPVNIFLDSDDHVK Lys2 8.5 ‐31.4 ‐7.1 >1 GSK3A UniRef100_P4984DIKPQNLLVDPDTAVLK Lys2 37.0 13.4 2.3 >1 GSK3B UniRef100_P4984DIKPQNLLLDPDTAVLK Lys2 14.9 13.6 20.4 >1 HER2/ErbB2 UniRef100_P0462GIWIPDGENVKIPVAIKVLR Lys1 ‐1.6 ‐3.0 ‐29.8 >1 HPK1 UniRef100_Q929DIKGANILINDAGEVR Lys2 29.8 0.0 ‐22.1 >1 IKKa UniRef100_O151DLKPENIVLQDVGGK Lys2 9.5 12.8 27.1 >1 IKKb UniRef100_O149DLKPENIVLQQGEQR Lys2 20.7 ‐19.4 ‐1.7 >1 ILK UniRef100_Q134WQGNDIVVKVLK Lys1 ‐19.6 ‐1.9 8.4 >1 ILK UniRef100_Q134ISMADVKFSFQCPGR Protein Kinase Domain ‐53.8 5.8 26.9 >1 IRAK1 UniRef100_P516 AIQFLHQDSPSLIHGDIKSSNV Lys2 ‐13.4 0.6 ‐7.9 >1 IRAK4 UniRef100_Q9NWDIKSANILLDEAFTAK Lys2 19.7 3.4 ‐0.3 >1 JAK1 domain2 UniRef100_P2345IGDFGLTKAIETDKEYYTVK DFG Motif ‐47.7 ‐49.9 ‐44.7 >1 JNK1, JNK2, JNK3 UniRef100_P4598DLKPSNIVVK Lys2 ‐13.2 ‐4.4 ‐19.0 >1 KHS1 UniRef100_Q9Y4NVHTGELAAVKIIK Lys1 9.5 ‐1.6 ‐8.6 >1 KHS2 UniRef100_Q8IV NVNTGELAAIKVIK Lys1 ‐34.5 4.1 ‐1.5 >1 LATS2 UniRef100_Q9NRDIKPDNILIDLDGHIK Lys2 ‐17.2 ‐24.1 ‐7.4 >1 LKB1 UniRef100_Q158DIKPGNLLLTTGGTLK Lys2 26.2 17.6 12.0 >1 LOK UniRef100_O948DLKAGNVLMTLEGDIR Lys2 ‐56.1 2.0 23.8 >1 94

MAP2K1, MAP2K2 UniRef100_P3650DVKPSNILVNSR Lys2 15.3 15.9 2.7 >1 MAP2K1, MAP2K2 UniRef100_P3650KLIHLEIKPAIR Lys1 8.5 30.6 ‐2.1 >1 MAP2K3 UniRef100_P4673DVKPSNVLINK Lys2 14.0 ‐2.0 ‐8.5 >1 MAP2K4 UniRef100_P4598DIKPSNILLDR Lys2 21.7 13.5 17.0 >1 MAP2K6 UniRef100_P5256DVKPSNVLINALGQVK Lys2 7.8 6.9 8.5 >1 MAP2K7 UniRef100_O147DVKPSNILLDER Lys2 6.2 ‐34.3 ‐59.9 >1 MAP3K2 UniRef100_Q9Y2ELAVKQVQFDPDSPETSKEVNLys1 1.2 ‐19.4 ‐0.6 >1 MAP3K2, MAP3K3 UniRef100_Q9Y2DIKGANILR Lys2 34.1 6.3 ‐2.5 >1 MAP3K4 UniRef100_Q9Y6DIKGANIFLTSSGLIK Lys2 5.7 ‐59.5 ‐75.4 >1 MAP3K5 UniRef100_Q996DIKGDNVLINTYSGVLK Lys2 ‐12.0 ‐3.7 ‐30.7 >1 MARK1 UniRef100_Q9P0DLKAENLLLDGDMNIK Lys2 11.9 9.3 ‐5.3 >1 MARK2, MARK3 UniRef100_P2744DLKAENLLLDADMNIK Lys2 ‐3.3 26.3 12.4 >1 MARK3 UniRef100_P2744EVAIKIIDKTQLNPTSLQK Lys1 18.1 30.5 26.3 >1 MARK3, MARK4 UniRef100_Q96L EVAIKIIDK Lys1 ‐14.6 ‐16.8 2.0 >1 MAST1, MAST2 UniRef100_Q6P0DLKPDNLLITSMGHIK Lys2 ‐93.1 ‐19.8 ‐2.5 >1 MAST3 UniRef100_O603DLKPDNLLITSLGHIK Lys2 27.8 2.7 ‐6.9 >1 MAST4 UniRef100_O150DLKPDNLLVTSMGHIK Lys2 ‐9.6 18.9 27.9 >1 MER, TYRO3 UniRef100_Q064KIYSGDYYR Activation Loop ‐29.4 8.4 1.2 >1 MET UniRef100_P0858DMYDKEYYSVHNK Activation Loop ‐46.9 ‐5.8 ‐19.2 >1 MLKL UniRef100_Q8NBAPVAIKVFK Lys1 ‐2.0 18.5 10.3 >1 MSK1 domain1 UniRef100_O755DIKLENILLDSNGHVVLTDFGLSLys2 29.5 27.4 1.8 >1 MSK2 domain1 UniRef100_O756DLKLENVLLDSEGHIVLTDFGLSLys2 27.2 19.4 ‐18.1 >1 MST1 UniRef100_Q130ETGQIVAIKQVPVESDLQEIIK Lys1 25.5 21.7 6.4 >1 MST2 UniRef100_Q131ESGQVVAIKQVPVESDLQEIIKLys1 33.5 15.6 ‐9.0 >1 MST3 UniRef100_Q9Y6DIKAANVLLSEHGEVK Lys2 22.5 13.6 ‐6.0 >1 MST4, YSK1 UniRef100_O005DIKAANVLLSEQGDVK Lys2 45.4 35.0 7.7 >1 NDR1 UniRef100_Q152DIKPDNLLLDSK Lys2 6.5 ‐16.1 ‐2.0 >1 NEK1 UniRef100_Q96PDIKSQNIFLTK Lys2 ‐0.1 ‐5.2 ‐8.3 >1 NEK3 UniRef100_P5195SKNIFLTQNGK Activation Loop 8.3 ‐30.3 2.1 >1 NEK4 UniRef100_P5195DLKTQNVFLTR Lys2 ‐5.1 ‐32.7 ‐9.6 >1 NEK6, NEK7 UniRef100_Q8TDDIKPANVFITATGVVK Lys2 9.4 ‐2.0 ‐9.0 >1 NEK7 UniRef100_Q8TDAACLLDGVPVALKK Lys1 ‐8.0 ‐19.5 2.7 >1 NEK8 UniRef100_Q86S DLKTQNILLDK Lys2 ‐6.5 ‐9.5 ‐15.4 >1 NEK9 UniRef100_Q8TDDIKTLNIFLTK Lys2 12.8 ‐12.8 ‐5.5 >1 NuaK1 UniRef100_O602VVAIKSIR Lys1 1.4 23.7 ‐9.9 >1 OSR1 UniRef100_C9JIGDVKAGNILLGEDGSVQIADFG Lys2 85.1 82.8 81.4 <0.1 p38a UniRef100_Q165QELNKTIWEVPER Protein Kinase Domain 28.0 28.2 47.3 >1 PCTAIRE2, PCTAIRE3 UniRef100_Q005SKLTENLVALKEIR Lys1 21.3 14.3 ‐7.5 >1 PDK1 UniRef100_O155EYAIKILEK Lys1 28.8 3.9 34.0 >1 PFTAIRE1 UniRef100_O949LVALKVIR Lys1 78.6 67.4 36.0 0.20 PHKg2 UniRef100_P1573ATGHEFAVKIMEVTAER Lys1 ‐31.1 2.6 23.6 >1 PIK3C3 UniRef100_Q8NETEDGGKYPVIFKHGDDLR ATP ‐42.1 ‐18.2 ‐52.1 >1 PIK3CD UniRef100_O003VNWLAHNVSKDNRQ ATP 7.4 0.2 ‐21.6 >1 PIP4K2A UniRef100_P4842AKELPTLKDNDFINEGQK ATP ‐0.8 21.7 15.5 >1 PIP4K2B UniRef100_P7835AKDLPTFKDNDFLNEGQK ATP ‐15.6 0.1 ‐10.4 >1 PIP4K2C UniRef100_Q8TBTLVIKEVSSEDIADMHSNLSNYATP ‐46.7 1.2 14.9 >1 PITSLRE UniRef100_P2112DLKTSNLLLSHAGILK Lys2 ‐41.0 ‐30.2 ‐41.6 >1 PKR UniRef100_P1952DLKPSNIFLVDTK Lys2 ‐0.5 ‐10.8 ‐2.1 >1 PLK1 UniRef100_P5335CFEISDADTKEVFAGKIVPK Lys1 26.1 32.3 18.5 >1 PRPK UniRef100_Q96S FLSGLELVKQGAEAR ATP Loop ‐24.0 ‐35.8 ‐26.4 >1 RAF1 UniRef100_P0404DMKSNNIFLHEGLTVK Lys2 ‐39.7 24.3 33.9 >1 RSK1 domain1, RSK2 domaUniRef100_P518 DLKPENILLDEEGHIK Lys2 32.7 30.2 38.5 >1 RSK2 domain1 UniRef100_P518 DLKPENILLDEEGHIKLTDFGLS Lys2 25.3 29.9 32.8 >1 RSK2 domain2 UniRef100_P518 DLKPSNILYVDESGNPESIR Lys2 21.8 1.5 12.4 >1 RSK3 domain1 UniRef100_Q153DLKPENILLDEEGHIKITDFGLSKLys2 43.7 39.7 38.3 >1 RSKL1 UniRef100_Q96S VLGVIDKVLLVMDTR ATP ‐25.4 ‐0.2 27.0 >1 SGK UniRef100_O001HKAEEVFYAVKVLQK Lys1 ‐38.3 ‐39.6 ‐19.9 >1 SGK3 UniRef100_Q96BFYAVKVLQK Lys1 36.0 11.6 ‐2.2 >1 SIK UniRef100_P5705TQVAIKIIDK Lys1 51.4 13.5 25.1 0.95 SLK UniRef100_Q9H2DLKAGNILFTLDGDIK Lys2 22.0 ‐21.2 ‐13.3 >1 smMLCK UniRef100_Q157QGIVHLDLKPENIMCVNK Lys2 ‐38.0 ‐0.2 32.1 >1 SRPK1, SRPK2 UniRef100_P7836FVAMKVVK Lys1 ‐35.8 11.9 6.2 >1 STLK3 UniRef100_Q9UEDLKAGNILLGEDGSVQIADFGVLys2 84.7 79.4 79.1 <0.1 STLK5 UniRef100_Q7RTYSVKVLPWLSPEVLQQNLQGYActivation Loop 13.6 18.1 26.1 >1 95

TAO1, TAO3 UniRef100_Q7L7 DIKAGNILLTEPGQVK Lys2 26.6 ‐6.3 ‐7.5 >1 TAO2 UniRef100_Q9ULDVKAGNILLSEPGLVK Lys2 32.6 12.8 ‐27.8 >1 TEC UniRef100_P4268YVLDDQYTSSSGAKFPVK Activation Loop 28.9 ‐26.9 ‐39.3 >1 TLK1 UniRef100_Q9UKYLNEIKPPIIHYDLKPGNILLVDGLys2 ‐13.2 1.3 ‐3.9 >1 TLK2 UniRef100_Q86UYLNEIKPPIIHYDLKPGNILLVNGLys2 5.2 24.1 11.4 >1 ULK1 UniRef100_O753DLKPQNILLSNPAGR Lys2 12.1 ‐3.1 ‐1.8 >1 VRK2 UniRef100_Q86YMLDVLEYIHENEYVHGDIKAANLys2 ‐26.4 2.2 ‐41.9 >1 Wnk1, Wnk2, Wnk3 UniRef100_Q9Y3DLKCDNIFITGPTGSVK Lys2 ‐40.2 ‐29.0 ‐20.4 >1 YANK3 UniRef100_Q86UDVKPDNILLDER Lys2 0.6 12.1 13.9 >1 ZC1/HGK, ZC2/TNIK, ZC3/MUniRef100_O958DIKGQNVLLTENAEVK Lys2 38.0 11.1 ‐15.5 >1

Table S2. Kinase targets of SU9516 in human DMD myotubes. The KiNativ assay was performed to determine the kinase targets of SU9516, the inhibiton of which may lead to increased levels of α7 integrin.

Labeling Site Key Conserved Lysine Lys1 1 Conserved Lysine Lys2 2 ATP Loop ATP binding loop Activation Loop Activation loop ATP ATP site in non-canonical kinase (e.g. lipid kinase) Protein Kinase Domain Other lysine within kinase domain, possibly not in ATP binding site

Labeling of residue outside of the protein kinase domain, Other possibly not in ATP binding site

>90% Inhibition 75 - 90% Inhibition 50 - 75% Inhibition 35 - 50% Inhibition No change >100% increase in MS signal (>2 fold increase) ND Not determined Data points inhibited >35% & not considered significant are left uncolored

96

Chapter 3

Sunitinib promotes myogenic regeneration via transient SHP-2 inhibition/STAT3 activation and prevents Duchenne muscular dystrophy disease progression

97

ABSTRACT

Duchenne muscular dystrophy (DMD) is a lethal, muscle degenerative disease causing premature death of affected children. It is characterized by mutations in the dystrophin gene, resulting in a loss of dystrophin protein. Loss of dystrophin causes an associated reduction in proteins of the dystrophin glycoprotein complex (DGC), leading to contraction-induced sarcolemmal weakening, muscle tearing, fibrotic infiltration and rounds of degeneration and failed regeneration affecting satellite cell populations. The α7β1 integrin has been implicated in increasing myogenic capacity of satellite cells therefore restoring muscle viability, increasing muscle force and preserving muscle function in dystrophic model mice. In this study, we show that an FDA-approved small molecule, Sunitinib, is a potent α7β1 integrin enhancer capable of promoting endogenous myogenic regeneration by stimulating satellite cell activation and increasing myofiber fusion via inhibition of SHP-2 and consequential activation of the STAT3 pathway. Treatment with Sunitinib in mdx mice demonstrated decreased membrane leakiness and damage owing to myofiber regeneration and enhanced support at the extracellular matrix. Decreased myofiber damage translates into a significant increase in muscle force production. This study identifies an already FDA approved compound, Sunitinib, as a possible DMD therapeutic with the potential to cover a wide variety of muscle dystrophies.

Contributions to Chapter 3: Author performed all studies in Chapter 3. 98

INTRODUCTION

Duchenne muscular dystrophy (DMD) is one of the most common X-linked neuromuscular diseases, with an incidence of 1 in 5000, leading to premature death of affected children 4. DMD is characterized by the loss of dystrophin, a

427kDa protein found at the sarcolemma of skeletal, cardiac and vascular smooth muscle 3,21. Structurally, dystrophin is essential to anchor intracellular actin filaments and sarcolemmal proteins to promote myofiber stability 23,326. The loss of dystrophin in DMD leads to the absence of dystrophin-associated proteins that results in altered muscle cell signaling, contraction induced muscle degeneration and replacement with fibrotic and fatty tissue31,99,327. Clinical features of DMD include gross motor delays, loss of ambulation leading to wheelchair confinement, respiratory insufficiency requiring ventilator assistance and dilated cardiomyopathy beginning during the second decade of life and premature death 4,5.

Currently, there are limited treatment options available for DMD patients.

Therapeutic options include corticosteroids (Prednisone) and glucocorticoids

(Deflazacort), both used to decrease inflammation and suppress the immune response 328,329. Recently, the exon skipping drug eteplirsen (Exondys 51) was given Food and Drug Administration (FDA) approval 118. Although showing promising results, eteplirsen is only applicable to 14% of the DMD population with the specific exon 51 mutation. Therefore, it is still essential to develop therapies targeting pathways viable to all DMD patients, regardless of mutation. 99

One of the hallmarks of DMD pathology are cycles of muscle degeneration and failed muscle regeneration that occur in the absence of dystrophin 31. This faulty regeneration has been attributed to a number of factors, one of them being decreased satellite cell (SC) capacity. SCs are the essential precursors to myogenesis and in DMD, while increased in number, they have been shown to be impaired due to faulty asymmetric division and interrupted SC niches, leading to improper muscle regeneration 105,233,330,331. Therapeutic interventions aimed at enhancing SC capacity could prove significantly beneficial to DMD pathology.

Numerous studies have indicated that signal transducer and activator of transcription 3 (STAT3) activation via the interleukin-6 (IL-6) cytokine is important for SC proliferation and self-renewal in response to resistance exercise and muscle injury 332–337. Activated STAT3 can directly affect the expression of the myogenic regulatory marker MyoD1 and promote myoblast differentiation 338–341.

Additionally, STAT3 inhibition promotes enhanced symmetric SC proliferation and increased SC engraftment into injured muscle 342,343. Together these studies suggest that transient STAT3 activation is important for both proliferation of SCs and differentiation into mature myofibers.

Several studies have identified the α7β1 integrin as a positive modifier of

DMD disease pathology in different mouse models utilizing various transgenic and

AAV delivery techniques 84,85,345,86,87,89–91,93,94,344. More recently, our lab demonstrated that treatment with an α7β1 integrin enhancing small molecule,

SU9516, increased muscle force generation and decreased disease pathology in mdx mice 346. This improvement could be attributed, in part, to the role α7β1 100 integrin plays in promoting myogenic capacity of SC following eccentric exercise

283.

Sunitinib is structurally related to SU9516, which has previously been shown to increase α7 integrin levels and correct DMD phenotype in mdx mice, and is currently used as an FDA approved, multi receptor tyrosine kinase (RTK) inhibitor for the treatment of renal cell carcinoma 347,348. Sunitinib has also been implicated in modulating the STAT3 pathway in cancer 349. In the present study, we show for the first time that Sunitinib treatment promotes increased expression of α7 integrin via transient inhibition of SHP-2/ERK1/2 and activation of the STAT3 pathway in a skeletal muscle cell line and in vivo, in the mdx mouse model of DMD.

Treatment with Sunitinib promotes SC activation and myogenic regeneration, leading to significantly improved muscle disease pathology and functional skeletal muscle force production. Together, our results provide evidence that Sunitinib can be re-purposed into the first small molecule therapy targeting muscle regeneration for the treatment of DMD.

MATERIALS AND METHODS

Study Approval

All animals were treated according to the rules and regulations specified in the

University of Nevada Reno Institutional Animal Care and Use Committee (IACUC).

At the end of the study, the mice were euthanized by cervical dislocation under 101 anesthesia and all skeletal muscles were harvested, the diaphragms were harvested for contractile studies.

Cell Culture

C2C12 cell line was originally purchased from ATCC and grown in media containing DMEM (Life Technologies) supplemented with 20% fetal bovine serum

(FBS) (Atlanta Biologicals), 1% penicillin/streptomycin (P/S) (GIBCO) and 1% L- glutamine (GIBCO). Myoblasts were grown until 70% confluent and differentiated into myotubes in media containing DMEM (Life Technologies) supplemented with

2% horse serum (HS) (Atlanta Biologicals) and 1% insulin-transferrin-selenium

(ITS) (GIBCO) with media changed daily for 120 hours, cells were incubated at

37°C with 5% CO2. Cells were treated with varying concentrations of Sunitinib

(Sigma) diluted in 1% DMSO (Sigma) or 1% DMSO alone with a maximum concentration of DMSO at 0.1%.

Animals

Previous studies 349 showed Sunitinib is soluble in a solution of 0.1% methylcellulose (Sigma) at 25mM. Initial, 5-day dosing study determined optimal

Sunitinib dose for α7B integrin enhancement to be 1mg/kg. Four-week-old, male mdx mice were administered Sunitinib via oral gavage at 1mg/kg, three times per week until 12-weeks of age (See study approval section at end of methods).

Immunoblotting 102

Protein was extracted from cell pellets or whole tissue using radioimmunoprecipitation assay buffer (RIPA) containing 1:500 dilution of protease inhibitor cocktail and 1:100 dilution of NaF and Na3VO4 phosphatase inhibitors.

Protein quantification was performed via BCA (Thermo Scientific), separated by

SDS-PAGE and transferred onto nitrocellulose membrane. Detection of α7B was performed as previously described 350. pSTAT3 (D3A7; 1:250), STAT3 (79D7;

1:1000), pERK (1:1000), ERK (1:1000), pSHP-2 (3751; 1:250), SHP-2 (D50F2;

1:1000) (Cell Signaling) were incubated overnight and followed by Alexa Fluor 680 or 800 conjugated goat anti-rabbit (Invitrogen) or HRP conjugated anti rabbit (Cell

Signaling) detected using pierce ECL western blotting substrate (Thermo

Scientific). Protein quantity was normalized to GAPDH (1:1000, Cell Signaling) or swift stain (G-biosciences) and imaged using LI-COR imaging system. Protein quantification was performed using Fiji (ImageJ).

Immunofluorescence

Cells: C2C12 cells were grown in chamber slides, fixed with 4% paraformaldehyde

(PFA) and blocked in 1% BSA containing 0.1% Tween and 0.05% Triton-X100 for

1 hour at room temperature (RT). Cells were then incubated overnight with MF20 monoclonal antibody (1:40, Developmental Studies Hybridoma Bank (DSHB)) against myosin heavy chain, followed by a 1 hour incubation with fluorescein isothiocyanate (FITC) -anti mouse secondary (1:200, BD Biosciences). Nuclei

DAPI label contained in Vectashield (Vector Labs) used to mount chamber slides. 103

Tissue: Post-harvest fresh hemi-diaphragms were embedded in a 2:3 (v/v) optimum cutting temperature (OCT) compound to 30% sucrose/PBS and cryosectioned. 10µm sections were immunostained for α7 integrin as previously described 345 followed by 1 hour incubation with FITC-anti rabbit (1:200, BD

Biosciences). Sections were labeled with Alexa Fluor 647 conjugated wheat germ agglutinin (WGA) (1:250) for 20mins and mounted with vectashield containing

DAPI for centrally located nuclei. Immunostaining for embryonic myosin heavy chain (1:40, DSHB) and Pax7 (1:50, DSHB – 30min permeabilization in 0.2% triton) were performed using Mouse on Mouse (M.O.M) detection kit (Vector Labs), followed by 1 hour incubation with FITC-anti mouse and 20 minute incubation with

WGA-647. All tissues were imaged using the Olympus Fluoview FV 1000 laser confocal microscope.

Ex vivo Contractility

Mice were put under anesthesia using isoflurane (1.5% isoflurane and 400 mL O2

/min) and placed supine on a temperature controlled platform to maintain their body temperature at 370C. Diaphragm muscle strip was dissected in oxygenated physiological salt solution (PSS) (pH 7.6, 30oC) and strung up with rib cage portion facing up on computer-controlled dual mode Aurora Scientific, Inc. 300B servomotor as previously described 351. The following protocol was performed: 1) determination of optimum length (L0) following 3 isometric twitches and 3 tetani

(150Hz). 2) Force frequency protocol will follow frequencies of 1,30,50,80,100,120 and 150Hz for 900ms each with 4 min intervals. After all protocols are completed 104 muscle was be stripped of rib cage and tendons and blotted for a final muscle weighing.

Forelimb Grip-Strength

Forelimb grip-strength measurements were blinded and obtained using a computerized grip-strength meter (Colombus Instruments) according to Treat-

NMD guidelines. Measurements were performed once after the 8 week treatment regime to prevent adaptation.

Whole Muscle Evans Blue Dye Assay

Evans blue dye (EBD) assay was performed as previously described 352. Briefly,

EBD (Sigma) was dissolved in phosphate buffered saline (PBS) at 10mg/ml and injected intraperitoneally at 5ul/g. Tissue was collected 24 hours post-injection, weighed and incubated for 2 hours at 55°C in 1 ml formamide (Sigma). Absorbance was read at 620 nm by spectrophotometer.

TaqMan RT-PCR

Tibialis anterior muscle was homogenized in Trizol reagent (Ambion) and processed for RNA extraction. First-strand cDNA synthesis was performed using

VILO reaction mix (ThermoFisher) and real time qPCR reactions were performed on a 96-well plate using TaqMan Fast Advanced Master Mix and TaqMan Gene

Expression Assays for Itga7 (Mm00434400_m1), MyoD1 (Mm00440387_m1), 105

Myog (Mm00446194_m1) Pax7 (Mm01354484_m1). Reactions were run on the

Applied Biosystems 7900HT Fast System in triplicate.

Mass Spectometry

Diaphragm tissue samples were run on a 12% SDS-PAGE gel, stained with mass

spectrometry grade coomassie stain, band of interest was cut-out and sent to the

University of Nevada, Reno Nevada Mick Hitchcock Proteomics Center where

proteins were in-gel digested and processed using a TMT10plex kit (ThermoFisher

Scientific) and analyzed using an Orbitrap Fusion mass spectrometer.

Statistics

GraphPad Prism software was used for statistical calculations. Student’s t-test was

used to compare means between two groups and one-way ANOVA was used to

compare means between three or more groups. All ANOVA calculations were

followed by Tukey’s post-hoc test. Means considered statistically significant when

p<0.05.

RESULTS

Sunitinib treatment increases α7β1 levels via MyoD1 and Myog transcription factors

A previous study demonstrated the beneficial effects of treatment with the small molecule SU9516 on DMD pathology via enhanced α7B integrin expression

346. Sunitinib is an FDA approved, structurally related compound of SU9516 (Fig. 106

1a) expected to have similar effects on DMD disease progression. To test this idea, optimal Sunitinib treatment dose was determined by performing a dosing curve consisting of 5 day treatment in mdx mice followed by assessment of α7B integrin levels in diaphragm muscle. Initially one animal per dosing group was treated with varying concentrations of Sunitinib and assessed for protein levels of α7B integrin.

Compared to vehicle, mdx mice treated with 0.5 mg/kg and 1mg/kg gave an increase in α7B integrin levels that appeared lowered at 5 mg/kg and 10 mg/kg, suggesting lower doses of Sunitinib produce more of an effect on α7B integrin expression (Fig. S1a). To further determine optimal dosing with more accuracy, a total number of 4 animals per treatment group were treated with Sunitinib for 5 consecutive days. Results show that maximum ~1.5-fold increase in α7B integrin expression was observed at 1mg/kg Sunitinib (Fig.1b) when compared to vehicle treated muscle. Additionally, a significant increase in α7B’s binding partner, β1D, was also observed at 1mg/kg (Fig. S1b), suggesting this to be the optimal dose for

Sunitinib treatment and therefore the dose set up for the long term study. This effective concentration was significantly lower than its structurally related compound SU9516 (Chapter 2 - effective at 5mg/kg). To study the effect of

Sunitinib on disease pathology, the treatment schedule developed consisted of 3 days on 1mg/kg Sunitinib and 4 days off, weekly treatments, beginning at 4 weeks of age and ending at 12 weeks in the mdx mouse model of DMD (Fig.1c). This treatment schedule was developed based on the robust and rapid increase in α7B integrin (Fig. 1b) and on potential downstream targets. The 1mg/kg Sunitinib treatment produced a significant increase in α7B integrin transcript (Fig. 1F) and 107

Figure 1. Sunitinib treatment increases α7B integrin via activation of Myod1 and Myog transcription factors. (A) Structural similarities between the known 108 integrin α7 enhancing molecule SU9516 and Sunitinib. (B) Dose response curve performed on N=4 mdx mice with daily 1mg/kg-10mg/kg Sunitinib treatment for a total of five days showing optimal α7B integrin enhancing dose at 1mg/kg. (C)

Schematic representation of the 8 week Sunitinib treatment plan and final muscle assessments. (D and E) Western blot analysis and quantification of diaphragm

α7B integrin levels showing a 1.5-fold increase in α7B expression with Sunitinib treatment. (F) RT-qPCR analysis of α7B integrin transcript levels showing ~1.4- fold increase with Sunitinib treatment. (G and H) RT-qPCR analysis of α7B upstream transcription factors and differentiation markers MyoD1 and Myog showing a ~1.8-fold and ~5.8-fold transcript increase, respectively, with Sunitinib treatment. Statistical significance of mean ± SEM; *p < 0.05, **p < 0.01.

109

Figure 2. Sunitinib improves specific muscle contractile function and overall muscle strength. Muscle isometric contractility and strength assessments performed on 12 week old mdx mice. (A) Single 1Hz pulses generated isometric twitch force outputs showing the severe decline in diaphragm muscle twitch between WT (N=5) and mdx vehicle treated muscle (N=11); Sunitinib treated muscle (N=11) twitch is significantly increased compared to vehicle treated. (B)

Isometric tetanic stimuli performed at 100Hz on diaphragm muscle showing a significant decline in tetanic force output between WT (N=5) and vehicle treated muscle (N=11), Sunitinib treatment (N=11) increased isometric tetanic force compared to vehicle treatment. (C) Isometric force performed at increasing 110 stimulation frequencies; Sunitinib treatment increased force production in the 50-

150Hz frequency stimulations when compared to vehicle treated muscle. (D)

Muscle exhaustion depicted as decreased forelimb grip-strength is apparent in vehicle treated mdx mice compared to WT mice at trials 5-6. No significant change in forelimb force is observed between WT and Sunitinib treated mdx mice during the six trials; Sunitinib treated mdx mice are stronger than the vehicle treated in trials 4-6 suggesting less muscle exhaustion. Data assessed for significance using

1-way ANOVA and statistical significance of mean ± SEM; WT vs. mdx-vehicle treatment #p <0.05, ##p < 0.01, ###p < 0.001; WT vs. mdx-Sunitinib treatment ++p

< 0.01, +++p < 0.001; mdx-vehicle treatment vs. mdx-Sunitinib treatment *p < 0.05,

**p < 0.01.

111 protein expression in mdx diaphragm muscle compared to vehicle treated muscle

(Fig.1D, E). Additionally, transcript levels of two known α7B integrin regulating transcription factors, MyoD1 and Myog 353,354, were also significantly up-regulated in the tibialis anterior (TA) muscle of Sunitinib treated mdx mice (Fig.1G, H).

Together these results suggest that Sunitinib can more potently exert the same integrin enhancing effects as the structurally related compound SU9516, at significantly reduced concentrations.

Sunitinib improves mdx diaphragm muscle function

Loss of dystrophin in DMD renders muscles fragile and unable to exert normal amounts of force. Regeneration of new muscle fibers is expected to yield an increase in muscle force production. To test whether Sunitinib treatment improved muscle contractility, ex vivo isolated muscle isometric contraction studies were performed on mdx diaphragm muscle. Isolated, isometric twitch (1Hz) force production in vehicle treated mdx diaphragm muscle (2.722 ±

0.75 mN/mg) is considerably lower than that of wildtype muscle (6.014 ± 0.93 mN/mg) (Fig. 2A). Sunitinib treatment produced a significant increase in twitch force (3.689 ± 0.29 mN/mg) compared to vehicle (Fig. 2A). Specific tetanic force, measured at peak 150 Hz stimulation, was also increased with Sunitinib (13.02 ±

1.38 mN/mg) compared to vehicle treated diaphragm (10.47 ± 2.31 mN/mg) (Fig.

2B). Sunitinib enhanced specific force production at increasing stimulations ranging from 50 to 150Hz when compared to vehicle treated muscle (Fig. 2C). To assess overall muscle strength, forelimb grip-strength was performed post 8-week 112

Figure 3. Sunitinib promotes muscle repair and improves markers of DMD disease progression. (A) Immunohistochemistry on diaphragm muscle shows 113 increased number of eMyHC (green) positive fibers in Sunitinib treated muscle (B)

Quantification of eMyHC positive muscle fibers normalized to total fiber numbers of a whole mdx diaphragm muscle section (N=4); Sunitinib treatment causes ~10% increase in eMyHC+ fibers compared to vehicle treatment. (C and D) Centrally located nuclei counts performed on WGA (grey) and DAPI (blue) immunolabeled mdx diaphragm muscle sections (N=6); Sunitinib treatment increased CLNs by

~6% compared to vehicle treated. (E) Myofiber size distribution of Sunitinib and vehicle treated mdx diaphragm sections, measured using minimum Feret’s diameter of whole 10µm sections (N=4); Sunitinib treated muscle shows a more even distribution of fiber sizes and a shift towards higher percentage of large fiber sizes compared to vehicle treated. (F) Evans blue dye fiber damage assessment shows decreased infiltration of the dye in Sunitinib treated mdx diaphragm muscle section and total gastrocnemius (GA) muscle compared to vehicle treated (N=4).

(G) Hydroxyproline assay was performed to quantify the amount of fibrotic infiltration in the mdx GA muscle (N=4); treatment with Sunitinib prevents fibrosis as shown by a 2-fold decrease in collagen content in the mdx GA muscle, compared to vehicle treated. (H) STAT3 pathway is activated in response to

Sunitinib treatment 1 hour post final dose (6 week total dosing) in mdx diaphragm.

Data assessed for significance using unpaired t-test and statistical significance of mean ± SEM; *p < 0.05, **p < 0.01; ***p<0.001.

114

Sunitinib or vehicle treatment. Results show that mdx forelimb muscle force significantly decreased from wildtype in the last two grip-strength trials, whereas none of the trials shows significant difference in force between wildtype and

Sunitinib treated mice (Fig. 2D). Sunitinib treated mdx forelimb strength was significantly increased compared to mdx forelimb strength in the last three grip- strength trials (Fig. 2D). These results suggest Sunitinib treatment shows significant improvements in muscle strength and function shown by isolated muscle force contraction studies and prevents the severe muscle fatigue observed in mdx mice.

Sunitinib promotes myogenesis in the mdx mouse

We next determined whether the increase in α7B integrin observed with

Sunitinib treatment translated into improved DMD disease pathology. Diaphragm muscle sections from 8-week, Sunitinib treated and vehicle treated mdx mice were analyzed for expression of a developmental form of myosin heavy chain (MyHC). Embryonic myosin heavy chain (eMyHC) is transiently expressed in developing, fetal muscle fibers and after muscle injury, in newly regenerated, adult fibers 355. Our results show that vehicle treated mdx diaphragm contains ~4% eMyHC expressing fibers (Fig. 3A, B) due to the hallmark bouts of degeneration/regeneration in DMD muscle. Expression of eMyHC was increased to 12% in Sunitinib treated diaphragm muscle, indicating that Sunitinib treatment is capable of promoting regeneration of newly formed muscle fibers in vivo.

Diaphragm muscle was also assessed for centrally located nuclei (CLN) as further 115 evidence of new fiber formation. Sunitinib treatment significantly increased CLNs by ~5% (Fig. 3C, D). Minimum Feret’s diameter showed a shift in fiber size.

Diaphragms from vehicle treated mice have an uneven distribution, with a higher percentage of small fibers in the 20-25µm range compared to Sunitinib fiber diameters showing a more even distribution and a higher percentage of larger fibers in the 30-50µm range (Fig. 3E). Newly generated fibers would expectedly decrease the amount of total muscle damage. To assess the amount of leakiness in a whole mdx muscle, an Evans blue dye (EBD) assay was performed. Results from the EBD assay show that Sunitinib treatment decreased the amount of muscle damage as determined by decreased numbers of positive myofibers in the diaphragm muscle sections and whole gastrocnemius muscle of treated mdx mice

(Fig. 3F). Muscle damage allows for robust infiltration of fibrotic and fatty tissue which become indicators of DMD disease progression 99,100. The amount of fibrotic infiltration in whole muscle, quantified via hydroxyproline colorimetric assay, was also shown to be decreased in gastrocnemius muscle with Sunitinib treatment (Fig.

3G). To elucidate the pathway by which Sunitinib promoted muscle regeneration, we next performed protein analysis on diaphragm tissue 1-hour post treatment on pathways postulated to be regulated by Sunitinib. Our results indicated that

Sunitinib promoted the activation of the STAT3 pathway, in vivo, in the diaphragm muscle of mdx treated mice (Fig. 3H). Taken together these results show that

Sunitinib treatment promotes muscle regeneration in vivo through STAT3 activation, thus decreasing muscle fiber damage and replacement of muscle with fibrotic/fatty tissue. 116

Sunitinib promotes satellite cell proliferation and myofiber fusion

To determine the mechanism by which Sunitinib promotes muscle fiber

regeneration, we next analyzed SC populations in dystrophic muscle. Pax7 is a

transcription factor expressed in quiescent and activated SCs 229,356. Pax7 transcript was significantly increased in Sunitinib treated tibialis anterior (TA) muscles compared to vehicle treated animals (Fig. 4B). Accordingly, Pax7 labeling in diaphragm muscle sections showed Sunitinib treated mice exhibit increased numbers of SCs compared to muscle treated with vehicle (Fig. 4A, C). This suggests Sunitinib treatment is promoting the proliferation of SCs required for myogenic regeneration.

Next, we determined whether Sunitinib treatment promoted myofiber fusion.

To measure fusion, C2C12 myoblasts were treated under differentiating conditions with increasing doses of Sunitinib. Treatment with Sunitinib results in larger fibers containing increased numbers of nuclei (Fig. 4D). Sunitinib doses as low as 200nM are capable of promoting ~1.7-fold increase in the myofiber fusion with maximum increase observed at 500nM (Fig. 4E). Together these results show that Sunitinib treatment promotes terminal differentiation of myotubes without depleting the SC pool.

Sunitinib inhibits SHP-2 - ERK1/2 activation and promotes STAT3 phosphorylation

To elucidate the mechanism by which Sunitinib acts to promote myogenic differentiation, the C2C12 cell line was used. A previous study showed Sunitinib 117

Figure 4. Sunitinib treatment promotes satellite cell proliferation and myoblast fusion. (A) Immunohistochemistry performed on diaphragm muscle 118 sections of mdx mice treated with vehicle or Sunitinib. Higher numbers of satellite cells are present in Sunitinib treated diaphragm; satellite cells are identified

(arrows) by their location around myofibers (α7B integrin (red)), Pax7+ cells

(green) and co-localization with nuclear DAPI (blue) stain. (B) Increased Pax7 transcript levels observed in Sunitinib treated tibialis anterior (TA) whole muscle.

(C) Quantification of satellite cell numbers, determined by counting Pax7+ cells per total fiber counts (10 panels per tissue, magnification 40X, N=3); Sunitinib treatment significantly increased satellite cell numbers. (D) C2C12 myoblasts under differentiating conditions 48-hour post DMSO or Sunitinib treatment at varying concentrations immunolabeled for myosin heavy chain (MHC – red) and nuclear DAPI (blue). (E) Fusion index determined by counting number of nuclei within MHC+ fibers and normalizing to total nuclei counts per 10X panel (N=3);

Increased myofiber fusion observed at all three Sunitinib concentrations with

500nM showing the highest fusion index compared to DMSO-vehicle treated cells.

Data assessed for significance using 1-way ANOVA and statistical significance of mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

119 can modulate the STAT3 pathway in cancer 349. Additionally, studies have shown a direct, inhibitory interaction between activated ERK1/2 and STAT3 357,358.

ERK1/2 is a downstream target of activation of SHP-2 by Jak2. C2C12 myoblasts were treated with Sunitinib and cells harvested at varying time points after treatment. Our results show treatment with 500nM Sunitinib caused inhibition of

SHP-2 activation within 5 minutes post-treatment, continuing for up to 1 hours (Fig.

5A, B). ERK1/2 activation is decreased starting at 15 minutes post Sunitinib treatment and lasting up to 2 hours (Fig. 5C, D). STAT3 activation is observed starting at 1.5 hours post Sunitinib treatment and lasting at least up to 3 hours (Fig.

5E). Additionally, α7B integrin expression was increased in myoblasts treated with

Sunitinib after 48 hours (Fig. 5F). Next we assessed whether an optimal dose of 5

µM SU9516 led to inhibition of ERK1/2 phosphorylation activation of STAT3 and in C2C12 myoblasts. Contrary to our expectations, we did not observe alteration in either pSTAT3 (Fig. S3A, B) or pERK1/2 (Fig. S3C, D). Together, these results further support the idea that Sunitinib exerts its effects predominately on early myogenic cells such as SCs and myoblasts by inhibition of SHP-2 - ERK1/2 signaling followed by activation of STAT3. 120

Figure 5. Sunitinib inhibits SHP-2 - ERK1/2 and activates the STAT3 pathway in C2C12 cell line. C2C12 myoblasts treated with 500nM Sunitinib in triplicate

(N=3) and taken at different time points to assess SHP-2 - ERK1/2 inhibition and 121

STAT3 activation. (A) Quantification of SHP-2 phosphorylation shows significant inhibition at 5 mins lasting up to 15 min. (B) Quantification of SHP-2 phosphorylation shows significant inhibition continuing from 30 mins to 1 hours.

(C) Quantification of ERK1/2 phosphorylation shows significant inhibition starting at 15 min post treatment and lasting up to 1 hour. (D) Quantification of ERK1/2 phosphorylation showing continued inhibition at 1.5 hours with recuperating phosphorylation after 2 hours of treatment. (E) Quantification of STAT3 phosphorylation showing activation after 1.5 hours of treatment. (F) Quantification of α7B integrin shows a 1.5-fold increase in expression 48 hours post treatment.

Data assessed for significance using 1-way ANOVA and statistical significance of mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

122

Figure 6. Proposed mechanism of SHP2 - ERK1/2 inhibition and STAT3 activation by Sunitinib. In response to muscle injury, gp130, mediated by Jak2 can activate both ERK1/2 and STAT3. (A) Upon gp130 receptor dimerization in response to growth factor binding, constitutively bound Jak2 is activated and trans- phosphorylates gp130 at several tyrosine residues (Y). P-Y759 recruits SHP-2 which is phosphorylated by Jak2 and signals via Grb2-Sos-Ras-Raf and MEK to activate ERK1/2. Once activated, ERK1/2 has been shown to directly phosphorylate STAT3 at a serine residue, thus preventing phosphorylation at tyrosine residues and nuclear translocation 357. ERK1/2 becomes the predominant signaling pathway promoting the proliferation of myogenic cells. (B) Sunitinib inhibits the activation of ERK1/2, potentially by: 1) preventing the association of

SHP-2 with gp130; 2) preventing the phosphorylation of SHP-2 by Jak2. Non- 123 phosphorylated ERK1/2 no longer phosphorylates STAT3 at its serine residue, thus allowing tyrosine phosphorylation and activation of STAT3 by Jak2. Dimerized

STAT3 translocates into the nucleus where it promotes the transcription of the transcription factor MyoD1 which can sub sequentially promote the transcription of myogenin (Myog). Both MyoD1 and MYOG can then promote the transcription of integrin α7 (Itga7). Integrin α7 promotes differentiation and fusion of myofibers, thus enhancing regeneration of muscle.

DISCUSSION

Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disease characterized by rounds of muscle degeneration and regeneration with eventual failure of muscle repair. In the present study, we provide evidence of halted DMD disease progression in mdx mice that received long term treatment with Sunitinib. Marketed under the name SUTENT®, Sunitinib was originally developed for the treatment of renal cell carcinoma (RCC) and gastrointestinal stromal tumors (GIST). Sunitinib is a multi-receptor tyrosine kinase (RTK) inhibitor with binding affinities for c-KIT, FLT3, RET, VEGFR and PDGFR 348. Optimal antitumor dosing schedule for Sunitinib in human trials was determined to be

50mg/day, required to reach circulating plasma levels of 50-100ng/ml for highest

RTK inhibition with manageable side effects, equivalent to 40mg/kg/day in mice

347,359,360. Studies have been conducted on pediatric patients with refractory solid tumors demonstrating that a 15mg/day is a more suitable dose for children 361,362. 124

For the purposes of our study, initial dosing studies with α7B integrin protein expression as the outcome measure, determined the optimal dose (1mg/kg) to be

40-fold lower than the antitumor dose. This suggests the myogenic effects observed are possibly due to targeting of a novel pathway not previously described for Sunitinib.

One of the hallmarks of DMD pathology are cycles of muscle degeneration and regeneration that occur in the absence of dystrophin 31. Muscle degradation allows for infiltration of inflammatory cells and replacement of muscle tissue with fibrotic and fatty tissue 99,100. DMD disease pathology also presents with lower SC capacity due to interrupted SC niches, resulting in impaired muscle regeneration

104,105,330,363. Therapeutic interventions aimed at enhancing the proliferative capacity of endogenous SCs could prove significantly beneficial to DMD pathology.

To this extent, numerous studies have indicated that STAT3 activation via the interleukin-6 (IL-6) cytokine is important for SC proliferation and self-renewal in response to resistance exercise and muscle injury 332–337,364. Other studies show that activated STAT3 can directly affect the expression of the myogenic regulatory marker MyoD1 and promote myoblast differentiation 338–341. Additionally, STAT3 inhibition was shown to promote enhanced symmetric SC proliferation and increased SC engraftment into injured muscle 342,343. Together these studies indicate that transient STAT3 activation might allow controlled cycles of SC proliferation and differentiation.

Small molecule therapeutics are ideal for transient activation of a specified pathway. In this study, we show that Sunitinib is capable of activating the STAT3 125 pathway in cell culture and in the diaphragm of mdx mice. Phosphorylation of

STAT3 allows for its dimerization and translocation into the nucleus where phospho-STAT3 dimers directly promote MyoD1 expression (Fig. 6b) 339,340.

Analysis of transcript levels in the gastrocnemius muscle of treated mdx mice indicated significant up-regulation of MyoD1 transcript as well as the differentiation marker MYOG, suggesting significant myogenic remodeling is occurring with

Sunitinib treatment. Studies have shown the direct interaction of MyoD1 and myogenin with the α7 integrin promotor, positively regulating α7 integrin transcription 353,354. Additionally, several studies have shown α7 integrin to be an important, positive DMD disease modifier 84–87,91,93,94,350, potentially due to enhancement of myogenic capacity and promotion of hypertrophy 283,344. This suggests that activation of the STAT3 pathway can directly lead to up-regulation of α7 integrin levels leading to myogenic hypertrophy.

To elucidate this pathway in vivo, α7 integrin transcript and protein expression levels in the mdx mouse diaphragm were analyzed and determined to be significantly up-regulated. To associate the increase in α7 integrin with increased hypertrophy the next step in our study looked at markers of muscle regeneration. We found that diaphragm muscle fibers of Sunitinib treated mice were expressing high levels of embryonic myosin heavy chain, a type of myosin found both developmentally and during adult myogenesis 355. Analysis of myofiber diameters revealed that treatment with Sunitinib caused a significant shift towards larger myofibers and a more even distribution of fiber sizes, suggesting regeneration is enhanced by Sunitinib. Additionally, during the course of the study, 126 a swift stain performed on diaphragm protein samples of treated and non-treated mdx mice revealed a novel band of approximately 400kDa observed in the diaphragm tissue of vehicle treated mdx mice that was significantly more robust in diaphragm muscle of the Sunitinib treated mice (Fig. S2A). A swift stain functions much as a coomassie stain, where it binds to all proteins found on the nitrocellulose membrane after a successful transfer. Therefore, to determine the nature of the newly found band, a fresh SDS-PAGE gel was run with the same protein samples, stained with mass spectrometry grade coomassie, cut from the gel and sent in to the Mick Hitchcock, Ph.D. Nevada Proteomics Center for enzymatic protein digestion and single band Liquid Chromatography/Mass Spectometry (LC/MS) to determine the band identity. Mass spectrometry results revealed with over 95% confidence that the main component of the analyzed band was the protein titin along with proteins commonly found bound to titin such as myosin-1 and obscurin

(Fig. S2B). Quantification of the titin band (containing also attached proteins) shows that Sunitinib treatment is promoting the expression of titin almost 30-fold higher than vehicle treated diaphragm muscle (Fig. S2C). Titin is a 700kDa, and therefore largest, muscle specific protein that is expressed in the early phase of muscle differentiation as the first sarcomeric protein that is detected during myofibrillogenesis. Additionally, titin is important as a scaffold during the supramolecular organization of the sarcomere 365–367. These results provide additional support to the effect of Sunitinib on the regeneration and differentiation of skeletal muscle in mdx mice. 127

We hypothesized that the observed increase in myogenic regeneration

occupied the interstitial space left behind by degenerating fibers before the

precocious deposition of fibrotic and fatty tissue. This was supported by a decrease

in fibrotic content in Sunitinib treated mdx gastrocnemius and all-around less leaky

muscle fibers as shown by decreased Evans blue dye infiltration. Together this

enhancement of muscle fiber stability leads to the increased force production of

the isolated diaphragm muscle and increased forelimb strength of treated mdx

mice. These results suggest that Sunitinib promotes muscle regeneration via

activation of STAT3 and downstream up-regulation of α7 integrin, leading to

increased fusion of myofibers allowing replacement of degenerating muscle and

increase muscle force.

The activation of STAT3 in response to muscle injury and resistance

exercise occurs due to production of the IL-6 cytokine. In fact, both human primary

myoblasts, mouse myogenic cells and murine in vivo muscle have recently been

shown to produce IL-6 suggesting intrinsic control of muscle production and a role

for IL-6 in myogenesis. IL-6 knockout mice present with blunted hypertrophic

muscle growth and decreased myonuclear accretion as well as decreased satellite

cell proliferation in response to muscle overload 332,334,335. The canonical pathway

for the IL-6 cytokine involves the binding to membrane bound or soluble gp130

receptors, recruitment and auto-phosphorylation of JAK2 which in turn

phosphorylates downstream targets SHP-2 - ERK1/2 and STAT3 (Fig. 6) 368. Due to Sunitinib’s inherent nature as a kinase inhibitor, a direct interaction with STAT3 128 is unlikely. Therefore, it is important to determine what the possible targets of

Sunitinib potentially are by dissecting the STAT3 pathway upstream.

SHP-2 is a protein tyrosine phosphatase that upon gp130 receptor dimerization, can bind to gp130 receptors via its SH-2 domain, where it can then be phosphorylated by Jak2 which is also bound to gp130 receptor complex. Upon phosphorylation, pSHP-2 recruits Grb2 and Sos which then bind Ras-GDP, Ras-

GDP undergoes phosphorylation and activates downstream Raf, MEK and finally

ERK1/2. To determine the effect of Sunitinib treatment on SHP-2 phosphorylation,

C2C12 myoblasts were treated with Sunitinib. Treated myoblasts showed significant inhibition of SHP-2 as fast as 5 minutes post treatment that maintained up to at least one hour post-treatment.

The next step was to move downstream of the SHP-2 pathway to look at

ERK1/2 activation, which we hypothesized would also be inhibited. Sunitinib treatment shows downstream inhibition of ERK1/2, fifteen minutes post-treatment and lasting up to one and half hours. Activated ERK1/2 has been shown to directly interact with STAT3 by promoting phosphorylation at a STAT3 serine residue, thus preventing the tyrosine phosphorylation which activates and dimerizes STAT3 sending it into the nucleus 357,358. Therefore, we postulated that inhibition of

ERK1/2 would release the inhibitory effect it promotes on STAT3 and allow its subsequent activation. We observed STAT3 activation in treated C2C12 myoblasts after only one and a half hours post-Sunitinib treatment. The activation of STAT3 can be explained by the observed rapid and prolonged SHP-2 and

ERK1/2 inhibition which prevents phospho-ERK1/2 from interacting with and 129 phosphorylating STAT3 at its inhibitory serine residue, thus allowing phosphorylation on tyrosine residues required for STAT3 activation (Fig.6B).

Finally, the last step was to correlate the activation of STAT3 to the increased expression of α7 integrin observed previously. C2C12 myoblasts were treated with Sunitinib and α7 integrin expression was determined to be increased expression fourty-six and a half hours post-STAT3 activation. Increased α7 integrin has previously been shown to regulate hypertrophy which explains the increased cultured myofiber fusion we observed. Additionally, treatment of myogenic cell lines demonstrates the direct effect of Sunitinib on these myogenic cells via STAT3 activation and increased α7β1 integrin expression.

The decision to perform a long-term study on dystrophic mice using

Sunitinib lie in the fact that it is structurally related to SU9516, previously shown to have beneficial effects. For this reason, studies were performed to test if SU9516 interacts with the same pathway defined with Sunitinib treatment. To this end,

C2C12 myoblasts were treated with 5 µM SU9516, however they did not show activation of the STAT3 pathway (Fig. S3A, B) nor was the inhibition of the ERK1/2 pathway observed (Fig. S3C, D). These results suggest that while structurally similar and both capable of α7 integrin enhancement, Sunitinib and SU9516 exert their effects via different mechanisms of action. This experiment also provides strong evidence that Sunitinib itself promotes the inhibition of SHP-2 - ERK1/2 and activation of STAT3, not its breakdown metabolite SU12662 348.

Constitutive activation of STAT3 would ultimately result in the exhaustion of the SC population. However, our results indicate higher numbers of SCs are 130 present in the diaphragm muscle of Sunitinib treated mdx mice, as well as increased Pax7 transcript levels in the tibialis anterior muscle. This can be explained by the transient nature of the treatment schedule in this study where the four days of off time (no treatment) allow for the inhibition of ERK1/2 to be reversed, in turn blocking STAT3 activation and allowing cells to re-enter the proliferative cell cycle. STAT3 has also been implicated in the proliferation of SCs in numerous studies, therefore, it is also possible that a threshold of STAT3 phosphorylation is required to remove SCs from the cell cycle, allowing symmetric division of SCs to occur prior to cell cycle withdrawal. We conclude that transient Sunitinib treatment not only promotes myogenic differentiation but allows SC proliferation and self- renewal. This suggests that the transient nature of our dosing schedule allows for the re-population of SC pools, asymmetric cell division and subsequent differentiation into mature myofibers.

Together our results show that the FDA approved drug Sunitinib is capable of promoting muscle regeneration via transient activation of STAT3 in the mdx mouse model of DMD. The significantly lower, 1mg/kg tri-weekly dose wouldn’t be expected to produce the same side effects observed with the higher 40mg/kg/day cancer doses and would therefore be deemed safer for pediatric use. In the future, it is important to determine the exact target of Sunitinib that is causing the inhibition of SHP-2 and consequential activation of STAT3, whether it is a direct inhibition of

SHP-2 or an upstream target 369,370. It’s been determined that a gain of function mutation in SHP-2 (PTPN11 in humans) is sufficient to promote DMD muscle pathology in an otherwise mildly dystrophic Becker’s muscular dystrophy (BMD) 131 patient, suggesting a detrimental role of constitutive SHP-2 activation further supporting inhibition by Sunitinib to be beneficial 371. Here, we identify Sunitinib as a potent skeletal muscle regenerative drug capable of halting disease progression that could be fast tracked into clinical trials for DMD as well other muscular dystrophies in which regeneration of muscle would be beneficial.

Figure S1. Determination of Sunitinib dosing schedule. (A) mdx mice (N=1) dosed with varying concentrations of Sunitinib (0.1mg/kg-10mg/kg) to determine which dose gives largest increase in α7B integrin protein levels, determined to be

1mg/kg. (B) mdx mice (N=4) treated with Sunitinib (1mg/kg-10mg/kg) to determine optimal increase in β1D integrin, confirmed to be 1mg/kg. 132

Figure S2. Increased expression of titin protein with Sunitinib treatment. (A)

Diaphragm muscle protein samples stained with Swift Stain highlighting a large molecular weight band with robust expression in Sunitinib treated samples. (B)

Mass spectrometry performed on single high molecular band cut out from SDS-

PAGE of the same diaphragm muscle samples treated with Sunitinib. Band determined to be titin protein with lower quantities of myosin-1, ryanodine receptors and obscurin. (C) Quantification of titin (and complexed proteins) using

Swift Stain; about 30-fold increase in titin and titin-complexed proteins observed in

Sunitinib treated samples. 133

Figure S3. SU9516 does not activate STAT3 nor inhibit ERK activation in

C2C12 myoblasts. Protein extracted from C2C12 myoblasts treated with either

DMSO for 60 minutes or 5uM SU9516 for 30, 60, 90, or 120 minutes was assessed using western blotting for pSTAT3/STAT3 (A) and pERK/ERK (C) levels. Blots were quantified for pSTAT3/STAT3 (B) and pERK/ERK (D). Data was assessed for significance between treatment groups using 1-way ANOVA. No significance was observed.

134

Chapter 4

Cardiac Dystrophy and the Role of α7 Integrin

135

ABSTRACT

DMD is characterized by loss of the X-linked dystrophin protein known to be expressed in skeletal, cardiac and smooth muscle. Skeletal muscle atrophy is the major, visible sign of DMD, where patients find themselves confined to wheelchairs early on in life. There exists, however, a visually hidden and deadly side to DMD residing in the heart of affected patients manifesting as cardiomyopathies. Most commonly, patients will present with dilated cardiomyopathies in which the left ventricular wall has undergone significant enlargement. The role of dystrophin in cardiac muscle significantly overlaps with its role in skeletal muscle. In the absence of dystrophin, the heart suffers myocardial atrophy coupled to myocardial remodeling, dilation and fibrosis.

Together fibrotic and fatty infiltration and myocardial hypertrophy give rise to severe arrhythmias and eventual cardiac failure, leading to the sudden death of patients.

The α7β1 integrin complex has been extensively studied in relation to skeletal muscle and its compensatory effect in the absence of dystrophin.

Mutations in the Itga7 gene have been highlighted for their contribution to congenital muscular dystrophies. In recent years, the level of involvement of α7 integrin in the heart has been questioned. A patient case study provides insight into the role of α7 integrin in congenital fiber type disproportion (CFTD), a form of congenital myopathy. This opens up the possibility that mutations in the Itga7 gene could be a direct cause of congenital myopathies and arrhythmias in patients. In 136 our study, we will discuss data indicating that Itga7 null mice, expressing no α7 integrin protein, present with cardiac conduction abnormalities. Possible α7 integrin roles and cardiac binding partners in the myocardium are discussed as well. Additionally, the establishment of α7 integrin as a modulator of the myocardium led us to question the potential for our recently identified α7 integrin enhancing small molecule, Sunitinib, to provide benefit to the dystrophic heart.

Contributions to Chapter 4: Author performed electrocardiography, echocardiography, heart histology and immunoblotting.

INTRODUCTION

Clinical manifestations

As previously mentioned, DMD is an X-linked disease characterized by mutations in the Dmd (dystrophin) gene that cause the loss of dystrophin protein.

Dystrophin is expressed in skeletal, cardiac and smooth muscle where it serves to anchor the sarcolemma to intracellular components such as actin providing support against mechanical stressors. Clinical manifestations of cardiac dystrophy present as dysfunctions in the left ventricle of the heart. These dysfunctions develop before the age of six in 25% of DMD patients, however they do not clinically manifest until later in life due to a general lack of mobility. The most common dysfunction in patients presents as dilated left ventricular walls that can 137

no longer effectively pump blood, leading to cardiac failure 372,373. Cardiac

arrhythmias are also a common feature of cardiac dystrophy, they are assumed to

be related to ventricular myocardial dilation. General arrhythmias occur in 44% of

DMD patients; clinically significant arrhythmias such as supraventricular

arrhythmias (SVT) and ventricular arrhythmias (VT) were present in 10% of DMD

patients and are significantly associated with cardiac dysfunction 374. Sinus tachycardia (increased heart rate) is commonly found in teenage patients with

DMD. Increased heart rate elevates the risk of developing cardiomyopathies, evidenced by abnormal electrocardiograms (ECG) readings preceding the onset of cardiomyopathy in DMD patients 375. The most common ECG findings were the

presence of a short PR interval and right ventricular hypertrophy (RVH) 376. In an

ECG reading, a short PR interval corresponds to faster conduction of the sinoatrial

(SA) nodal impulse. The SA impulse bypasses the AV node sending the signal

directly between the atria and ventricle. RVH is associated with poor pulmonary

circulation, which in DMD could be related to decreased function of the diaphragm

and intercostal muscles. These results suggest that RVH and arrhythmias precede

the onset of dilated cardiomyopathies in DMD patients.

Current therapeutics for cardiomyopathy in DMD

Therapeutic interventions mentioned previously for DMD focus on skeletal

muscle histological markers as signs of improvement in disease progression.

Although cardiac failure is one of the defining factors of mortality in DMD patients,

it has taken a backseat during the development of DMD therapies. Therefore, it is 138

crucial to start evaluating novel therapies, as well as older “established” therapies

such as corticosteroids for their potential effects on cardiac function. Studies are

now corroborating the effects of corticosteroid use on the delayed development of

cardiomyopathies, adding further commendation to the effectiveness of the

therapy 377. The standard of care for non-genetic cardiomyopathies consists of the

use of angiotensin-converting enzyme (ACE) inhibitors and β-adrenergic blockers.

The use of these therapies has been shown to improve left ventricular function and

size in both DMD and BMD patients presenting with cardiomyopathies and are

therefore the main form of DMD cardiac management 372,378. Current forms of

exon-skipping drugs, gene therapy, stem cell therapy and pharmaceutical

intervention have the potential to provide cardiac benefit to DMD patients and are

currently under investigation.

Molecular mechanisms of cardiac dystrophy

In the absence of dystrophin, the cardiomyocyte membrane becomes

fragile and unable to fully support heart contraction or promote lateral force

transduction 379. This fragility causes microtears in the membrane and an ever

increasing number of secondary mechanistic defects, all contributing to cardiac

failure in DMD patients.

In dystrophic skeletal muscle, it is generally accepted that resting

intracellular calcium (Ca2+) levels are increased, causing the activation of

proteases and caspases that further aggravate disease pathology 34,380. This increase in resting intracellular Ca2+ has also been reported in cardiomyocytes. 139

Studies have implicated several different mechanisms of Ca2+ entry, including: 1) influx through microtears, 2) influx through stretch-activated channels (SACs), namely TRPC1 and 3) influx of sodium (Na+) ions through activation of non-specific channels, inducing the Na+ - Ca2+ exchange (NCX) channel to reverse its flux directionality, promoting the influx of Ca2+; thus contributing to increased intracellular resting Ca2+ 381,382.

These alterations in Ca2+ influx are coupled to increases in sarcoplasmic reticulum (SR) protein expression and post-translational modifications of ryanodine receptors (RyR), both of which can produce significant changes to calcium induced calcium release (CICR). In the presence of increased cytosolic

Ca2+ concentrations, the open probability of RyRs is higher, thus increasing the probability of diastolic CICR which affects the after-depolarization and pacemaker activity of the heart 383. This could account for the arrhythmias described in DMD patients, as disrupted EC-coupling is one of the first cardiac manifestations found in mdx pups 384. In addition, elevated cytosolic Ca2+ promotes increased levels of reactive oxygen species (ROS) production in dystrophic muscle. Increased ROS cause oxidation of RyRs, further potentiating the aforementioned disrupted CICR

385. This is additionally supported by studies where the use of ROS scavengers was shown to normalize intracellular Ca2+ levels 386. Furthermore, RyR nitrosylation and phosphorylation of the macromolecular complex bound to RyRs has been associated with dystrophic cardiac pathology 384. These results encourage the development of potential therapeutic therapies for DMD cardiac 140 phenotype aimed at L-type Ca2+, NCX channels or proteins targeting RyR post- translational modifications.

Increases in intracellular Ca2+ also have severe effects on mitochondrial homeostasis. In response to the increase in Ca2+, mitochondria undergo a process called mitochondrial permeability transition (MPT), which is mediated my

Cyclophilin D. During this process, mitochondria form large pore complexes spanning both inner and outer membranes that cause the loss of intermembrane contents and mitochondrial swelling. Studies have successfully reversed this process by both knockdown and pharmacological inhibition of Cyclophilin D.

Ultimately, if left unreversed, MPT leads to mitochondrial rupture, cell necrosis and death, adding to the list of events that promote myocardial necrosis 387.

The large list of molecular events that occur in response to increased intracellular Ca2+ all contribute to the progression of cardiac dystrophy described in DMD patients. In addition, fibrotic tissue replacement of necrotic myocardial cells further inhibits cardiac signal propagation and pacemaker activity, causing arrhythmias in DMD patients. While studies have gathered a lot of information on potential players of cardiac dystrophy, a lot of work is still required to dissect out the essential players and find therapies that target both skeletal and cardiac muscle.

The DGC in cardiac muscle

In a similar manner to skeletal muscle, dystrophin associates with the same series of glycoproteins to form the DGC complex in cardiac tissue. In both skeletal 141 and cardiac muscle, the DGC is localized within the costameres, structures found beneath the sarcolemmal membrane and associated with the Z-line. The Z-line is the most important structural anchor of the sarcomere where contractile force is produced and it is also a known signaling hot-spot 30,388. The roles of costameres include the transduction of bi-directional force from the sarcomeres to the sarcolemma as well as bi-directional signaling from the ECM to the sarcomeres and vice-versa 389. The mechanical role of the costameres is surely attributed to the presence of the DGC, much like in skeletal muscle. In the absence of dystrophin, membrane instability leads to the contraction induced sarcolemmal damage of cardiomyocytes. Further proof of the importance of dystrophin in the heart is evidenced proteolytic cleavage of dystrophin protein by enteroviral protease 2A, which has been implicated in viral myocarditis, thus supporting the essential role of dystrophin in cardiac muscle 390.

Integrin-vinculin-talin complex

The integrin-vinculin-talin complex is the second major complex present at the costameres of striated muscle and has been shown to co-localize with the DGC

388. Talin and vinculin are both tethered to the costamere via their interaction with integrins present at the sarcolemma 390,391. Vinculin is essential to embryonic development, global vinculin knockout mice die in utero and present with severely reduced myocardium at embryonic day 8 to 10 (E8.0-10). This suggests that interaction of vinculin with integrins is essential for cross membrane signaling involved in cell-cell interaction, locomotion and proliferation. Additionally, studies 142

have determined cardiac specific vinculin overexpression can aid in myofilament

organization, leading to improved contraction in myosin deficient fly hearts 392.

Vinculin is therefore essential to myocardial organization and could be a new target

for cardiomyopathy therapies.

Integrins are heterodimeric, transmembrane proteins found at the

sarcolemma of striated muscle, known to be involved in bi-directional signaling

events across the membrane. The α and β subunits come together to form integrin

heterodimers where the β1 subunit can bind 9 different α subunits and once

associated, can provide different affinity for binding partners. In cardiac muscle the

sequential expression of integrin isoforms during development is different from that

of skeletal muscle. The β1 subunit isoforms β1D and β1A are co-expressed in

developing embryos. Later during development the β1A isoform expression slowly

decreases, allowing the β1D isoform to be dominantly expressed in adult

cardiomyocytes. The β1D subunit is expressed in developing hearts before the α7

subunit, during that time it is likely bound to α5 or α6A subunits. In adult

cardiomyocytes the predominant integrin heterodimer is α7β1 and it preferentially

binds laminin protein located in the ECM 393.

To characterize the role of β1 integrin in cardiac development,

cardiomyocytes were retrieved from adrenergically stimulated neonatal rat

ventricular tissue and tested for their ability to provide a hypertrophic response.

This response was only obtained when expressing or overexpressing the β1 integrin subunit, while the response was subdued when integrin signaling was inhibited 394. This suggests β1 integrins are involved in cardiomyocyte function and 143 are therefore essential to heart development and remodeling. In a separate experiment, the expression of a β1 integrin loss-of-function chimera produced conduction changes in the heart, indicating an additional role for β1 integrin in the electrical conduction of the heart 395. Additionally, the conditional deletion of β1 integrin in an mdx background leads to severe cardiac dystrophy with extensive calcification, increased fibrotic replacement and myocardial atrophy. This cardiac dysfunction is significantly more severe than that found in the mdx mouse alone.

Deletion of β1 integrin also leads to decreased responsiveness to adrenergic stimulation and higher sensitivity to isoproterenol induced hypertrophy/cardiac failure 396. These results suggest an important role for β1 integrin in both cardiac development and function, making it a viable therapeutic target to treat dystrophic cardiomyopathies.

It is known that α7β1 integrin is up-regulated in mdx mice. This could provide a mechanism of compensation for the lack of dystrophin in cardiomyocytes, therefore producing a delayed onset of cardiomyopathy in mdx mice which is only observed at 21 months of age. Mutations in Itga7 are shown to cause a type of congenital muscular dystrophy 86. However, the role of α7 integrin in the heart has not been well described. While several studies have been performed on β1 integrin, possibly due to its expression profile preceding that of

α7 integrin in the heart, very few studies have elucidated the role of α7 integrin in the DMD cardiac phenotype. In a clinical report study, whole genome sequencing was performed on an Italian family characterizing two digenic, missense mutations on two different genes shown to promote a severe cardiac phenotype. These two 144

genes are myosin heavy chain 7B (MYH7B) and α7 integrin (Itga7). Segregation

of the mutations within the family suggested a role for α7 integrin in CFTD, a type

of congenital cardiomyopathy 397. This case suggests that α7 integrin could have

a role in cardiac dystrophy that should be further investigated.

Differences between murine and human hearts

The major obstacle in the cardiac research field is the non-existence of a

perfect cardiac animal model. While the sequence of electrical signals and the

overall function of the heart are the same, the frequency of cardiac contractions is

very different between model species. In a nutshell, the larger the heart the lower

the frequency of cardiac contraction. With this in mind, it follows that the heart of

species similar in body-mass weight to humans would share similar mechanics of

heart contractions. This makes swine and sheep models the most appealing

animal models to study the cardiac function. Unfortunately, this comes with a

serious monetary disadvantage in the daily maintenance of these large breed

animals in comparison to smaller breeds such as mice, rats and rabbits 398. In the case of DMD, an additional limitation is that dystrophic animal models are mostly murine with the exception of a couple canine breeds. Additionally, due to the incredibly large cost and ethical concerns surrounding the use of the most common dystrophic dog models, the golden retriever muscular dystrophy (GRMD) dog and the Cavalier King Charles spaniel, research on cardiac dystrophy in DMD is mostly carried out in dystrophic mouse models. 145

When analyzing data from murine cardiac muscle there are several aspects to take into consideration. More previously mentioned, mouse hearts contract at significantly higher rates than human hearts, making the ventricular action potential duration much shorter, with a rapid repolarization. If analyzing cardiac muscle histology, one must take into account the difference in myofilament expression profiles, with the mouse heart containing mostly fast α-myosin heavy chain (MHC) and human heart containing mostly the slower β-MHC. Myofilament kinetics differ substantially between the fast α-MHC and the much slower β-MHC, consistent with cardiac contraction rates. Additionally, studies have found that mouse cardiac troponin I (the sarcomeric Ca2+ regulator) has higher phosphorylation levels, rendering myofilaments more sensitive in mice than in humans, possibly due to the requirement for fast contraction of mouse cardiomyocytes. Human hearts also possess a greater ability to increase their cardiac response in the event of exercise than mice. Cardiac output for humans can increase by as much as 10-fold (65-79 bpm to 173-188 bpm) whereas mice can only increase their cardiac output by less than 33% 398. These differences in mouse and human cardiac muscle must be kept in mind when analyzing cardiac data taken from dystrophic mouse hearts.

In addition to the various differences between murine and human hearts, dystrophic murine mouse models, namely mdx mice, do not follow the same time course for development of cardiomyopathies as DMD patients. The mdx mouse model of DMD only shows signs of cardiomyopathy at 21 months of age 399. Due to the increased maintenance cost of sustaining dozens of mice for close to two years, the mdx/utrn-/- mouse line was developed. This mouse model is missing both 146

dystrophin and the postulated compensatory protein in mice, utrophin. In the

absence of both proteins, mice develop cardiomyopathies at 8 to 11 weeks of age

as opposed to the 21 months required for the mdx mouse model to develop cardiac

symptoms, making it a more appealing mouse model 400. However, the mdx/utrn-/-

mouse is not without its drawbacks, while more appealing in terms of money and

time, the mdx/utrn-/- mouse model does not recapitulate human DMD

cardiomyopathies at the molecular level as it is missing two significant

sarcolemmal proteins. Still, for the time being, mdx/utrn-/- is considered the best

mouse model for studying dystrophic cardiomyopathies.

In the present study, we attempt to characterize the role of α7 integrin in the

myocardium of mice. We used the previously developed Itga7 null mice, where

exon 1 of the Itga7 gene is replaced with lacZ DNA 401. Using this mouse line we

characterized some histological changes in the heart tissue, such as α7 integrin

binding partner localization and fibrosis in Itga7-/- mice. Additionally, we

characterized cardiac abnormalities using ECGs collected in aged Itga7-/- mice.

Although requiring further research, we demonstrate that in the absence of α7 integrin, mice develop cardiac abnormalities similar to those found in congenital cardiomyopathies. We further question whether pharmacological up-regulation of

α7 integrin in dystrophic mouse models (mdx, mdx/utrn+/-) could provide benefit to

cardiac dystrophy. Results show that in fact, increasing levels of α7 integrin in the

heart of dystrophic muscle is consistent with reduced amounts of fibrotic infiltration.

Additionally, our studies provide preliminary results on the use of isoproterenol to

induce cardiomyopathies in mdx and mdx/utrn+/- dystrophic mouse models. 147

Together our study provides insight into the role of α7 integrin in murine hearts and suggests a therapeutic role for α7 integrin in the heart of dystrophic mice.

MATERIALS AND METHODS

Mice and genotyping

Itga7 null mice were generated by replacing exon 1 of the mouse Itga7 gene with the lacZ cDNA. Heterozygous Itga7 mice were crossed to obtain homozygous

-/- Itga7 mutants and wild-type (WT) controls. Mice were anesthetized with CO2 and sacrificed by cervical dislocation. Genotyping was performed with the following primers: a7PF10 (5’ TGAAGGAATGAGTGCACAGTGC 3’), a7exon1R1 (5’

AGATGCCTGTGGGCAGAGTAC 3’), and bgalR2 (5’ GACCTGCAG-

GCATGCAAGC 3’). Mdx-utrn-/- mice (breeding pair) were bought from Jackson

Laboratories and back crossed to mdx mice to obtain mdx-utrn+/- genotype. All procedures involving mice were performed under the approved protocol 000399 from the Institutional Animal Care and Use Committee of the University of Nevada,

Reno.

Immunofluorescence

Cryosections from mouse muscle tissue were obtained with 10 μm of thickness, fixed with 4% PFA and permeabilized with 0.2% Triton-X for 20 mins. The sections were then incubated overnight at 4°C with the following primary antibodies: Integrin

α7B (Song et al., 1993), Integrin β1D 401, laminin (pan antibody against skeletal 148 muscle laminin isoforms) (Sigma L9393; 1:400), laminin α2 (Sigma L0663; 1:100);

Connexin 43 (Thermo Fisher Scientific CX-1B1; 1:200). The sections were then washed using 0.1 M phosphate buffered saline (PBS) pH 7.2. After washing with

PBS, sections were incubated with secondary antibodies conjugated with FITC and TRITC (Jackson Immuno Research; 1:200) for 1 hour at room temperature and mounted with Vectashield Antifade Mounting Medium with 4',6-Diamidino-2-

Phenylindole, Dihydrochloride (DAPI). Images for stained mouse muscle tissue sections were acquired on an Olympus IX81 confocal microscope. The acquired images were analyzed in Fiji version 1.49.

Immunoblotting

Protein was extracted from whole heart tissue using radioimmunoprecipitation assay buffer (RIPA) containing 1:500 dilution of protease inhibitor cocktail and

1:100 dilution of NaF and Na3VO4 phosphatase inhibitors. Protein quantification was performed via BCA (Thermo Scientific), separated by SDS-PAGE and transferred onto nitrocellulose membrane. Detection of α7B and β1D was performed as previously described 350. Protein quantity was normalized to GAPDH

(1:1000, Cell Signaling) or swift stain (G-biosciences) and imaged using LI-COR imaging system. Protein quantification was performed using Fiji (ImageJ).

Histology

Sirius Red staining was performed on 10µm mouse cardiac muscle cryosections.

Sections were hydrated in a series of 100%, 95% and 80% ethanol gradient 149 incubations of 3 minutes each. The sections were then washed in water and stained with Gills Hematoxylin (Fisher Scientific; S5400-1D) for 10 minutes. After washing, the sections were incubated in Scott’s solution for 3 minutes, washed once again and incubated in 0.1% Sirius Red/ Picric Acid (Rowley Biochemical;

SO-674) for 30 minutes. The sections were then washed twice for 5 minutes in acidified water and dehydrated in a series of 80%, 95% and 100% ethanol gradient incubations for 10 minutes each. The dehydration was followed by a 5 minute incubation with Xylene and mounted with DEPEX medium.

Echocardiography

Left ventricle echocardiography evaluation of mdx-utrn+/- mice following isoproterenol induced cardiac stimulation was performed under 1-2.5% isofluorane anesthesia using the Vevo® 2100 Imaging System.

Electrocardiography

Non-invasive recording of electrocardiography (ECG) was performed in conscious, non-anesthetized mice using ECGenieTM (Mouse Specifics, Boston, MA) as previously described23. Briefly, mice were placed on the ECGenie platform containing embedded electrodes in the platform floor and allowed to acclimate to environment for 10 minutes. Signals were digitized with a sampling rate of 2,000 samples/sec and only continuous recordings of 5 secs or more were used for analysis. Each signal was then analyzed using EZCGTM, a software that incorporates Fourier analysis and linear time-invariant filtering of digital 150 frequencies below 3 Hz and above 100 Hz to minimize background environmental noise. The software then uses an algorithm to find the peak of each R-wave and calculate heart rate and plots P, Q, R, S and T waves allowing for rejection of unfiltered noise. Three separate readings of 5 secs or more accounting for >30 peak readings per mouse were averaged.

Statistical Analysis

GraphPad Prism software was used for statistical calculations. Student’s t-test was used to compare means between two groups. Means considered statistically significant when p<0.05.

RESULTS

Characterization of the Itga7-/- cardiac phenotype:

Cardiac abnormalities are present in Itga7-/- mouse hearts.

To characterize any potential cardiac abnormalities in the Itga7-/- mouse and due to the lack of data determining onset of cardiomyopathies in these mice, we decided to age our mice out to 12 months of age. Unexpectedly, around 70% of our mice were found deceased prior to the 12-month mark, for the remaining three mice, ECG readings were taken at 10 months of age and recordings were performed on non-anaesthetized mice. Average compilation of ECG wave readings from wildtype and Itga7-/- mice are depicted showing apparent differences 151 in R wave magnitudes and P to R intervals (Fig. 1A and B). ECG readings were analyzed by EZCGTM software incorporating Fourier analysis. Readings were obtained in awake, free-moving mice with no heart rate variability reported between wildtype and Itga7-/- mice at the time of reading (Fig. 1C). Analyzed ECG reading results indicate decreased PQ and PR intervals in the Itga7-/- mice compared to wildtype counterparts also recorded at 10 weeks of age (Fig. 1D and

E). Ventricular depolarization, as determined by the QRS interval, remains constant between wildtype and Itga7-/- hearts (Fig. 1F). However, there is a decline in the amplitude of the R wave itself within the QRS value accompanied by a shorter SR amplitude (Fig. 1G and H). Taken together these results demonstrate that aged Itga7-/- mice present with significant cardiac abnormalities.

Fibrotic content in Itga7-/- myocardium is not significantly altered.

Replacement of necrotic myocardial cells with fibrotic tissue is a major player in DMD cardiomyopathy pathology. Due to the cardiac abnormalities identified in the Itga7-/- mouse hearts, fibrotic accumulation was assessed in heart sections containing the Purkinje fibers. To determine if the amount of fibrotic tissue infiltration is altered in wildtype and Itga7-/- hearts, cryosections were stained with

Sirius red. Visually, the myocardium of wildtype hearts (Fig. 2A) and that of Itga7 -

/- hearts (Fig. 2B) have the same negligible levels of Sirius red stain and therefore equivalent collagen content. This suggests that the cardiac changes occurring in the Itga7-/- hearts are not due to the disruption of myocardial cell to cell connections. 152

Figure 1. Electrocardiogram assessed cardiac changes in Itga7-/- mice. EKG readings were performed on an N=3 of both wildtype and Itga7-/- mice with over 25

EKG signals analyzed per mouse. (A and B) Average EKG wave compilation of the >25 signals obtained during EKG readings. (C-H) Analysis results of total EKG readings on wildtype and Itga7-/- mice showing constant heart rates between sample groups and no change in QRS values. Itga7-/- mice display decreased PQ,

PR, mean R amplitude and mean SR amplitude suggesting cardiac abnormalities.

153

Loss of α7 integrin does not alter the localization of connexin 43.

Connexin 43 (Cx43) is the most abundant gap junction channel expressed in cardiomyocytes. It is preferentially expressed at the intercalated discs where cardiomyocte-cardiomyocyte communication occurs. Cx43 is responsible for the regulation of cardiac rhythm, vascular tone and endothelial function. Ischemia- induced arrhythmias, hypertrophic cardimyopathies and ischemic cardiomyopathies have all been associated with the mislocalization of Cx43 along the myocyte membrane (Michela, 2015). Laminin signaling via β1 integrin has been shown to affect L-type Ca2+ current in atrial myocytes, suggesting a role for integrins in the autonomic regulation of myocytes (Wang, 2000). Therefore, we hypothesized that α7 integrin might be involved in the anchoring of Cx43 at the intercalated discs and that in the absence of α7 integrin, Cx43 would be mis-placed at the myocyte membrane.

To this end, hearts sections of both Itga7-/- and wildtype hearts were probed for the presence of both α7B and β1D integrin. As expected, expression of α7B was present in wildtype hearts but not detected in Itga7-/- hearts

(Fig. 3A and F). Surprisingly, the expression of β1 integrin in Itga7-/- remained unchanged, suggesting a possible compensatory role of other integrins (α5, α6)

(Fig. 3B and G). Likewise, assembly of laminin at the basal lamina of cardiac tissue was not altered (Fig. 3C and H) and no difference in α2 laminin was detected between Itga7-/- and wildtype hearts (Fig. 3D and I). These results suggest the loss of α7 integrin does not significantly change the localization of its binding partners, 154 although overall levels of binding partner protein expression still need to be determined.

Next, we identified the localization pattern of Cx43 at the intercalated discs and in the subendocardium-myocardium interface. Unlike cardiomyocytes,

Purkinje fibers express Cx43 throughout their membrane, therefore acting as a detection method for Purkinje fibers themselves. Cx43, and therefore Purkinje fibers, at the subendocardium-myocardium interface were not altered in Itga7-/- hearts compared to wildtype hearts, suggesting normal development of Purkinje fibers in the absence of α7 integrin (Fig. 3E and J). Longitudinal sections of the heart, where the intercalated discs can be discerned, show co-localization of Cx43 and α7 integrin at the intercalated discs in wildtype hearts (Fig. 3K-M). However, in the absence of α7 integrin, Cx43 can still be found at the intercalated discs, albeit Cx43 expression appears to be downregulated (Fig. 3N-P). Together these results suggest that α7 integrin is not involved in the anchoring of Cx43, however, co-localization of Cx43 and α7 integrin does not rule out its potential involvement in the function of Cx43. Additionally, overall Cx43 expression levels in the heart need to be assessed.

The potential of α7 integrin therapies in cardiac dystrophy:

Treatment with Sunitinib decreases fibrosis in the mdx mouse heart.

We have identified the involvement of α7 integrin in the maintenance of cardiac rhythm using the Itga7-/- mouse model. Abnormalities in the ECG 155

Figure 2. Levels of fibrosis remain unchanged in Itga7-/- mice. Representative figure of N=1 for fibrotic content within wildtype (A) and Itga7-/- (B) hearts, assessed using Sirus Red stain and determined to be unchanged between the two groups.

156 recordings of Itga7-/- mice, such as shortened R wave amplitudes, are consistent with left ventricular cardiac abnormalities. We therefore hypothesized that increasing α7 integrin expression in the heart of dystrophic mice, specifically dystrophin null mice, could prevent the progression of cardiac dystrophy.

To this end, we turn to the previous study conducted using Sunitinib treatment in mdx mice (Chapter 3). The hearts of all the mice were collected at the end of the study and were retrieved to characterize fibrosis levels. Replacement of necrotic myocytes with fibrotic tissue is a significant player in the development of cardiomyopathies in DMD patients as it interferes with proper cardiac conduction.

While mdx mice do not follow the human DMD cardiac dystrophy timeline and only develop cardiomyopathies after almost two years of age, interestingly, they do develop significant accumulation of fibrosis early on in life. In our previous study, treatment with Sunitinib was shown to both increase levels of α7 integrin in the diaphragm muscle of mdx mice and decrease levels of fibrosis in the gastrocnemius muscle (refer to Chapter 3). To determine the levels of fibrosis in the heart of these twelve week old mdx mice, heart sections were stained for Sirius red. Fibrosis is not present in wildtype mice (Fig. 4A), however significant levels of fibrotic replacement are depicted in the heart of vehicle treated mdx mice (Fig. 4B).

Treatment with Sunitinib appears to significantly reduce the levels of fibrosis in the heart of mdx mice (Fig. 4C). To properly quantify the total amount of fibrosis present in the whole heart, a hydroxyproline assay was performed. Levels of fibrosis present in the entire mdx mouse hearts are significantly decreased with

Sunitinib treatment (Fig. 4D). These results suggest Sunitinib could function to 157 prevent myocyte necrosis and therefore collagen replacement, possibly via increased α7 integrin levels.

Sunitinib treatment increases α7 integrin in the hearts of mdx-utrn+/- mice

To further study the importance of α7 integrin in the dystrophic heart, the mdx-utrn+/- mouse model was used as it exhibits an intermediary cardiac phenotype in comparison to the mdx and the mdx-utrn-/- models. The mdx-utrn+/- mice exhibit lowered utrophin expression, allowing faster cardiac disease progression, yet still present in a more similar manner to DMD patient cardiac dystrophy (Grady, 1997). Hence, the mdx-utrn+/- mice have the advantage of displaying earlier cardiac symptoms compared to the mdx model which does not exhibit cardiac symptoms until 21 months of age. As we know, utrophin is a positive modifier of DMD disease progression and a model mouse not expressing both dystrophin and utrophin could have a phenotype that is not consistent with patient DMD disease pathology and would be considered too dissimilar from the natural disease progression.

To determine whether Sunitinib increases the expression of α7 integrin in the heart, mdx-utrn+/- mice were treated with Sunitinib daily for 2 weeks starting at

5 weeks of age. Levels of α7 integrin were increased 1.5-fold in the heart of

Sunitinib treated mice compared to their vehicle treated counterparts (Fig. 5A).

Additionally, the major β-subunit found in cardiac muscle, β1D, shows a trend towards increasing, albeit not significantly (Fig. 5B). These results suggest that the

158

Figure 3. Histological analysis of WT and Itga7 null cardiac muscle. (A and F)

Transversal view of immunostaining for integrin α7B, (B and G) integrin β1D (C and H) laminin (pan antibody) and Myosin Heavy Chain, (D and I) laminin α2 and

(E and J) laminin and connexin 43 in (A-E) WT and (F-J) Itga7 null cardiac muscle.

Immunostaining for integrin α7B reveals the presence of integrin α7 in WT cardiac muscle (A) and its absence in Itga7 null cardiac tissue (E), whereas integrin β1D staining is present in the cardiomyocyte basal lamina both in WT and Itga7 null 159

cardiac muscle (B,G). Laminin and laminin α2 immunostaining reveals normal

laminin distribution in the absence of integrin α7 (C, D, H, I). There are no

considerable differences in connexin 43 distribution in WT (E) and Itga7 null (J)

cardiac muscle. Longitudinal view of immunostaining for integrin α7 (K, L, N, O) and connexin 43 (K, M, N, P) in WT (K-M) and Itga7 null (N-P) cardiac muscle.

160

anti-fibrotic benefits observed in mdx mice with Sunitinib treatment could be due

to increased levels of α7 integrin.

Mimicking DMD cardiomyopathy using β-adrenergic stimulation

Currently, no good animal models are available to study the development of

cardiac dystrophy that naturally occurs in DMD patients. The mdx mouse model

does not develop dystrophic cardiomyopathy until 21 months of age, thus not

correlating with the development of cardiomyopathy in patients (10 to 15 years of

age) (McNally, 2006; Wasala, 2013). For this reason, new models of dystrophic

cardiomyopathy were developed, such as the mdx-utrn+/- and mdx utrn-/- models, the latter presenting with severe myocyte necrosis at 8 to 11 weeks of age 400.

However, as previously mentioned, this mouse model does not recapitulate natural

disease progression as it is missing two major ECM complexes, the DGC and

UGC, the latter being present in DMD patients. Recently in the field, the use of β-

adrenergic stimulation in dystrophic mice has produced cardiomyopathies similar

to those found in DMD patients and in a shorter timeline amenable to the study of

potential treatments 402,403. Here we present preliminary data demonstrating that

the use of β-adrenergic stimulation via low-dose, consistent, isoproterenol dosing

can produce significant cardiomyopathy in the heart of mdx-utrn+/- mice. First, it is

worth mentioning that our 2-week isoproterenol treatment of 12-14 week old mdx

mice via intraperitoneal injection at 0.8 mg/day 403 did not induce significant

conduction changes as measured by ECG when compared to wildtype mice (Table

1). However, changes were observed in the study conducted by Spurney, et.al, 161

Figure 4. Sunitinib treatment decreases fibrosis in mdx mouse hearts. mdx mice were treated with Sunitinib intermittently (see Fig. 1c, Chapter 3) for 8 weeks, cardiac sections were stained with Sirius red at 12 weeks. Wildtype mice exhibit no fibrotic infiltration (A) while the mdx mice show significant fibrosis in the right ventricle of the heart, which appears significantly reduced in the Sunitinib treated mdx mouse hearts (C). Hydroxyproline quantification of total fibrosis levels in mdx- vehicle and mdx-Sunitinib treated hearts show Sunitinib treatment significantly decreases fibrosis infiltration (D). *p<0.05 162

Figure 5. Sunitinib treatment increases α7β1 integrin in the heart. mdx-utrn+/-

mice treated with Sunitinib daily for 2 weeks show increased levels of α7B protein

(A) as well as increased, if not significantly, of β1D integrin. *p<0.05

2011 after 4 weeks of isoproterenol at 0.5mg/kg/day, suggesting 2-week

isoproterenol treatment is not sufficient to induce cardiomyopathies in younger

mdx mice. To determine whether we could induce cardiomyopathy in dystrophic

mice in a shorter span of time, our study involved the treatment of mdx-utrn+/- mice with isoproterenol (iso- mdx-utrn+/-) at 0.8mg/day for 2 weeks starting at 10 weeks 163

of age. Isoproterenol was contained within an osmotic pump inserted sub-

cutaneously in the mice, unfortunately, inappropriate pump insertion resulted in the

death of several mice. Of the three surviving mice, results show significant cardiac

alterations all pointing toward the development of cardiomyopathies. Visually, the

heart of the mdx-utrn+/- is much larger and heavier than the mdx control hearts.

Normalized to mouse weight, the iso-mdx-utrn+/- mouse heart is about 1.1-1.4 mg/g

heavier than the mdx control hearts (Fig. 6). This suggests significant remodeling

of the heart and potential ventricular hypertrophy. Echocardiograms were

performed on the mice to determine ventricular wall size (Fig. 7A). Results are

consistent with an enlarged left ventricular wall in the iso- treated mdx-utrn+/-

mouse hearts (Fig. 7B). Fractional shortening in the iso- treated mdx-utrn+/- heart is severely reduced suggesting the hypertrophic left ventricle can no longer efficiently pump blood (Fig. 7C). This is consistent with a significant decrease in the ejection fraction of the left ventricle (Fig. 7D). Finally, fibrotic content in the hearts was assessed using Sirius red staining. Fibrosis levels in the 12-week old mdx mouse are shown to be higher than its wildtype counterpart where no fibrosis is apparent (Fig. 8A and B). However, collagen content appears to be further increased in the iso- mdx-utrn+/- mice suggesting myocardial necrosis (Fig. 8C).

These results are all indicative of β-adrenergic stimulation induced

cardiomyopathy in the mdx-utrn+/- mouse heart after a two-week treatment with

isoproterenol. This suggests this animal model can be used to induce

cardiomyopathies in 2 weeks, similar to those found in patients and treatment

options can be assessed in an attainable time frame 164

DISCUSSION

The DMD patient population has a high incidence of cardiomyopathies that contribute to their morbidity. The type and progression of cardiomyopathies in

DMD patients is different from other genetic, non-ischemic cardiomyopathies.

Conduction abnormalities are apparent at 10 to 15 years of age in DMD patients and disease progression is very rapid. This fast progression is due to the many ways in which Ca2+ ions can enter myocytes in the absence of dystrophin and the large list of detrimental effects this has on cell survival. Additionally, due to Ca2+ induced myocardial necrosis, replacement with fibrotic tissue further inhibits cardiac conduction, promoting the fast progression of cardiac failure. Current therapeutic interventions are not targeted specifically at dystrophic cardiomyopathies but used as generalized cardiomyopathy treatments. Therefore, therapies aimed specifically at dystrophic cardiomyopathies are of high importance as they could mean the difference between life and death for DMD patients. In this study, we present evidence that α7 integrin is a positive cardiac disease modifier.

The most convincing evidence of the importance of α7 integrin in cardiac muscle comes from the results obtained from the ECG evaluations on Itga7-/- mice. The

ECG presented decreased PR intervals in the Itga7-/- mice compared to wildtype hearts, suggesting rapid atrioventricular conduction, as well as a decreased R wave amplitude, suggesting impaired 165

Table 1. β-adrenergic challenge in mdx mice. Analyzed EKG values for wildtype and isoproterenol treated mice show that 2-week daily treatment with isoproterenol at 0.8 mg/day did not induce cardiac conduction changes in 14-week old mdx mice.

Figure 6. β-adrenergic challenge increases mdx-utrn+/- heart size. mdx-utrn+/- mice were treated with 0.8 mg/day isoproterenol for 2 weeks. Isoproterenol treated mice exhibited enlarged heart size and weight compared to the mdx control hearts.

166

Figure 7. Echocardiogram assessment on mdx-utrn+/- isoproterenol treated mice. Echocardiograms were obtained to determine ventricular wall enlargement in 12-week old, mdx control and mdx-utrn+/- mice (A). Left ventricular wall size was increased in the mdx-utrn+/- stimulated with isoproterenol compared to the mdx control heart (B). Fractional shortening and ejection fraction were both reduced in mdx-utrn+/- isoproterenol treated mouse heart compared to mdx control heart (C and D).

167

Figure 8. Fibrosis is elevated in mdx-utrn+/- isoproterenol treated hearts.

Sirius red stain was performed on heart sections showing that 12-week old wildtype hearts exhibit no fibrotic infiltration (A) while there is an increase in fibrosis in the right ventricle of the 12-week old mdx control mouse (B) and significant increases in fibrosis are observed in the mdx-utrn+/- isoproterenol treated mouse

(C).

168 ventricular depolarization. Interestingly, these results are consistent with studies describing similar cardiac abnormalities in the presence of CFTD. One such study describes a patient suffering from CFTD, at 1 year of age, presenting with rapid atrioventricular conduction, while another 13 year old patient had developed dilated cardiomyopathy 404. Additionally, another study reported mutations in the

Itga7 gene as one cause of CFTD 397. CFTD is a form of myopathy characterized by consistent type 1 fiber hypotrophy in relation to fiber type 2. Taken together, these studies suggest that mutations in the Itga7 gene can cause CFTD which later develops into cardiac abnormalities and possibly dilated cardiomyopathy.

Therefore, the next step in our characterization of the Itga7-/- mouse heart should involve histological characterization of fiber type hypotrophy, giving further insight into the mechanistic function of α7 integrin within the heart.

In an attempt to shed light on the cause of cardiac abnormalities present in the Itga7-/- mouse heart, the next step in our study identified the co-localization and potential modulating role of α7 integrin on gap junction channels. The most abundant gap junction channel in the heart is Cx43 and our study demonstrates that its localization to the intercalated discs in the heart is not altered in the absence of α7 integrin. However, at first sight it does appear that Cx43 levels are reduced in the Itga7-/- mouse heart. Therefore, in the future of this study the quantification of total Cx43 channel expression in wildtype and Itga7-/- mouse hearts should be assessed. Additionally, there are several other ion channels expressed in cardiac muscle that could be responsible for the cardiac abnormalities observed. Of particular interest is the Nav1.5 channel, responsible for the action potential 169 conduction throughout the myocardium via the influx of Na+ ions. The expression and function of Nav1.5 was determined to be altered in the absence of dystrophin protein and the DGC complex in dystrophic mice 405. Due to the association of the

DGC to the integrin-vinculin-talin complex it is possible that the Nav1.5 channel is also altered in response to the absence of α7 integrin protein in the heart.

Therefore, future studies should characterize the Nav1.5 channel for its localization and expression levels as another potential candidate in the alteration of cardiac abnormalities observed in the Itga7-/- mouse heart.

The changes in cardiac electrical signaling in the Itga7-/- mouse heart are evidence of a role for α7 integrin in cardiac function. We therefore hypothesized that increasing the levels of α7 integrin in the heart of dystrophic mdx mice could provide benefits to cardiac disease progression. For this, we turned to the hearts of mdx mice from our previous study (refer to Chapter 3), where the mice were treated for an 8-week period with the α7 integrin enhancing drug, Sunitinib. No functional studies could be performed on these hearts, however, histological changes to the heart, namely fibrotic tissue replacement, could be assessed. The amount of fibrosis in the hearts of 12-week old, Sunitinib treated mdx mice werefound significantly lowered in comparison to their vehicle treated counterparts. Due to the amount of available hearts, we were not able to assess the increase in total α7 integrin post-treatment in these mice. However, previously

Sunitinib treated (daily for 2 weeks) mdx-utrn+/- mouse hearts showed a significant increase in the levels of α7 integrin. This led us to believe that the decrease in

170 fibrosis observed in the mdx treated mouse hearts could be due to the up- regulation of α7 integrin via Sunitinib.

In our previous study, we determined that Sunitinib exerts is effects via activation of the STAT3 pathway in skeletal muscle, leading to the up-regulation of pro-differentiation markers such as MyoD and myogenin (refer to Chapter 3).

Interestingly, several studies have studied STAT3 in the context of cardiac disease and determined the activation of STAT3 to be cardio-protective. Namely, cardiac overexpression of STAT3 induced the expression of cardiac protective factors in response to Doxorubicin induced cardiotoxicity 406. In reciprocal studies, the conditional ablation of STAT3 from cardiomyocytes left them susceptible to inflammation, fibrosis and heart failure in age related heart failure and ischemic injury models 407,408. Additionally, STAT3 signaling has been implicated in mesenchymal stem cell cardiac repair via IL-6 release 409. More recently, studies show that failing human cardiomyocytes exhibit impaired activation of STAT3 signaling 410 and that it is a critical regulator of β-adrenergic mediated cardiac stress adaptation, where loss of STAT3 causes pronounced cardiomyocyte hypertrophy, cell death and fibrotic replacement 411. Therefore, the beneficial anti- fibrotic effects observed in the Sunitinib treated mdx mice could be due to the activation of STAT3 in the heart. Future studies should determine the STAT3 profile in cardiomyocytes treated with Sunitinib as well as identify differences in cardiac conduction in Sunitinib treated hearts.

In order to set up a large study on the effects of Sunitinib in dystrophic hearts, the first step is to determine what dystrophic cardiac animal model to use. 171

As previously mentioned, there is currently no perfect animal model in which to

study cardiac dystrophy. The mdx mouse model develops cardiomyopathies too

late in the mouse life and the mdx-utrn-/- model is missing two large components of

the ECM that could produce significant changes in myocyte signaling. Studies

have recently turned to the use of isoproterenol to induce cardiomyopathies in

dystrophic mice. However, in the mdx mouse isoproterenol treatment must be

given daily, for 4 weeks straight, to produce significant cardiac effects. For these

reasons, we decided to characterize the extent of cardiomyopathy development in

the mdx-utrn+/- mouse in the presence of two week, low-dose, β-adrenergic

stimulation. Preliminary study results show that we are able to produce significant

cardiomyopathy in 2 weeks, with potential for a faster 1 week treatment as the

severity of cardiomyopathy observed was high. Induction of cardiac abnormalities

is evidenced by increased heart size, increased left ventricular volume, lowered

ejection fractions and increased fibrotic replacement, using the mdx-utrn+/- mouse model and daily isoproterenol treatments. This model of dystrophic cardiomyopathy can be used to test therapies, such as Sunitinib, within a reasonable timeline and without the monetary cost of mouse maintenance for up to two years.

In the future, both the Itga7-/- and mdx-utrn+/- mouse models can be used to

provide a better understanding of the role of α7 integrin in cardiac remodeling. The

effect of α7 integrin on cardiac channel kinetics can be determined using

cardiomyocyte isolation and the patch clamp technique as well as Ca2+ indicators.

This information can then be used to discover therapeutics such as small molecule 172 therapies aimed at α7 integrin enhancement and/or STAT3 activation, aimed specifically at slowing down the progression of dystrophic cardiac conduction abnormalities and cardiomyopathies.

173

Chapter 5

Conclusions and Future Directions in the Development of Small Molecule

Therapies for DMD

174

CONCLUSIONS AND FUTURE DIRECTIONS

DMD is a rare, X-linked neuromuscular disorder affecting circa 1 in 5,000 male children worldwide. It is characterized by severe muscle wasting evidenced by positive Gower’s sign early on in life, later developing into significant motor delays, ventilation deficits and cardiopulmonary failure leading to the untimely death of patients. Two centuries have passed since the clinical description of the first patient with DMD and another three decades since the discovery of the protein dystrophin as the DMD disease culprit. To date, there is no cure for this disease and treatments are equally scarce, consisting mainly of treatment with corticosteroids for the management of inflammation. However, patient care has substantially improved, patients are now met with a team of doctors each specialized in one particular area of the disease. Pulmonary care has been greatly upgraded with the use of nocturnal ventilators and patients now live well into their twenties and thirties. Additionally, patients are receiving angiotensin-converting enzyme (ACE) and β-adrenergic inhibitor treatments to slow down the progression of cardiomyopathies. However, while all these treatments increase survival rates in patients, they also advise a new need for improvements in the quality of life of

DMD patients and ultimately a cure for this devastating disease.

One recent and exciting advancement in the treatment of DMD is the newly

FDA approved, exon-skipping drug Eteplirsen (Exondys 51). Patients receiving this treatment presented with BMD-like phenotype and have significantly increased survival rates. However, as previously mentioned, it is only applicable to 14% of 175 the DMD population bearing the specific exon 51 mutation. Nonetheless, this new therapeutic targets the primary cause of DMD disease pathology which is the absence of dystrophin protein. Currently under phase I clinical trials, is another therapy targeting the primary cause of the disease, the AAV delivery of several different configurations of µ-dystrophin. The main obstacle to overcome using AAV delivery of µ-dystrophin is the innate immunity of patients to the AAV vectors as most would have been exposed to it at some point in their life. In fact, a phase I/II clinical trial held by Solid Biosciences to test SGT-001, an AAV delivered µ- dystrophin therapy, was put on hold in 2017 by the FDA due to hospitalization of the first patient dosed. The trial later had its clinical hold removed in 2018 as the company modified treatment to address the adverse effects observed 412. Once this obstacle of innate AAV immunity has been overcome, µ-dystrophin therapies will certainly be at the top of the DMD therapeutic chain. Also at the very top of this chain is the development of the new CRISPR-Cas9 technology for the correction of the dystrophin reading frame. This therapeutic strategy will surely be in clinical trials in the very near future as it has already proven successful in the GRMD canine model of DMD 227. As is the case with µ-dystrophin, CRISPR-Cas9 technology is delivered using AAV vectors and therefore might undergo some of the similar obstacles. Additionally, neither gene editing technique has yet successfully transduced satellite cell populations and would therefore require multiple rounds of treatment, increasing the financial burden of the disease on families and the risk for immune responses to the AAV vector. For these reasons, future studies using gene editing techniques would benefit from redirecting their 176 focus on AAV vector delivery into satellite cells. Transduced satellite cells would then produce adult muscle fibers already expressing the truncated forms of dystrophin provided by either µ-dystrophin or CRISPR-Cas9 techniques, thus requiring only one dose of treatment. Nonetheless, together, both gene editing therapies are at the cusp of becoming the preferred treatment option for DMD patients.

Gene editing techniques such as exon-skipping and CRISPR-Cas9 are highly concentrated on the “hotspot” genomic area in the dystrophin gene and while this is the most common genetic modification encountered in DMD patients, several other mutations, as well as novel mutations, do still occur. Therefore, the development of novel therapies targeting secondary causes of DMD disease are still well underway. These encompass therapies aimed at stabilizing the sarcolemma of skeletal muscle by acting as the structural anchor replacement between the ECM and intracellular actin in the absence of dystrophin. Such therapies include Laminin-111 (embryonic form of laminin), TAT-utrophin and

MG53 all shown to provide structural support to the sarcolemma of mdx mice.

Therapies such as IGF-1, Galectin-1, biglycan and Wnt7a are all targeted at promoting the regeneration of skeletal muscle, Galectin-1 also showing some anti- inflammatory potential. Currently, all these protein therapies are in the pre-clinical stages of investigation. Regenerative therapies, such as stem cell therapy, are currently in the initial phases of clinical trials where several pre-myogenic cell lines, such as mesoangioblasts and MSCs, have shown significant improvements in both canine and human tissue and/or patients, making them viable future treatment 177 options. This dissertation was focused on the development of novel drug therapies capable of stabilizing the sarcolemma by targeting α7 integrin enhancement.

Interestingly, as the study progressed it was determined that enhancing α7 integrin has a significant effect of the regeneration of muscle and the activation of satellite cell populations.

This study began with the development of a novel cell-based assay (α7+/LacZ myotubes) used to screen hundreds of thousands of compounds for their α7 integrin enhancing capabilities. Using this assay, we discovered a handful of small molecules capable of promoting increased levels of α7 integrin. The most robust compound, SU9516, is the topic of Chapter 2 in this dissertation. SU9516 was originally described as a selective CDK2 inhibitor and tested for its effectiveness in halting colon carcinoma cells 313. Selective inhibition of CDK2 using eight other

CDK inhibitors did not increase α7 integrin expression, therefore eliminating CDK2 as the molecular target of SU9516. Treatment of both C2C12 and human DMD patient myotubes indicated significant increases in α7 integrin protein, suggesting

SU9516 is acting through a pathway previously undescribed for this molecule. In addition, we demonstrate that daily treatment with 5mg/kg of SU9516 increases

α7 integrin expression in mdx mouse skeletal muscle tissue, which suggests that

SU9516 is hitting the target in vivo as well. The mdx mice treated with SU9516 also presented increases in isolated skeletal muscle force production as well as forelimb grip-strength force, increased centrally located nuclei and decreased levels of fibrosis as determined by Sirius red stain. The increased force production was associated with a high increase in the expression of embryonic myosin heavy 178 chain, suggesting SU9516 is promoting high levels of muscle regeneration in the diaphragm muscle of mdx mice. These in vivo studies suggest daily treatment with

SU9516 up-regulates the expression of α7 integrin translating into increases in skeletal muscle force production of the diaphragm and forelimb muscles of treated mdx mice.

Since selective CDK2 inhibition was eliminated as the molecular target for the observed muscle effects, we sought to determine previously undescribed targets for SU9516. Our study shows that in mdx mice and in human DMD patient myotubes, SU9516 is a potent inhibitor of the p65-NF-κB pathway. Whether this is a direct inhibition or a secondary effect of the primary target inhibition is unknown, however, inhibition of NF-κB by SU9516 explains the decrease in collagen infiltration in the mdx mouse skeletal muscle. A KiNativ assay was performed on human DMD patient myotubes to determine novel molecular targets of SU9516.

As a positive control for KiNativ assay function, the results confirmed that SU9516 is a selective CDK2 inhibitor, as previously described, suggesting the validity of the assay in determining molecular targets. Additionally, STE20/SPS1-related proline-alanine-rich protein kinase (STK39/STLK3/SPAK) and the SPAK homolog oxidative stress response -1 (OSR1) were both identified in the KiNativ assay with inhibition levels of ~80% at 0.1µM SU9516. To determine whether the inhibition of these kinases resulted in increased α7 integrin levels we used the SPAK/OSR1 activity repressor STOCK1S-50699. Indeed, treatment with STOCK1S-50699 in

α7+/LacZ myotubes resulted in about a 1.5-fold increase in α7 integrin levels, the same fold increase described for SU9516. These results suggest that inhibition of 179

SPAK/OSR1 causes the downregulation of stress responses modulated by the p65-NF-κB pathway. However, these results do not provide a direct relationship between the SPAK/OSR1 inhibition and the observed increase in α7 integrin expression thus warranting further investigation into the relevance of the

SPAK/OSR1 pathway with regards to DMD disease pathology.

The discovery of SU9516 and its ability to slow down DMD disease progression in the mdx mouse model was an exciting new step towards the development of small molecule therapies for DMD patients. However, due to the toxicity levels observed with the use of SU9516 both in cells and in vivo, our expedition continued, leading us to the discovery of an SU9516 analog and the topic of Chapter 3 in this dissertation.

Sunitinib is a structural analog of SU9516 that is currently marketed under the name SUTENT® and used for the treatment of RCC, GIST and currently being reviewed as a potential treatment for specific solid tumors. Sunitinib was initially developed as a multi-RTK inhibitor, targeting VEGFR, PDGFR, c-Kit, RET and

FLT3 receptors, making it a potent antiangiogenic drug. Due to its structural analogy to SU9516 we hypothesized that Sunitinib could perform in a similar manner by halting DMD disease progression without the toxic effects observed with SU9516. The most important point to keep in mind throughout this study is that Sunitinib was being used at doses that are about 40-fold lower than the antiangiogenic doses used for cancer treatment. Additionally, Sunitinib exerts its effects at doses 5-fold lower than SU9516 and daily treatment is not required, with the treatment schedule for this study set at tri-weekly dosing with 4 days of “off” 180 time. Initial dosing studies based on α7 integrin endpoint expression determined optimal Sunitinib dosing to be 1mg/kg (compare at 40mg/kg for cancer studies in mice and 5mg/kg SU9516), giving a 1.5-fold increase with merely 5 days of treatment.

Sunitinib is currently FDA approved for the treatment of RCC and GIST, therefore, our studies began with the in vivo treatment of mdx mice and the assessment of disease progression. An 8-week treatment with Sunitinib demonstrated significant up-regulation of both α7 integrin transcript and protein levels. The direct interaction of Sunitinib with the α7 integrin promotor is not likely, due its inherent nature as a kinase inhibitor. For this reason, we looked at transcript levels of two known α7 integrin promotor enhancers, MyoD and myogenin and determined that both were up-regulated with Sunitinib treatment. This suggests that treatment with Sunitinib is acting via upstream pathways to enhance α7 integrin expression.

We determined that Sunitinib treatment significantly increases force production of both isolated diaphragm muscle and forelimb force as measured by grip-strength, suggesting that like SU9516, increased α7 integrin translates into increased skeletal muscle force production. Increased force production can be achieved in two ways, increased skeletal muscle volume or stabilization of already present skeletal muscle tissue. Therefore, we assessed regeneration markers after 8 weeks of treatment and determined that Sunitinib is causing a significant increase in two regenerative markers, eMyHC and centrally located nuclei. This led us to believe that Sunitinib is acting via a pathway that causes skeletal muscle 181 hypertrophy by up regulating α7 integrin, as has previously been described 283.

Additionally, we performed a fusion assay showing that Sunitinib treatment promotes the fusion of myoblasts to form myotubes thus supporting the regenerative hypothesis.

The next question to be answered was whether this giant push towards regeneration was negatively affecting and/or depleting the satellite cell pool in the treated mdx muscle. To assess satellite cell numbers, we looked at whole tibialis anterior Pax7 transcript levels and Pax7 protein expression within the diaphragm muscle of mdx mice treated with Sunitinib. Surprisingly, we found that levels of

Pax7 and therefore satellite cells were increasing with Sunitinib treatment, suggesting that the pathway involved or potentially the treatment schedule allows control of both symmetric and asymmetric division of satellite cells. In this study, we also provide evidence that Sunitinib activates the STAT3 pathway both in mdx mouse skeletal muscle and in cultured C2C12 myoblasts. STAT3 has been implicated in the proliferation and differentiation of satellite cells, thus explaining the expansion in the satellite cell pool observed with Sunitinib treatment. It is quite unlikely that Sunitinib is exerting its activating effects directly on STAT3 due to its nature as an inhibitor. Several studies have implicated the cytokine IL-6 in the hypertrophy of skeletal muscle via the Jak2/STAT3 pathway. Therefore, our study looked upstream of STAT3 at the canonical IL-6/Jak2/STAT3 pathway as described in Chapter 3, Fig. 6.

C2C12 myoblasts treated with Sunitinib show activation of STAT3 at about one and half hours post treatment. We looked at a timeline of Sunitinib treatment 182 to determine if upstream targets of the IL-6 pathway are affected. Our study indicates that ERK1/2 is inhibited fifteen minutes post Sunitinib treatment and upstream of ERK1/2, SHP-2 is shown to be inhibited five minutes post treatment.

Our results are in accordance with Sunitinib, directly or indirectly, inhibiting SHP-2 and shutting down the Grb2-Sos-Ras-MEK cascade, thus inhibiting phosphorylation of ERK1/2. ERK1/2 has been shown to directly interact with and inhibit STAT3, therefore, once inhibited by Sunitinib, ERK1/2 can no longer inhibit

STAT3 phosphorylation at tyrosine residues, allowing the activation of STAT3, nuclear translocation and downstream expression of MyoD and myogenin, both known positive transcription factors for the α7 integrin promotor. The up-regulation of α7 integrin would then cause the fusion of myoblasts to form myotubes and therefore promote the regeneration of skeletal muscle responsible for the observed increased force production with Sunitinib treatment.

Interestingly, mutations that cause the constitutive activation of SHP-2

(PTPN11 in humans) are consistent with the development of Noonan syndrome, where patients present with short stature, congenital heart defects, skeletal malformations and bleeding problems 413. A unique case describes a 9-year old boy genetically diagnosed with BMD but presenting with both DMD features and disease progression. Further, whole-genome evaluation determined that the boy had coinheritance of Noonan syndrome and BMD, suggesting that the activation of PTPN11 aggravates muscular dystrophy disease 371. Mutations that cause constitutive activation of PTPN11 function as negative muscular dystrophy disease modulators, allowing faster progression of the disease. Studies have also 183 determined that conditional ablation of PTPN11 in satellite cells drives them into quiescence and severely perturbs skeletal muscle repair, as satellite cells are no longer activated to differentiate into adult muscle 414. This is consistent with the pathway shown in Chapter 3, Fig. 6, where activation of ERK1/2, required for satellite cell proliferation, is only activated downstream of PTPN11/SHP-2, therefore, in the absence of PTPN11/SHP-2 satellite cells are never activated and driven to proliferate or differentiate into new muscle. Together these studies suggest that PTPN11/SHP-2 is required for satellite cell activation and, like STAT3, cannot be constitutively activated or inhibited but must cycle through “on and off” phases. These phases are controlled by the presence of growth factors, in this case the cytokine IL-6, expressed only when regenerative pathways are active.

However, if modulated by a small molecule compound such as Sunitinib, the cycles of satellite cell proliferation and differentiation could potentially be aimed towards regeneration of new muscle, while still allowing the repopulation of the satellite cell pool.

In this study, we describe a new pathway involved in the modulation of satellite cell activation and muscle regeneration. We describe the interaction between SHP-2/ERK1/2 and STAT3 and their effect in the regeneration of skeletal muscle in the mdx mouse model of muscular dystrophy treated with Sunitinib.

While we have shown inhibition of SHP-2 five minutes post treatment we still cannot definitively say that Sunitinib is directly inhibiting SHP-2, as it could be interacting with a nearby pathway that communicates with the IL-6/Jak2/STAT3 pathway. Further studies are required to determine the exact target of Sunitinib, 184 allowing pharmacokinetic studies to be performed in order to determine the optimal dose of Sunitinib treatment resulting in the best regenerative capabilities.

Nonetheless, the pathway responsible for the satellite cell modulation and regeneration observed during the study has been identified. As Sunitinib is an already FDA-approved drug, it could be fast tracked into Phase II clinical trials and potentially provide the first regenerative small molecule therapy for DMD patients.

Almost all DMD patients eventually exhibit signs of cardiomyopathies, the most common being dilated cardiomyopathies that cause increased left ventricular wall thickness and abnormal blood pumping. It has not yet been determined whether cardiac arrhythmias occur pre or post-cardiomyopathy, however sinus tachycardia is commonly found in DMD teenagers suggesting arrhythmias might precede the onset of cardiomyopathies. Myocardial necrosis in the absence of dystrophin can be considered the primary cause of cardiac dysfunction and is promptly replaced by fibrotic tissue causing further cardiac damage. Interestingly, in both mdx mice and in DMD patients increased fibrosis seems to accumulate first in the right ventricle of the heart. This is potentially due to pulmonary problems consistent with decreased diaphragm and intercostal muscle function. Decreased right ventricular function could be the cause of arrhythmias that later on promote the development of left ventricular dilated cardiomyopathies and cardiac failure.

However, due to the lack of movement of DMD patients, it is sometimes difficult to obtain ECG readings when cardiac conduction abnormalities are beginning to present. Current therapies for dilated cardiomyopathies in DMD patients are the same as for general non-ischemic dilated cardiomyopathies, consisting of 185 treatment with ACE and β-adrenergic inhibitors. Aside from that, there is currently no viable treatment for the heart defects observed in the DMD patient population.

The two previous chapters in this dissertation have described small molecule therapies aimed at increasing α7 integrin protein expression. This leads us to question whether α7 integrin has a role in dystrophic hearts and whether our

α7 integrin centered therapies have an effect on the hearts of treated dystrophic mice, bringing us to Chapter 4. In this study, we have partially described the role of α7 integrin in the heart using the Itga7-/- (α7 integrin null) mouse line. Several studies have characterized the role of β1 integrin as being essential to heart development and remodeling in the event of stress. The role of α7 integrin in cardiac muscle has never been described aside from one study suggesting its involvement in the development of CFTD, a type of congenital cardiomyopathy 397.

Being that α7 integrin is only expressed postnatally, heart development abnormalities are unlikely to occur and the Itga7-/- mouse line is not embryonic lethal. Due to the lack of literature on the Itga7-/- mouse with regards to the heart we made an educated decision to age our mice to 12 months, allowing the development of a cardiac phenotype, if one existed. To our surprise, about 80% of our Itga7-/- mouse colony died between 10 to 12 months of age. During these months the mice never appeared morbid nor did they show any signs of disease, appearing deceased in their cages. This suggested the involvement of cardiac failure in their death and prompted us to lower our study age to 10 months.

ECG evaluation of three Itga7-/- mice identified significantly decreased PR values, coupled to shorter mean R amplitudes compared to their wildtype 186 counterparts. Shorter R waves are suggestive of left ventricular contraction abnormalities, potentially due to fibrotic accumulation. However, Sirius red stain performed to look at fibrotic content showed no accumulation in the Itga7-/- hearts.

These results suggest these mice have severe cardiac abnormalities by 10 months of age in the absence of α7 integrin that is not associated with the accumulation of fibrosis. Our study attempted to find the reasoning for cardiac abnormalities in the absence of α7 integrin, hypothesizing that α7 integrin could serve as a sort of anchor for gap junction channels. However, this was not the case, while we do show that α7 integrin and connexin 43 do co-localize, no abnormalities in connexin

43 localization were observed in the absence of α7 integrin.

As previously mentioned, intracellular Ca2+ is responsible for the downstream string of events that lead to myocardial fibrosis in the absence of dystrophin protein. If α7 integrin serves a similar purpose at the sarcolemma, future studies should characterize Ca2+ signaling in isolated cardiomyocytes from Itga7-/- hearts. This would determine if increased intracellular Ca2+ is responsible for the abnormalities observed in the Itga7-/- hearts. Additionally, echocardiograms of

Itga7-/- must be obtained to determine if the arrhythmias observed in the EKG are coupled to cardiomyopathies or other structural abnormalities in the Itga7-/- hearts.

Our study merely characterized gap junctions, as they are responsible for regulating cardiac rhythm, in the future, other ion channels such as Nav1.5, NCX and L-type Ca2+ channels should be characterized in the absence of α7 integrin as their location and/or function could be altered, therefore altering RyR kinetics and signal conduction. Finally, it is also possible that cross membrane signaling 187 supported by α7 integrin with regards to cell survival, proliferation and differentiation is absolutely required for the maintenance of cardiomyocytes.

Therefore, future studies should characterize α7 integrin signaling pathways in cardiac muscle.

Nonetheless, these results suggest α7 integrin has a significant role in cardiac function and maintenance. For this reason, we postulated that our α7 integrin enhancing small molecules, namely Sunitinib, could provide amelioration of cardiac manifestations in treated mdx mice. In fact, Sunitinib treated mdx hearts have significantly decreased collagen accumulation around the right ventricle, suggesting increased α7 integrin prevents fibrotic accumulation in the hearts of

Sunitinib treated mice. As mentioned before, several studies have also determined that activation of the STAT3 pathway is cardioprotective and while no correlation has been made to the increase in α7 integrin, this is certainly a possibility as we have shown that activation of STAT3 leads to increased α7 integrin expression.

The main obstacle to studying cardiac pathology with regards to DMD is the lack of a good animal model. The mdx mouse model only develops cardiomyopathies at 21 months of age, making it very financial and time consuming. The mdx-utrn-/- mouse model was developed to better mimic the timeline of human cardiac pathology in a murine model. In fact, the mdx-utrn-/- mouse develops severe cardiomyocyte necrosis at 8 to 11-weeks of age making it a more accommodating model. However, molecularly, the mdx-utrn-/- mouse model is missing two major sarcolemmal proteins, dystrophin and utrophin, the latter having been identified as a positive disease modifier for DMD and therefore this 188 model also does not recapitulate DMD cardiac disease. It is also worth mentioning that mdx-utrn-/- mice develop severe skeletal muscle contractures that make them not amenable to echocardiography. For these reasons, in this study, we looked into using the mdx-utrn+/- mouse model which is missing dystrophin protein but still has one functional copy of the utrophin gene, thus giving lowered yet present levels of utrophin protein.

We postulated that we could use β-adrenergic stimulation in the mdx-utrn+/- mouse to promote cardiomyopathies, as has been previously performed in mdx mice 402,403, allowing the study of potential therapies such as Sunitinib. We had previously attempted to induce cardiac arrhythmias in relatively young mdx mice using a 2-week isoproterenol treatment which was unsuccessful. Therefore we turned to the mdx-utrn+/- model and successfully induced both arrhythmias and cardiomyopathies with continuous, daily isoproterenol challenge for 2-weeks at 0.8 mg/day. Mice developed significant left ventricular dilation, decreased ejection fractions and significant fibrosis compared to the mdx control mouse hearts. Next, we determined that Sunitinib treatment in mdx-utrn+/- mice induces increased expression of α7 integrin. Therefore, we provide evidence that the mdx-utrn+/- model, in response to consistent 2-week β-adrenergic challenge, can be used to study the effects of the drug Sunitinib in a dystrophic heart context. In the future, this model can be used to induce cardiomyopathies in mice as young as 12 weeks of age without compromising utrophin expression as a disease modifier. Future studies are required to determine whether the decrease in fibrosis observed with 189

Sunitinib treatment translates into halted cardiac disease progression as assessed by ECG, echocardiograms and Ca2+ handling.

In this dissertation, we have identified two α7 integrin enhancing drugs,

SU9516 and Sunitinib, both capable of halting skeletal muscle disease progression in the mdx mouse model of DMD. We also provide evidence that while structurally similar, they do so by different mechanisms of action. While SU9516 seems to be exerting its effects via NF-κB and SPAK/OSR1 it does not inhibit the

ERK1/2/STAT3 pathway. Sunitinib on the other hand inhibits SHP-2/ERK1/2 proliferative pathway and activates the STAT3 differentiation pathway. In addition, we characterize a new mouse model with which to study dystrophic dilated cardiomyopathies, the mdx-utrn+/- model with two week isoproterenol challenge.

We also determine the potential for Sunitinib, via activation of STAT3 pathway and increased α7 integrin, to reduce fibrosis in dystrophic hearts making it very appealing as a novel drug therapy for DMD patients. As it is also FDA- approved it could be fast tracked into clinical trials for DMD and other dystrophies benefiting from muscle regeneration.

As this dissertation is being written, minimal treatments and zero cures are available for DMD patients, however significant strides towards development of therapies are being made.

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Appendix A

Additional Publications

191

Human Molecular Genetics, 2017, Vol. 26, No. 11 2018–2033

doi: 10.1093/hmg/ddx083 Advance Access Publication Date: 7 March 2017 Original Article

ORIGINAL ARTICLE Impaired fetal muscle development and JAK-STAT activation mark disease onset and progression in a mouse model for merosin-deficient congenital muscular dystrophy Andreia M. Nunes1,2, Ryan D. Wuebbles2,†, Apurva Sarathy2,†, Tatiana M. Fontelonga2, Marianne Deries1, Dean J. Burkin2 and Solveig Thorsteinsdottir 1,3,*

1Departamento de Biologia Animal, Centro de Ecologia, Evoluc¸ao~ e Alterac¸oes~ Ambientais, Faculdade de Ciencias,^ Universidade de Lisboa, 1749-016 Lisbon, Portugal, 2Center for Molecular Medicine, University of Nevada School of Medicine, Reno, NV 89557, USA and 3Instituto Gulbenkian de Ciencia,^ 2780-156 Oeiras, Portugal

*To whom correspondence should be addressed. Tel: þ351 217500212; Fax: þ351 217500028; Email: [email protected]

Abstract Merosin-deficient congenital muscular dystrophy type 1A (MDC1A) is a dramatic neuromuscular disease in which crippling muscle weakness is evident from birth. Here, we use the dyW mouse model for human MDC1A to trace the onset of the disease during development in utero. We find that myotomal and primary myogenesis proceed normally in homozygous dyW/ embryos. Fetal dyW/ muscles display the same number of myofibers as wildtype (WT) muscles, but by E18.5 dyW/ muscles are significantly smaller and muscle size is not recovered post-natally. These results suggest that fetal dyW/ myo- fibers fail to grow at the same rate as WT myofibers. Consistent with this hypothesis between E17.5 and E18.5 dyW/ muscles display a dramatic drop in the number of Pax7- and myogenin-positive cells relative to WT muscles, suggesting that dyW/ muscles fail to generate enough muscle cells to sustain fetal myofiber growth. Gene expression analysis of dyW/ E17.5 muscles identified a significant increase in the expression of the JAK-STAT target gene Pim1 and muscles from 2-day and 3- week old dyW/ mice demonstrate a dramatic increase in pSTAT3 relative to WT muscles. Interestingly, myotubes lacking in- tegrin a7b1, a laminin-receptor, also show a significant increase in pSTAT3 levels compared with WT myotubes, indicating that a7b1 can act as a negative regulator of STAT3 activity. Our data reveal for the first time that dyW/ mice exhibit a myo- genesis defect already in utero. We propose that overactivation of JAK-STAT signaling is part of the mechanism underlying disease onset and progression in dyW/ mice.

† These authors contributed equally to this work. Received: August 8, 2016. Revised: January 30, 2017. Accepted: March 2, 2017

VC The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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Introduction mononucleated muscle cells necessary for cell-mediated hyper- trophy of myofibers. We show for the first time that MDC1A starts Merosin-deficient congenital muscular dystrophy (CMD) type before birth in dyW/ mice and that the onset of the disease in 1A (MDC1A), or laminin-a2 CMD (LAMA2-CMD), is a devastating utero is marked by impaired fetal myogenesis. neuromuscular disease in which patients demonstrate hypo- tonia from birth. Human MDC1A was initially described by Tome´ et al. and is caused by mutations in the LAMA2 gene (1–3), Results which encodes the laminin a2 chain of laminins 211 and 221 (4). Myotomal and primary myogenesis proceed normally Laminin 211 is the predominant laminin isoform in the base- in dyW/ embryos ment membrane surrounding adult muscle fibers, while lam- inin 221 localizes specifically to neuromuscular junctions (5,6). Our first aim was to detect the stage of MDC1A onset during Laminin 211 is crucial for myofiber survival (7,8), and is involved mouse development. We first characterized myotome develop- in the regulation of the autophagy-lysosome pathway and the ment in E10.5 dyW/ embryos. Immunostaining (for n numbers; ubiquitin-proteasome system (9,10). see Supplementary Material, Tables S1 and S2) with a pan- It is generally believed that the absence of laminin 211 muscle laminin antibody demonstrates that the laminin matri- around myofibers in MDC1A patients causes a constant stress ces lining the dermomyotome and the myotome are normal in on these cells, which progressively damages them, inducing dyW/ embryos (Fig. 1A, A0,CandC0). The mouse Lama2 gene is muscle wasting, inflammation and fibrosis (11). Since infants normally expressed by myotomal myocytes (Fig. 2D and F) and are already affected at birth, the muscle weakness underlying laminin a2 chain localizes to the myotomal basement mem- the disease must already have arisen during development in brane in a similar pattern to laminin a1anda5 chains (Fig. 2A, utero. However, it is unclear when and how disease begins in B, E0 and G0). Laminin a1 and a2 chains are also detected in a MDC1A. patchy pattern among myotomal myocytes (Fig. 2A, E, E0, G and Mouse skeletal muscle development starts at E8.5 when G0) and this pattern is unperturbed in dyW/ embryos (Fig. 1A, Pax3- and/or Pax7-positive muscle stem cells at the edges of the A0, C and C0). Together these observations suggest that the dis- dermomyotome are induced to initiate the myogenic program tribution of laminin 111 and 511 is normal in dyW/ embryos. and differentiate into myotomal myocytes (12–14). The myo- Immunostaining for myosin heavy chain (MHC) on sections genic program involves the expression of one or more of the of E10.5 dyW/ embryos and their WT littermates shows that myogenic regulatory factors (MRFs), the transcription factors myotome morphology and size in dyW/ embryos is not signifi- Myf5, MyoD, Mrf4 and myogenin (15). From E11.5 until E14.5, cantly different from that of WT embryos (Fig. 1A, A00,C,C00 and these myocytes fuse with differentiating primary myoblasts giv- M; n ¼ 4 per genotype). Immunolabeling for Pax3, Pax7 and ing rise to primary (embryonic) myofibers of the trunk muscles, myogenin did not reveal any differences between WT and dyW/ while dermomyotome-derived Pax3-positive cells have embryos (Fig. 1E–G and I–K). TUNEL analysis showed no in- migrated and differentiate into the primary myofibers of limbs, crease in apoptosis in dyW/ myotomes (Fig. 1B and D)and tongue and diaphragm (16–20). Subsequently, from E14.5 until phospho-histone 3 (pH3) labeling was similar in WT compared birth, Pax7-positive muscle stem cells within the muscle with dyW/ embryos (Fig. 1H and L). We therefore masses undergo a second wave of differentiation into second- conclude that myotomal myogenesis proceeds normally in ary myoblasts. These myoblasts then use the primary myofibers E10.5 dyW/ embryos. as a scaffold and fuse with each other, generating secondary To analyze if primary myogenesis proceeds normally after (fetal) myofibers, and subsequently fuse with both primary and E10.5 in dyW/ embryos, we quantified the total number of pri- secondary myofibers, increasing their size (17,20). Thus fetal mary myofibers in three epaxial deep back muscles (see skeletal muscle grows both by addition of new myofibers Materials and Methods) of E17.5 dyW/ fetuses and their WT lit- (hyperplasia) and by fusion of myoblasts to existing myofibers, termates, stained with an antibody to slow myosin. This quanti- increasing their size (cell-mediated hypertrophy). fication shows that WT and dyW/ embryos form the same Laminins 111 and 511 are thought to play a role in early number of primary myofibers (Fig. 1N;n¼ 36 per genotype). In stages of myogenesis in the somites (21) and laminins 211, 411 fact, our analysis of laminin deposition during primary myogen- and 511 are found in fetal muscles (5). However, little is known esis (E11.5–E13.5) in normal embryos supports this hypothesis. about the exact assembly dynamics and specific roles of the dif- Immunostaining with pan-muscle laminin antibody (Fig. 2H ferent laminin isoforms during myogenesis. It has been sug- and H0)(23,24) and with antibodies for a2(Fig. 2I), a1 and a5 gested that a4- and a5-laminins can compensate for the chains (data not shown) failed to detect a laminin-containing absence of a2-laminins during myogenesis in utero (5,22), but basement membrane around primary myotubes. These results this hypothesis has not been formally tested. indicate that primary myogenesis is normally laminin inde- Here, we perform a detailed analysis of the assembly dy- pendent (see Fig. 3P). namics of the different laminin isoforms during normal mouse These data show that myotome development and primary W/ skeletal muscle development. We then use the dyW mouse model myogenesis are not significantly affected in dy embryos. for MDC1A to study the effect of laminin a2-chain deficiency on skeletal muscle development in vivo. We demonstrate that Laminins 411 and 511 line myofibers and Pax7-positive myotomal and primary myogenesis proceed without defects W/ W/ cells in fetal dy muscles in dy embryos. However, during secondary myogenesis, dyW/ muscles exhibit impaired growth, fail to maintain the nor- We next determined the normal dynamics of laminin assembly mal number of Pax7-positive muscle stem cells and experience a during fetal muscle development by immunohistochemistry. dramatic drop in the number of myogenin-positive myoblasts. Laminin assembly around myofibers starts at E14.5 (Fig. 3A), i.e. This dyW/ muscle defect correlates with an overactivation of precisely at the beginning of secondary myogenesis (17). In JAK-STAT signaling as well as a dysregulation of Myostatin sig- agreement with, and expanding on the data by Patton et al. (5), naling which we suggest hampers the amplification of the pool of we find that laminin assembly around myofibers involves 193

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Figure 1. Normal myotomal and primary myogenesis in dyW/ embryos. Transverse sections of E10.5 WT (A and B, E–H) and dyW/ embryos (C and D, I–L). (A–A00 and C–C00) Immunohistochemistry for pan-muscle laminin (yellow arrows) and MHC (blue arrows) in WT and dyW/ embryos. (B and D) Apoptosis (TUNEL assay) in WT and dyW/ embryos (yellow arrows in B and D). (E–G and I–K) Immunostaining for Pax3 (E and I), Pax7 (F and J) and myogenin (G and K) in WT (arrows in E–G) and dyW/ (arrows in I–K) embryos. (H and L) pH3 immunostaining (arrows) in WT (H) and dyW/ (L) embryos. (M) Quantification of myotome cross sectional area in E10.5 WT and dyW/ embryos. (N) Quantification of slow-myosin positive myofibers in E17.5 WT and dyW/ epaxial muscles. Data in M and N are represented as mean 6 SEM. See Supplementary Material, Table S2 for n numbers. NT, neural tube. Dorsal is on the left. Scale bars: 50 lm. 194

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Figure 2. First phase of laminin assembly during mouse myogenesis: the myotome. (A–G0) Transverse sections of CD-1 E10.5 embryos at forelimb (A–E0) and hindlimb (F–G0) levels stained by immunofluorescence (A–C, E, E0, G and G0)orbyin situ hybridization (D and F). (A–C) Immunostaining for laminin a1 (A), a5 (B) and a4 (C) chains. (D and F) In situ hybridization for Lama2. (E, E0, G and G0) Immunostaining for a2 laminin and MHC with DNA (DAPI) staining (E and G) and grayscale image of a2 laminin immunostaining (E0 and G0). (H and I) Transverse sections of CD-1 E12.5 embryos at forelimb level stained by immunofluorescence. (H and H0) Immunostaining for lam- inin (pan-muscle laminin antibody) and MHC, with DNA staining (H), and grayscale image of immunostaining with pan-muscle laminin antibody (H0). (I) Immunofluorescence for laminin a2 chain. See Supplementary Material, Table S1 for n numbers. FL, Forelimb; HL, Hindlimb; NT, neural tube; DRG, dorsal root gan- glia; MHC, myosin heavy chain. Dorsal is on the left, except (G) and (G0) where dorsal is down. Scale bars: 50 lm (A–D,F,H–I); 25 lm (E,E0,G,G0). laminins containing the a2, a4 and a5 chains (Fig. 3B–D), while 3O, O0 and O00). Thus during normal fetal myogenesis, laminins laminin a1 is absent (data not shown; see Fig. 4C). The myofiber 211, 411 and 511 are gradually assembled around myofibers (see basement membrane is discontinuous at E14.5 (Fig. 3A–D), but Fig. 3P) and by E17.5 most Pax7-positive muscle stem cells reside then grows progressively in subsequent stages (E15.5: Fig. 3E–H underneath the myofiber basement membrane. and M; E17.5: Fig. 3I–L). Pax7-positive muscle stem cells are re- It has previously been suggested that laminin a4anda5 ported to enter their niche under the myofiber basement mem- chains may play a role in compensating for the absence of a2 brane at around E16.5 (25,26). Consistent with this notion, we chain laminins during development (5,22). Indeed, at E17.5 observed a progressive increase in laminin coverage near Pax7- these two laminin chains are detected around both WT and positive cells between E15.5 (Fig. 3N, N0 and N00) and E17.5 (Fig. dyW/ myofibers (Fig. 4A, B, E and F). The laminin a1 chain, 195

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Figure 3. Second phase of laminin assembly during mouse myogenesis: secondary myogenesis. (A–L) Transverse sections of CD-1 E14.5 (A–D), E15.5 (E–H) and E17.5 (I– L) fetuses at forelimb level showing epaxial muscles stained by immunohistochemistry for pan-muscle laminin (A, E and I), laminin a2 (B, F and J), a4 (C, G and K) and a5 (D, H and L) chains. (M) 3D reconstruction of whole mount E15.5 epaxial muscles showing double immunohistochemistry for laminin a2 and MHC. (N–O00) Immunohistochemistry on transverse sections of E15.5 (N and N00) and E17.5 (O and O00) epaxial muscles with pan-muscle laminin and Pax7 antibodies with DNA (DAPI) staining. (P) Schematic representation of laminin assembly dynamics over time during myotomal, primary and secondary myogenesis. See Supplementary Material, Table S1 for n numbers. TS, transversospinalis; LG, longissimus; IL, iliocostalis; MHC, myosin heavy chain. Dorsal is on the left. Scale bars: 50 lm (A–O); 25 lm(N0,N00,O0 and O00).

normally absent in WT muscles, is not upregulated in fetal compared with WT fetuses. Double immunostaining for Pax7 dyW/ muscles (Fig. 4C and G). Thus, in terms of laminin a- and laminins shows that Pax7-positive cells are in close contact chain immunoreactivity, fetal WT and dyW/ muscles only dif- with laminin a4 and a5 chains in both WT and dyW/ muscles fer in that the a2 chain is absent in the dyW/ (see inserts in Fig. (Fig. 4K–N00). Moreover, Pax7-positive cells in dyW/ muscles at 4C and G), as previously reported for adult dyW/ muscles PN2 remain covered by laminins (Fig. 4O, O0 and O00). We con- (27,28). The localization of the a7 subunit of the a7b1 integrin clude that fetal dyW/ muscles contain laminins 411 and 511 as (Fig. 4D and H) and the a-subunit of dystroglycan (Fig. 4I and J), well as the a7b1 integrin and dystroglycan in a pattern very both laminin receptors, is also not affected in dyW/ when similar to the one observed in the WT. 196

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Figure 4. Laminins 411 and 511 line integrin a7b1- and a-dystroglycan-positive myofibers and Pax7-positive cells in dyW/ muscles. (A–J) Immunostaining for laminin a4 (A and E), a5 (B and F), a1 (C and G), a2 (inserts in C and G) chains, integrin a7 subunit (D and H) and a-dystroglycan (I and J) in epaxial muscles of E17.5 WT (A–D and I) and dyW/ (E–H and J) fetuses. (K–N00) Double immunostaining for Pax7 and laminin a4 (K, K00, M and M00) and laminin a5 (M, M00, N, and N00) on transverse sections of epaxial muscles of E17.5 WT (K–N00) and dyW/ (M–N00) fetuses. (O–O00) Double immunostaining for Pax7 and pan-muscle laminin on transverse sections of PN2 dyW/ epaxial muscles. n3 fetuses/pups per genotype/stage and staining, except for a7 integrin (n¼2 per genotype) and laminin a2(n¼1 per genotype). LNa2, laminin a2. Dorsal is to the left and medial is up. Scale bars: 100 lm (A–J); 50 lm (K–O00). dyW/ fetuses display impaired muscle growth development of dyW/ muscles with that of WT muscles at E15.5–E18.5 and PN2 (Fig. 5; Supplementary Material, Fig. S1). We next asked whether laminins 411 and 511 are able to compen- Overall muscle morphology, as viewed by MHC and pan-muscle sate for the lack of functional laminin 211 during fetal and early laminin immunostaining, appears to be normal in dyW/ when postnatal stages of development. To this end we compared the compared with WT fetuses and PN2 pups (Supplementary 197

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Material, Fig. S1A–J). High-magnification images of pan-muscle cells/myofiber (Supplementary Material, Table S3). This reduc- laminin staining demonstrate that, despite the absence of lam- tion in the number of Pax7- and myogenin-positive cells in inin 211 in dyW/ fetuses (insert in Fig. 4G) the pattern of total dyW/ muscles is not due to cell death (Fig. 5O–R), nor to a sig- laminininWTanddyW/ fetuses and PN2 pups is indistinguish- nificant reduction in the number of cells undergoing mitosis able (Fig. 5A–J). Measurements of the area of three epaxial muscle (Fig. 5S; P ¼ 0.515; n ¼ 4–5 per genotype). groups (transversospinalis, longissimus and iliocostalis; see In normal WT muscles, the number of Pax7- and myogenin- Materials and Methods) revealed that fetal WT and dyW/ positive cells goes down between E18.5 and PN2 (Fig. 5M and N; muscles do not differ in size at E15.5 and E16.5, but that from gray lines). Our quantitative data show that the number of W/ E17.5 onwards dy muscles are smaller than WT muscles (Fig. Pax7-positive cells in the muscles of dyW/ PN2 pups is similar W/ 5K). This difference is significant for E18.5 where dy muscles to WT pups at PN2 (Fig. 5M; P ¼ 0.394; n ¼ 4 per genotype), sug- 2 have a mean area of 497 170 6 26 028 lm compared with 607 gesting that the number of Pax7-positive cells diminishes preco- 2 274 6 26 626 lm for WT muscles (Fig. 5K; P ¼ 0.023; n ¼ 4–5 per ciously in dyW/ relative to WT muscles. Furthermore, the W/ genotype) and remains significant for PN2 where dy muscles number of myogenin-positive cells, although more variable 2 have a mean area of 707 547 6 52 802 lm compared with among individuals of both genotypes, is also very similar in 2 975 901 6 56 748 lm for WT muscles (Fig. 5K; P ¼ 0.021, n ¼ 3–4 dyW/ relative to WT muscles at PN2 (Fig. 5N; P ¼ 0.528; n ¼ 3 per per genotype). In spite of this difference in muscle area, the num- genotype). To validate our cell count data at PN2, we isolated W/ ber of myofibers present in WT and dy fetuses at E15.5–E18.5 PN2 epaxial muscles and assessed Pax7 and myogenin W/ does not differ (Fig. 5L) and, although dy have on average protein levels. Interestingly, Pax7 and myogenin protein levels slightly fewer myofibers at PN2, this difference did not reach stat- in dyW/ PN2 muscles are increased 1.5- and 1.6-fold, respect- P ¼ n ¼ istical significance (Fig. 5L; 0.088; 3–4 per genotype). ively, compared with WT pups (Fig. 5T). W/ We conclude that although dy fetuses generate a normal Together these data demonstrate that dyW/ fetal muscles number of myofibers during fetal development, dyW/ muscles undergo a precocious drop in the number of Pax7- and fail to grow normally, being significantly smaller than WT myogenin-positive cells, which correlates with the significant muscles from E18.5 onwards. These results suggest that laminin reduction in cross-sectional area observed in E18.5 dyW/ 211 is essential for the normal growth of fetal muscles and that muscles. At PN2 the numbers of Pax7- and myogenin-positive laminin 411 and 511 are unable to compensate for its absence. cells are similar in WT and dyW/ muscles and Pax7 and myogenin protein levels are actually increased in dyW/ rela- Fetal dyW/ muscles fail to fully expand their pool of tive to WT muscles. However, regardless of this apparent recov- Pax7-positive muscle stem cells and generate fewer ery, the difference in cross-sectional area between WT and W/ differentiated myoblasts dy muscles does not diminish; rather it becomes larger indicating that increased Pax7 and myogenin protein levels To address the cellular mechanism behind the impaired muscle does not translate into an actual recovery (Fig. 5K). growth in dyW/ fetuses, we used immunohistochemistry for Pax7 and myogenin in sections of WT and dyW/ fetuses and PN2 pups to detect muscle stem cells and differentiated myo- dyW/ muscles display an overactivation of the blasts, respectively. During normal WT fetal myogenesis, there JAK-STAT signaling pathway is a steady increase in the number of Pax7-positive cells in the We next applied an RT-qPCR approach to assess for potential muscle masses between E15.5 and E17.5 (Fig. 5M; gray line), and changes in the following signaling pathways known to be the number of Pax7-positive cells stays stable between E17.5 modulators of muscle growth: Wnt/b-catenin (29,30), Notch (31– and E18.5 (Fig. 5M; gray line). This occurs even though this pool 34), JAK-STAT (35–37) and Myostatin (GDF8) (38–41). These ex- of cells also feeds into the myogenin-positive pool through dif- periments were done on muscles isolated from E17.5 fetuses, ferentiation (Fig. 5N; gray line). Quantification of the number of i.e. immediately before dyW/ muscles are significantly differ- Pax7- and myogenin-positive cells shows that these are at first normal in dyW/ muscles (Fig. 5M and N; black lines), but be- ent from WT muscles in terms of a cross-sectional area and the tween E17.5 and E18.5 there is a dramatic reduction in the num- number of Pax7- and myogenin-positive cells. ber of both Pax7- and myogenin-positive cells in dyW/ (Fig. 5M We did not detect significant differences in transcript levels W/ and N; black lines) compared with WT muscles (Fig. 5M and N; of the Wnt signaling target genes Axin2 and Wisp1 in dy gray lines). E18.5 dyW/ fetuses have a mean number of when compared with WT fetal muscles (Fig. 6A). Transcript lev- 348 6 52 Pax7-positive cells per section and thus display a 31% els of the Notch target genes Dll1, HeyL and Hey1 were also not W/ reduction in the number of these cells compared with WT different between dy and WT muscles (Fig. 6B). However, fetuses which have a mean number of 502 6 39 cells, this differ- transcripts for the JAK-STAT signaling target gene Pim1 were ence being statistically significant (Fig. 5M; P ¼ 0.047; n ¼ 5 per significantly increased (Fig. 6C; a 42% increase; P ¼ 0.007) in W/ genotype). Thus, whereas E18.5 WT muscles have on average dy muscles, while Bcl6 and Myc were unchanged (Fig. 6C). 0.19 Pax7-positive cells/myofiber per section, dyW/ muscles Finally, the Myostatin signaling target gene Akirin1, which is have 0.13 Pax7-positive cells/myofiber (Supplementary Material, negatively regulated by Myostatin (42), was significantly W/ Table S3). E18.5 dyW/ fetuses show a 47% reduction in the increased in dy compared with WT muscles (Fig. 6D; a 27% number of myogenin-positive cells compared with WT fetuses, increase; P ¼ 0.027). Cdkn1a, which encodes for p21 and can be as dyW/ muscles have an average of 113 6 12 myogenin-posi- regulated by Myostatin signaling (40), demonstrates a slight, but tive cells per section and WT muscles have an average of not significant, increase in transcript levels in dyW/ fetuses 212 6 21 cells and this difference is statistically significant (Fig. (Fig. 6D). Together, these results point to the possibility that an 5N; P ¼ 0.008; n ¼ 4 per genotype). E18.5 WT muscles therefore overactivation of the JAK-STAT signaling pathway and a down- have an average of 0.08 myogenin-positive cells/myofiber per regulation of the Myostatin signaling pathway in E17.5 dyW/ section, while dyW/ muscles have only 0.04 myogenin-positive muscles may underlie the myogenesis defect identified at E18.5. 198

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Figure 5. dyW/ fetuses display a myogenesis defect. (A–J) High magnification images of pan-muscle laminin immunostaining on transverse sections of WT (A–E) and dyW/ (F–J) epaxial muscles at E15.5 (A and F), E16.5 (B and G), E17.5 (C and H), E18.5 (D and I) and PN2 (E and J) showing that fiber shape and laminin coverage is un- affected in dyW/ compared with WT muscles. (K) Graph showing cross sectional area of epaxial WT (gray line) and dyW/ (black line) muscles from E15.5 to E18.5 and PN2. (L) Graph showing myofiber number in the same epaxial muscles from E15.5 to E18.5 and PN2. (M and N) Graphs showing the quantification of Pax7- (M) and myo- genin-positive (N) cells in fetal and PN2 WT (gray lines) and dyW/ (black lines) epaxial muscles . (O–R) TUNEL assay on transverse section of WT (O and Q) and dyW/ (P and R) muscles at E17.5 (O and P) and PN2 (Q and R). (S) Graph showing the number of pH3-positive cells in E17.5 WT and dyW/ muscles. (T) Western blot quantifica- tion of Pax7 and myogenin proteins in PN2 WT and dyW/ muscles. See Supplementary Material, Figure S1 for images of entire composite sections of epaxial muscle groups and Supplementary Material, Table S2 for n numbers for (A–S). Data in (K–N), (S and T) is represented as mean 6 SEM (*P < 0.05; **P <0.01). Scale bars: 50 lm (A–J); 100 lm (O–R).

To test this hypothesis further, we quantified the amount of Furthermore, pSTAT3 levels remain significantly higher in 3- phosphorylated STAT3 (Tyr 705) (pSTAT3) in E17.5 back muscles week old dyW/ compared with WT muscles (Fig. 6H; P ¼ 0.012). using SureFire analysis. This assay revealed that dyW/ back Together, these data demonstrate that dyW/ muscles display a muscles have, on average, more pSTAT3 compared with control significant increase in JAK-STAT signaling from very early on, muscles, but this difference was not statistically significant (Fig. possibly as early as E17.5, and that pSTAT3 activity remains sig- 6E; P ¼ 0.131; n ¼ 4–6 per genotype group). We then isolated pro- nificantly higher in the muscles of 3-week old dyW/ mice. tein from the back muscles of PN2 pups and performed Western Inflammatory cells and fibrotic tissues are known to secrete blot analysis for pSTAT3 (Tyr 705). The results revealed a 3.4- cytokines that may augment JAK-STAT signaling (43). However, fold and statistically significant (P ¼ 0.002) increase in pSTAT3 fetal and PN2 dyW/ muscles were morphologically normal at levels in dyW/ compared with WT muscles (Fig. 6F). all stages studied (Fig. 5A–J; Supplementary Material, Fig. S1A–J) 199

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Figure 6. Assessment of the mechanism underlying disease onset in dyW/ muscles. (A–D) RT-qPCR analysis of the expression of Wnt, Notch, JAK-STAT and Myostatin signaling pathway members and target genes in E17.5 WT and dyW/ epaxial muscles. (E) SureFire analysis of pSTAT3 (Tyr 705) signal in E17.5 control versus dyW/ muscles. (F) Western blot analysis of pSTAT3 (Tyr 705) and mature Myostatin in WT and dyW/ PN2 muscles. (G) Western blot analysis of pSTAT3 and mature Myostatin in cultured a7þ/þ and a7/ myoblasts and myotubes. (H) Western blot analysis of pSTAT3 levels in triceps muscle of 3-week old WT and dyW/ mice. Data represented as mean 6 SEM (*P <0.05; **P <0.01; ***P <0.001). and did not display any infiltration of macrophages at E17.5 or reason for the increased pSTAT3 activity detected in E17.5 and PN2 (as assessed by CD11b immunolabeling; data not shown) PN2 dyW/ muscles. nor any increase in Sirius Red staining at PN2 (data not shown). Interestingly, mature Myostatin protein levels were also sig- Thus, these results exclude inflammation and fibrosis as the nificantly increased (2.1-fold; P ¼ 0.049) in PN2 dyW/ muscles 200

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when compared with WT (Fig. 6F) indicating that, unlike at and is 75% of WT muscles at PN2, indicating that muscle growth E17.5 where our RT-qPCR results indicate a reduction in starts lagging behind at E18.5 and that the effect is more pro- Myostatin signaling, at PN2 mature Myostatin protein is upregu- nounced at PN2. Reduced myofiber cross sectional area is in- lated in dyW/ muscles. deed one of the hallmarks of disease in dyW/ animals as the Laminin 211 is known to bind to and signal through the a7b1 cross-sectional area of triceps brachii myofibers in 5-week old integrin receptor (44,45). To assess whether the a7b1 integrin dyW/ animals is, on average, 50% that of same stage WT modulates the JAK-STAT and/or Myostatin signaling pathways, muscle (47). Moreover, intramuscular injections of laminin 111 / we measured pSTAT3 and mature Myostatin levels in a7 and into tibialis anterior muscle of 3-week old dyW/ animals in- þ/þ control a7 myoblasts and myotubes. No significant differ- creases the cross sectional area of this muscle by 65% compared / þ/þ ences were detected between a7 and a7 myoblasts (Fig. with that of PBS injected control dyW/ animals (48). Together, 6G). Mature Myostatin levels were also not significantly differ- these observations demonstrate that the presence of laminin / þ/þ ent in a7 compared with a7 myotubes (Fig. 6G). However, 211 (or the closely related laminin 111) acts positively on muscle < W/ pSTAT3 levels were dramatically increased (3.1-fold; P 0.0001) growth. Here we demonstrate that dy muscle growth starts in a7/ myotubes compared with a7þ/þ myotubes (Fig. 6G). lagging behind already in utero, before any signs of muscle path- These results demonstrate that whereas the a7b1 integrin does ology are detected, which leads us to propose that the onset of not appear to affect JAK-STAT signaling in myoblasts, it is a po- MDC1A disease in dyW/ mice involves a defect in cell- tent negative regulator of JAK-STAT signaling in myotubes. mediated hypertrophy during fetal myogenesis. Together, these results implicate increased pSTAT3 activity in early stages of dyW/ pathology through a mechanism that seems to involve the a7b1 integrin on myotubes. Moreover, we Overactivation of JAK-STAT marks dyW/ disease onset also provide evidence for a dysregulation of Myostatin signaling Our results suggest that the STAT3 signaling pathway is overac- in dyW/ compared with WT muscles as Myostatin signaling is tivated in dyW/ muscles and that the link between laminin reduced in dyW/ fetal muscles masses but then mature 211 and STAT3 signaling lies in the a7b1 integrin, since integrin Myostatin levels are increased in these muscles after birth. a7-null myotubes in vitro show a dramatic upregulation of pSTAT3 compared with control myotubes. It has already been Discussion extensively demonstrated that integrin a7b1 signaling plays an MDC1A starts during development in utero in the dyW/ important role in disease progression of Lama2-deficient mice mouse model (7,49,50). Curiously, a7-null myoblasts do not show any increase in pSTAT3. However, myoblasts in vitro do not fully represent W/ Here we reveal for the first time that MDC1A in the dy the diversity of mononucleated muscle cell in vivo. Indeed, pre- mouse starts during development in utero. Although laminins vious studies have shown that JAK-STAT3 overactivation in sat- containing the a2-chain are first detected during myotome de- ellite cells promotes aging and impairs their expansion during velopment, this phase of myogenesis proceeds normally in muscle repair (36,37). Thus we cannot exclude the possibility W/ dy embryos. Laminin 111 and 511 are present during myo- that dyW/ fetuses display an increased STAT3 phosphoryl- tome development (this study, 5,21) and we find that both lam- ation in a subset of mononucleated muscle cells. Altogether, inin a1 and laminin a2 are detected within the E10.5 myotome, these results indicate that a major function of laminin 211 in suggesting laminin 111 is able to compensate for the absence of myofibers is to attenuate STAT3 signaling via the a7b1 integrin. laminin 211 in the myotome. Consistent with this hypothesis, E18.5 dyW/ fetal muscles have 31% fewer Pax7-positive overexpression of a laminin a1 transgene (46) and laminin 111 cells than WT muscles which demonstrates that these muscles protein therapy ameliorate pathology in laminin a2-deficient are unable to maintain normal levels of self-renewal of muscle mice (47,48). stem cells. We see no indication of cell death, nor differences in Strikingly, primary myogenesis appears to proceed in the pH3-positive cell numbers, excluding increased apoptosis or im- total absence of assembled laminins both in trunk and limbs paired proliferation as the reason for the drop in Pax7-positive (this study, 23) suggesting that this phase of myogenesis is cells. Muscle stem cell amplification is achieved by symmetric laminin-independent. Accordingly, we verified that primary divisions of Pax7-positive cells which originate two Pax7- myogenesis proceeds normally in dyW/ embryos. positive stem cells, whereas asymmetric divisions maintain one Laminin assembly around myotubes resumes at E14.5, at the Pax7-positive stem cell while simultaneously generating a com- very beginning of secondary (fetal) myogenesis, with the assem- mitted cell which later differentiates and fuses with the muscle bly of laminins 211, 411 and 511, but not laminin 111 (this study, 5). We find that these laminins progressively come to line myo- fiber (51–53). The 3D organization of the myofiber basement fibers and Pax7-positive muscle stem cells as they enter their membrane favors symmetric divisions of Pax7-positive cells niche at around E16.5 (25,26,49). Fetal myogenesis is character- (54). Furthermore, an intact laminin matrix in the Pax7-positive ized by intense muscle growth, which occurs in two ways: (1) by stem cell niche is essential for their self-renewal (55). Thus lam- the generation of secondary myofibers (hyperplasia) most of inin 211-deficiency is likely to alter the nature of the satellite which occurs until E18.0 (26) and (2) through growth of these cell niche as well as its 3D organization. Interestingly, satellite secondary myofibers, as well as the preexisting primary myofib- cells cultured on isolated myofibers under conditions ers, through fusion of myoblasts to these fibers (cell-mediated that inhibit JAK-STAT signaling show a significant increase in hypertrophy) (17,20). Our data show that WT and dyW/ fetuses the frequency of symmetric cell divisions and a decrease in display similar myofiber numbers. However, our data indicate asymmetric divisions (36). Thus we suggest a model that between E17.5 and E18.5, dyW/ fetuses exhibit an impair- where altered laminin composition and/or overactivation of ment in cell-mediated hypertrophy, which is not rescued by the JAK-STAT signaling in fetal dyW/ muscles increases the fre- presence of laminins 411 and 511. Our measurements show that quency of asymmetric cell divisions at the expense of symmet- the area of dyW/ muscles is 82% of that of WT muscles at E18.5 ric cell divisions, thus leading to a precocious reduction in the 201

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Figure 7. Model illustrating possible mechanism of disease onset during dyW/ fetal muscle development. Illustration of muscles in normal (WT; A and B) and Lama2- deficient (dyW/; C and D) mice at fetal E18.5 (A and C) and postnatal PN2 (B and D) stages. A close-up of the surface of a muscle fiber shows two muscle stem cell div- ision events (A0–D0) and the proposed results of these divisions (A00–D00)inWT(A0,A00,B0 and B00) and dyW/ (C0,C00,D0 and D00) muscles at E18.5 (A0,A00,C0 and C00) and PN2 (B0,B00,D0 and D00). Lama2-deficiency results in a significant reduction in the number of Pax7- and myogenin-positive cells at E18.5 compared with same stage WT muscles. This may occur due to an increase in the frequency of asymmetric cell divisions at the expense of amplifying symmetric divisions (A0 and C0), either because of altered laminin composition (absence of laminin 211) and/or an increase in pSTAT3 activity (A0 and C0). Consequently, E18.5 dyW/ muscles would have fewer Pax7- positive muscle stem cells than WT muscles (A00 and C00). We further propose that, due to an increased expression of Aikirin1 (A0 and C0), committed cells in fetal dyW/ muscles would display impaired expansion capacity, leading to fewer terminally differentiated myoblasts (A00 and C00; shaded cells in C00 would not be generated). Therefore, E18.5 dyW/ muscles have fewer myogenin-positive, terminally differentiated myoblasts than WT muscles (A00 and C00). In normal postnatal muscle, the fre- quency of asymmetric cell divisions increases and expansion of muscle stem cells and committed cells decreases. This is illustrated by showing only asymmetric cell divisions in WT (B0 and B00) and dyW/ muscles (D0 and D00) and fewer cell divisions of committed cells in both WT (B00) and dyW/ muscles (D00). Lama2-deficiency con- tinues to lead to increased levels of pSTAT3 post-natally and in addition leads to the concomitant increase in mature myostatin levels (B0 and D0). Although PN2 dyW/ muscles are significantly smaller than WT muscles they have similar numbers of stem cells and differentiated cells at this stage (B00 and D00). Thus the rate of asymmet- ric divisions appears to be similar in WT and dyW/ PN2 muscles. However, the joint overactivation of JAK-STAT and Myostatin signaling at PN2 could exert a progres- sive impairment on the division capacity of muscle stem cells and committed cells in dyW/ individuals (B00 and D00) with consequences for subsequent stages. This effect may later have a negative impact on the regeneration capacity of dyW/ muscles. number of Pax7-positive cells in dyW/ relative to WT muscles upregulation of Akirin1 (Mighty)indyW/ muscles at E17.5. (Fig. 7A and C). Akirin 1 is negatively regulated by Myostatin signaling and is Our results also show that E18.5 dyW/ fetal muscles have known to activate MyoD in satellite cells (40,42,57). Normally, an even more dramatic reduction in myogenin-positive cells. committed myoblasts can undergo one or two cell divisions be- This suggests that E18.5 dyW/ fetal muscles also experience an fore MyoD activates p21, inducing cell cycle exit (58). impaired generation of normal numbers of differentiated and Interestingly, Mstn/ fetuses exhibit a drop in the number of fusion-competent myoblasts. However, increased JAK-STAT sig- Pax7- and myogenin-positive cells at E18.5, which the authors naling leads to an increase in MyoD levels (35–37,56). A possible suggest is due to an overactivation of MyoD in committed cells, explanation may lie in the concomitant and significant leading to their faster exit from the cell cycle and formation of 202

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fewer differentiated and fusion-competent cells (41). We thus rather than putting deteriorating muscle fiber health as a first suggest that the drop in myogenin-positive cells observed in step in the disease, our data demonstrate that the primary de- E18.5 dyW/ muscles may be due to an attenuation of fect in dyW/ mice arises because of impaired in fetal myogene- Myostatin signaling which makes committed cells differentiate sis. Our results also uncovered the relevance of JAK-STAT and faster and without significant amplification (Fig. 7A and C). Myostatin pathways in dyW/ disease onset and progression, Taken together we propose a model for MDC1A onset (Fig. which emphasizes the importance of these pathways as targets 7A and C) where the absence of laminin 211 around fetal myo- for future therapies. Taken together, our study provides an im- fibers and/or an overactivation of JAK-STAT signaling shift the portant framework for future in utero therapies and highlights balance of muscle stem cell divisions to asymmetric divisions, the necessity for prenatal disease diagnosis and early leading to a reduction in the renewal rate of the Pax7-positive intervention. muscle stem cell pool. Furthermore, the simultaneous attenu- ation in Myostatin signaling inhibits the normal amplification Materials and Methods of committed cells, leading to a drop in the number of myogenin-positive cells, formation of fewer fusion-competent Mice and genotyping myoblasts and, consequently, an impairment in fiber growth. dyW mice (gift from Eva Engvall via Paul Martin; The Ohio State University, Columbus, OH, USA) have a LacZ-neo cassette in- dyW/ pups start life with significantly smaller muscles serted in the Lama2 gene, and homozygous animals produce a and do not possess the machinery to recover their size small amount of a truncated laminin a2 protein lacking the N- terminal LN domain (27,28). Heterozygous dyW mice were In agreement with the situation in human MDC1A patients who crossed to obtain homozygous dyW/ mutants and wild-type W/ have smaller muscles at birth (3), we found that dy pups are (WT) controls. Fetuses heterozygous for the dyW allele were in- born with significantly smaller muscles than WT pups. distinguishable from WT controls in all parameters measured W/ Although dy pups no longer differ from WT pups in the (data not shown) and were thus used as controls together with W/ number of Pax7- and myogenin-positive cells, dy pups have WT fetuses in the SureFire assay. Outbred Charles River CD-1 not recovered from the impaired growth during fetal myogene- mice (Envigo, Spain) were used to assess the distribution of lam- sis. In fact, the apparent recovery of Pax7-positive cell numbers inin variants during normal myogenesis (Figs 2 and 3). W/ in dy pups is not due to an increase in Pax7-positive cells in The day of the vaginal plug was designated embryonic day W/ dy muscles. Rather it is due to a decrease in the number of (E) 0.5 and embryos were staged as in Kaufman (64). Pax7-positive cells in WT muscles between E18.5 and PN2. Anesthetized pregnant females were sacrificed by cervical dis- Indeed, a gradual drop in Pax7-positive cell numbers is known location, uterine horns removed and placed in cold phosphate to occur during normal postnatal development with the pro- buffered saline (PBS) with Ca2þ and Mg2þ where embryos (E10.5– gressive increase in the frequency of asymmetric cell divisions E13.5) and fetuses (E14.5–E18.5) were dissected out. Post-natal (59). Moreover, muscle stem cells experience a change in iden- day 2 (PN2) pups were anesthetized by hypothermia and sacri- tity between fetal and postnatal stages of development, where ficed by decapitation. Embryos for in situ hybridization were col- their capacity for self-renewal goes down while the potency to lected in PBS. either differentiate or to recolonize the satellite cell niche in- Embryos, fetuses and pups from heterozygous dyW crossings creases (60). Our data show that fetal dyW/ muscles experi- were genotyped with the following primers: 50 ACTGCCCTTTC ence a premature decrease in the number of Pax7-positive cells TCACCCACCCTT 30,50 GTTGATGCGCTTGGGACTG 30 and 50 GTC suggesting that they make the transition to a postnatal identity GACGACGACAGTACTGGCCTCAG 30. too early. All procedures involving mice were performed under two PN2 pups show a significant increase in pSTAT3 activity (Fig. approved protocols: (3/2016) from the Animal Welfare Body of 7B and D), suggesting that continuous laminin 211-integrin a7b1 the Faculty of Sciences, University of Lisbon and (000404) from signaling is normally required to dampen this signaling path- the Institutional Animal Care and Use Committee of the way. Curiously, in contrast to the situation observed at E17.5 University of Nevada. where Myostatin signaling appears to be attenuated, dyW/ PN2 muscles display higher levels of mature Myostatin com- Cell culture pared with WT muscles (Fig. 7B and D). The reason for this is unclear, but a simultaneous increase in pSTAT3 and Myostatin Myoblasts isolated previously from gastrocnemius muscles of is characteristic of aged muscle, and is thought to be a major 10-day old a7bgalþ/þ and a7bgal/ mice (65) were cultured in factor in the observed loss of regeneration potential during six-well plates until confluence in Dulbecco’s modified Eagle muscle aging (61,62). Consistent with this notion, the muscles medium (DMEM) (GIBCO) supplemented with 20% fetal bovine of dyW/ mice have a dramatic reduction in regeneration cap- serum (Atlanta Biologicals), 0.5% chick-embryo extract (Seralab), acity (48,63). 2mML-glutamine (GIBCO) and penicillin/streptomycin (100 U/ W/ We therefore propose that dy muscles display an aged ml; GIBCO) at 37 C with 5% CO2. Myotubes were obtained by dif- phenotype where, at each time-point from fetal development to ferentiating a7bgalþ/þ and a7bgal/ myoblasts during 5 days in adulthood, the muscle environmental cues resemble that of DMEM supplemented with 1% horse serum (Atlanta Biologicals), substantially older muscles. 2mM L-glutamine (GIBCO) and penicillin/streptomycin (100 U/ ml; GIBCO). Conclusions In situ hybridization In the present study, we provide the first evidence of in utero de- fects in a mouse model for MDC1A. This marks a paradigm shift To determine the mRNA expression pattern of Lama2 (66), E10.5 in our understanding of MDC1A disease pathogenesis because embryos were fixed in 4% paraformaldehyde (PFA) in PBS for 203

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whole mount in situ hybridization as described previously (66). WesternSureTM PREMIUM Chemiluminescent Substract (LICOR) Briefly, embryos were hybridized overnight at 70 C and probe and detection with an Odyssey imaging system (Li-Cor localization was detected with alkaline phosphatase- Biosciences). Normalization was performed with glyceralde- conjugated anti-dioxygenin antibodies and NBT/BCIP (Roche) as hyde 3-phosphate dehydrogenase (GAPDH) and/or total STAT3. a substrate. The number of embryos used is listed in Quantifications were performed in Fiji (http://fiji.sc/Fiji). Supplementary Material, Table S1. Western blots on myoblasts and myotubes were done in tripli- cate. Muscle tissue from at least 5 PN2 and 3-week old mice per genotype were used. Antibodies used are listed in Immunohistochemistry Supplementary Material, Table S4. Embryos were fixed in 0.2% PFA in 0.12M phosphate buffer (PB) overnight at 4C. Fetuses and PN2 pups were fixed in 2% PFA in Real time quantitative RT-PCR PB for 2 days at 4C. Embryos, fetuses and PN2 pups were cryo- embedding and 12 lm cryosections were processed for immu- RNA was extracted from E17.5 back muscles with Trizol Reagent nohistochemistry essentially as in Bajanca et al. (67). However, kit (Ambion, Life Technologies). First-strand cDNA synthesis cryosections of fetuses and PN2 pups were incubated with 0.2% was performed using the SuperScript III First-Strand Synthesis Triton X-100 in PBS for 20 min at room temperature before System for RT-PCR kit (Ambion, Life Technologies). Real-Time blocking with 5% bovine albumin serum in PBS. Antigen re- qPCR reactions were performed in triplicates with 2 PowerUp trieval was done in E15.5-PN2 tissue by immersing sections in SYBR Green Master Mix (Life Technologies) and 500 ng RNA. Tris–EDTA (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20) buf- Transcript levels were normalized against Gapdh expession and fer, pH 9.0 at 95C for 20 min. When staining with monoclonal the fold change was calculated using DDCt method. Primers mouse antibodies, the Mouse-On-Mouse (MOM) kit (Vector used are listed in Supplementary Material, Table S5. Laboratories) was used. Antibodies used are listed in Supplementary Material, Table S4. The polyclonal antibody against EHS-laminin (i.e. laminin Image analysis and quantifications 111) (4) detects all laminins containing a1, b1orc1 chains (68). Sections processed for in situ hybridization were photographed Since all laminin isoforms in skeletal muscle contain at least using an Olympus DP50 camera coupled to an Olympus BX51 the c1 chain, we used this polyclonal anti-laminin antibody to microscope. Sections processed for immunohistochemistry detect all muscle laminins and designate it pan-muscle laminin were either imaged with a Hamamatsu Orca R2 camera coupled antibody. TUNEL assay was performed using DeadEnd to an Olympus BX60 fluorescence microscope, or images were Fluorometric TUNEL System (Promega). DNA was visualized acquired on an Olympus IX81 or Leica SPE confocal microscope with 4,6-diamidino-2-phenylindole (DAPI, 5 mg/ml, Sigma). The system. The acquired images were analyzed in Fiji version 1.49 number of embryos, fetuses and PN2 pups processed for immu- and imported to Amira V.5.3.3 (Visage Imaging, Inc.) software as nohistochemistry and/or TUNEL assay are listed in described previously (69,70). Supplementary Material, Tables S1 and S2. Transverse sections processed for immunohistochemistry were used for muscle cross-sectional area measurements and quantification of total myofibers, primary myofibers, Pax7- and Whole mount immunohistochemistry myogenin-positive cells. To ensure maximum standardization, Whole mount immunohistochemistry was performed as quantifications were always done in three specific epaxial described previously (69). Briefly, dissected fetal back muscles muscle groups (transversospinalis, longissimus and iliocostalis) were incubated with primary and secondary antibodies for 2 at forelimb level and three to five sections per staining and em- days at 4C and were washed for a full day after each antibody bryo/fetus/PN2 pup were used. Overlapping confocal images incubation. For nuclear staining fetal muscles were incubated covering the three specific epaxial muscles were obtained with with Topro3 (T3605; Molecular Probes; 1:400) and 10 mg/ml ribo- a20 lens and individual images were then stitched together nuclease A (55674; Calbiochem; 1:100) together with the second- into a large composite image (see Supplementary Material, Fig. ary antibodies. Antibodies used are listed in Supplementary S1) using the Fiji plugin Pairwise Stitching (71). Areas were Material, Table S4. measured using a drawing tool to line the muscles in Fiji and quantifications were performed using Fiji plugin Cell Counter (http://fiji.sc/Cell_Counter). We consider the sum of the number SureFire of Pax7- and myogenin-positive cells as a close estimate for the AlphaLISA SureFire Ultra kit for p-STAT3 (Tyr705) (ALSU-PST3- total number mononucleated muscle cells in the muscle masses A500) was performed according to the manufacturer’s instruc- (Supplementary Material, Table S3) (20). All measurements and tions (Perkin Elmer). Ten micrograms of protein extract were counts were done in a blinded fashion. used per sample for this assay.

Statistical analysis Western blotting Student’s t-test was used to test for differences between Protein extracts were collected in radioimmunoprecipitation mRNA, protein or pSTAT3 levels in muscle samples from WT W/ þ/þ / assay buffer (RIPA) with 5 mM NaF, 10 mM Na3VO4 and a prote- and dy fetuses and/or PN2 pups, and a7 and a7 cells, ase inhibitor cocktail (Thermo Scientific; 1:500) and stored at considering P < 0.05 as statistically significant. Student’s t-test 80C. After quantification using Pierce BCA protein assay kit was also used to test for differences in the number of Pax7- and (Thermo Scientific), protein extracts were separated with SDS 10 myogenin-positive cells in sections of WT and dyW/ muscles. or 12% polyacrylamide gel electrophoresis and transferred to Finally, differences in muscle cross-sectional area and myofiber nitrocellulose membranes. Signals were detected using numbers were tested using a nested ANOVA where individuals 204

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were nested within genotypes (WT or dyW/). As described system of developing, adult, and mutant mice. J. Cell Biol., above, all parameters were quantified in stitched composite 139, 1507–1521. images of three to five sections per individual, each section cov- 6. Patton, B.L. (2000) Laminins of the neuromuscular system. ering the same three muscle groups. Each stage was tested sep- Microsc. Res. Tech., 51, 247–261. arately. Statistical analyses were performed using GraphPad 7. Vachon, P.H., Xu, H., Liu, L., Loechel, F., Hayashi, Y., Arahata, Prism 5 and STATISTICA 12 software. K., Reed, J.C., Wewer, U.M. and Engvall, E. (1997) Integrins (a7b1) in muscle function and survival. Disrupted expression Supplementary Material in merosin-deficient congenital muscular dystrophy. J. Clin. Invest., 100, 1870–1881. Supplementary Material is available at HMG online. 8. Laprise, P., Valle´, K., Demers, M.J., Bouchard, V., Poirier, E.M., Ve´zina, A., Reed, J.C., Rivard, N. and Vachon, P.H. (2003) Merosin (laminin-2/4)-driven survival signalling: complex Acknowledgements modulations of Bcl-2 homologs. J. Cell. Biochem., 89, We thank Jeff Miner for generously sharing his anti-a5 and anti- 1115–1125. a4 laminin antibodies, Madeleine Durbeej for kindly giving the 9. Carmignac, V., Svensson, M., Ko¨ rner, Z., Elowsson, L., anti-a1 antibody and Patrıcia Ybot-Gonzalez for the Lama2 Matsumura, C., Gawlik, K.I., Allamand, V. and Durbeej, M. probe. The MF20, Pax3, Pax7 and IIH6 C4 antibodies were de- (2011a) Autophagy is increased in laminin a2 chain-deficient veloped by D.A. Fischman, C.P. Ordahl, A. Kawakami and muscle and its inhibition improves muscle morphology in a K.P. Campbell, respectively, and were obtained from the mouse model of MDC1A. Hum. Mol. Genet., 20, 4891–4902. Developmental Studies Hybridoma Bank, developed under the 10. Carmignac, V., Que´re´, R. and Durbeej, M. (2011b) Proteasome auspices of the NICHD and maintained by The University of inhibition improves the muscle of laminin a2 chain- Iowa, Department of Biology, Iowa City, IA 52242, USA. We also deficient mice. Hum. Mol. Genet., 20, 541–552. thank Ines^ Fragata, Jorge Palmeirim and Margarida Barbaro for 11. Gawlik, K.I. and Durbeej, M. (2011) Skeletal muscle laminin help with the statistical analysis, Ines^ Antunes for help in the and MDC1A: pathogenesis and treatment strategies. Skelet. laboratory, Patrıcia Gomes de Almeida for help with image pro- Muscle, 1, 1–13. cessing and for critically reviewing the manuscript and all 12. Venters, S., Thorsteinsdottir, S. and Duxson, M.J. (1999) Early members of our groups for suggestions and constant support. development of the myotome in the mouse. Dev. Dyn., 216, 219–232. Conflict of Interest statement. None declared. 13. Gros, J., Scaal, M. and Marcelle, C. (2004) A two-step mechan- ism for myotome formation in chick. Dev. Cell, 6, 875–882. Funding 14. Hollway, G. and Currie, P. (2005) Vertebrate myotome devel- opment. Birth Defects Res. C Embryo Today, 75, 172–179. This work was supported by Fundac¸ao~ para a Ciencia^ e a 15. Buckingham, M. (2006) Myogenic progenitor cells and skel- Tecnologia (FCT, Portugal) (project PTDC/SAU-BID/120130/2010, etal myogenesis in vertebrates. Curr. Opin. Genet. Dev., 16, SFRH/BD/86985/2012 scholarship to A.M.N and SFRH/BPD/65370/ 525–532. 2009 scholarship to M.D.), Association Franc¸aise contre les 16. Cinnamon, Y., Kahane, N. and Kalcheim, C. (1999) Myopathies (AFM) Te´le´thon (contract n 19959), CureCMD, Characterization of the early development of specific hypax- Struggle Against Muscular Dystrophy (SAM), NIH/NIAMS ial muscles from the ventrolateral myotome. Development, (R01AR064338-01A1), the University of Nevada, Reno (USA) and 126, 4305–4315. Mick Hitchcock Scholarship (to A.S. and T.F.). 17. Biressi, S., Molinaro, M. and Cossu, G. 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doi: 10.1093/hmg/ddx048 Advance Access Publication Date: 8 February 2017 Original Article

ORIGINAL ARTICLE PTRH2 gene mutation causes progressive congenital skeletal muscle pathology Jinger Doe1,†, Angela M. Kaindl2,3,†,*, Mayumi Jijiwa4, Michelle de la Vega4, Hao Hu5, Genevieve S. Griffiths4, Tatiana M. Fontelonga1, Pamela Barraza1, Vivian Cruz1, Pam Van Ry1, Joe W. Ramos4, Dean J. Burkin1 and Michelle L. Matter4,*

1Department of Pharmacology, University of Nevada School of Medicine, Reno, NV 89557, USA, 2Institute of Cell Biology and Neurobiology, 3Department of Pediatric Neurology, Charite´ –Universitatsmedizin,€ 13353 Berlin, Germany, 4The University of Hawaii Cancer Center, Honolulu, HI 96813, USA and 5Max Planck Institute for Molecular Genetics, 14195 Berlin, Germany

*To whom correspondence should be addressed at: University of Hawaii Cancer Center, 701 Ilalo Street, Honolulu, HI 96813, USA. Tel: 808 4413486; Fax: 808 5870742; Email: [email protected] (M.L.M.); Pediatric Neurology and Institute of Cell and Neurobiology, Charite´ - Universitatsmedizin€ Berlin, Augustenburger Platz 1, 13353 Berlin, Germany. Tel/Fax: þ49 30450566 112/920; Email: [email protected] (A.M.K.)

Abstract Peptidyl-tRNA hydrolase 2 (PTRH2) regulates integrin-mediated pro-survival and apoptotic signaling. PTRH2 is critical in muscle development and regulates myogenic differentiation. In humans a biallelic mutation in the PTRH2 gene causes infantile-onset multisystem disease with progressive muscle weakness. We report here that the Ptrh2 knockout mouse model recapitulates the progressive congenital muscle pathology observed in patients. Ptrh2 null mice demonstrate multiple degen- erating and regenerating muscle fibers, increased central nuclei, elevated creatine kinase activity and endomysial fibrosis. This progressive muscle pathology resembles the muscular dystrophy phenotype in humans and mice lacking the a7 integ- rin. We demonstrate that in normal muscle Ptrh2 associates in a complex with the a7b1 integrin at the sarcolemma and Ptrh2 expression is decreased in a7 integrin null muscle. Furthermore, Ptrh2 expression is altered in skeletal muscle of classical con- genital muscular dystrophy mouse models. Ptrh2 levels were up-regulated in dystrophin deficient mdx muscle, which correl- ates with the elevated levels of the a7b1 integrin observed in mdx muscle and Duchenne muscular dystrophy patients. Similar to the a7 integrin, Ptrh2 expression was decreased in laminin-a2 dyW null gastrocnemius muscle. Our data establishes a PTRH2 mutation as a novel driver of congenital muscle degeneration and identifies a potential novel target to treat muscle myopathies.

Introduction integrins play a critical role in the pathogenesis of muscular dystrophies (1). Muscular dystrophies are a group of genetic dis- Integrins are ab heterodimeric cell surface receptors that link eases that result in progressive muscle degeneration, functional the cytoskeleton to the underlying extracellular matrix (ECM) disability and premature patient death (1). For example, loss and activate downstream signaling pathways necessary for nor- of or mutations within the laminin binding a7 integrin gene mal development and skeletal muscle function. In muscle, cause a congenital muscular dystrophy with delayed motor

† The authors wish it to be known that, in their opinion, the first 2 authors should be regarded as joint First Authors. Received: December 7, 2016. Revised: February 1, 2017. Accepted: February 3, 2017

VC The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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milestones in humans and mice (2,3). Moreover, a skeletal muscular dystrophies, including centrally nucleated fibers (Fig. muscle-specific knockout mouse model of the downstream a7 1C) in triceps, tibialis anterior and gastrocnemius skeletal integrin effector serine/threonine integrin-linked kinase (ILK) muscle, elevated creatine kinase activity (Fig. 1D), increased results in a phenotype similar to the a7 integrin null mice (4) sug- fiber size variation (Fig. 1E) and endomysial fibrosis (Fig. 1F and gesting that integrin effector proteins play key roles in the G) in the null mice. Evans blue dye (EBD) uptake (11,12), a meas- pathogenesis of muscle myopathies. Not all genetic mutations ure of sarcolemmal defects, revealed multiple damaged myofib- that cause muscle myopathies have been identified. ers in the gastrocnemius muscle of Ptrh2 null mice, while no PTRH2 (also called BIT1; Bit-1) functions on an integrin- myofibers were EBD positive in control littermates (Fig. 1H and mediated signal transduction pathway to regulate cell survival I). Active regeneration was observed in Ptrh2 null skeletal and apoptosis (5–7). In cells attached to the ECM, PTRH2 associ- muscle by the presence of muscle fiber clusters expressing em- ates in a complex with focal adhesion kinase (FAK) and acti- bryonic myosin heavy chain (eMyHC), which is a marker of re- vates a pro-survival PI3K-AKT-NFjB-bcl-2 pathway (6). Loss of generation (13). There was a significant increase in myofibers integrin-mediated attachment, on the other hand, promotes the expressing eMyHC in Ptrh2 null muscle compared to age- association of PTRH2 with a pro-apoptotic Groucho-TLE com- matched littermate controls (Fig. 1J). Muscle regeneration in the plex and induces apoptosis (5). null mice was further supported by a significant increase of cen- Using whole exome sequencing, we recently identified a trally located nuclei (approximately 15%), an indication of newly homozygous nonsense mutation in the PTRH2 gene in two sib- formed muscle fibers (4) and by a 10-fold increase in myofibers lings of a consanguineous Turkish family (8). Both patients de- with diameters below 10 lm(Fig. 1C and E). Taken together, veloped a progressive congenital myopathy with delayed motor these data demonstrate the presence of small regenerating milestones and distal muscle weakness and wasting. Patients fibers by P7 in the Ptrh2 nulls. Furthermore, creatine kinase ac- were unable to roll from prone to supine until 8 (male II.1) or 12 tivity, which is a marker of muscle damage, was significantly (female II.4) months of age and did not walk or sit without as- increased in the null mice at P7 (Fig. 1D) supporting the occur- sistance until 18 (II.1) or 36 (II.4) months compared to healthy rence of muscle damage at this early age. aged-matched controls at 5, 5 and 10 months of age respect- ively. Recently, at 15 years-of-age, the older and more severely affected female patient was no longer mobile and became Ptrh2 associates with a7b1 integrin in skeletal muscle wheelchair dependent due to muscle weakness and ataxia (8). Because Ptrh2 functions on an integrin regulated pathway and the These findings point to a PTRH2 mutation as a novel driver in Ptrh2 null mouse phenotype described here is similar to that of a7 progressive human congenital muscle degeneration with integrin null mice that develop congenital muscular dystrophy delayed motor milestones. with delayed motor milestones, (3,14,15)wenextexamined Similar to the patients, Ptrh2 null mice appear healthy at whether Ptrh2 was in a complex with the a7b1 integrin. Indeed, birth, however, they develop a runting syndrome and die within Ptrh2 localized to the sarcolemma of myofibrils and co-localized the first two weeks of life. At this early age, the primary pheno- with the muscle-specific a7B integrin in WT gastrocnemius type is muscle weakness and epaxial muscle fiber diameters are muscle (Fig. 2A). To further test that Ptrh2 and a7B integrin are in smaller in the Ptrh2 null mice (9), suggesting impaired or a complex, we immunoprecipitated Ptrh2 from the gastrocnemius delayed muscle development. Indeed, Ptrh2 null mice exhibit a muscle lysate and examined for co-precipitation of the a7B integ- myopathy with hypotrophic myofibers (10). rin by immunoblotting. The a7B integrin was detected in Ptrh2 We report here that the Ptrh2 knockout mice recapitulate the immunoprecipitates, and in reciprocal co-immunoprecipitations muscle degeneration identified in patients with a PTRH2 gene Ptrh2 was detected in a7B integrin immunoprecipitates (Fig. 2B mutation. The mice develop a postnatal progressive muscle and C). Ptrh2 did not co-immunoprecipitate with either the alpha pathology with multiple degenerating and regenerating muscle 5 or beta 1 integrin (Fig. 2D). Taken together, Ptrh2 and the fibers, increased central nuclei, elevated creatine kinase activity muscle-specific a7B integrin associate in a complex in skeletal and endomysial fibrosis. Moreover, PTRH2 expression is differ- muscle. entially regulated in skeletal muscle of mouse models for con- We next examined whether loss of Ptrh2 altered a7integrinex- genital muscular dystrophy. pression and localization in skeletal muscle. Immunofluorescence and Western blot analysis demonstrated a significant decrease in Results a7 integrin protein expression in Ptrh2 null gastrocnemius muscle (Fig. 3A and B), suggesting that Ptrh2 positively regulates a7integ- Ptrh2 null mice develop congenital progressive muscle rin expression. In contrast, laminin a2 (a ligand for the a7integrin) pathology expression was similar in Ptrh2 null and WT skeletal muscle Similar to the human phenotype (8), Ptrh2 null mice did not ex- (Supplementary Material, Fig. S1), indicating Ptrh2 is not involved hibit any muscle weakness at birth, but became progressively in laminin matrix deposition in the basal lamina of muscle at P7. weaker within the first week. By postnatal day 7 (P7; Fig. 1A) Moreover, at P7 Ptrh2 null muscle showed no increase in inflam- these mice were unable to right themselves, displayed severe matory cell infiltrate as measured by CD4 and CD11 immunofluor- joint contractures, could no longer walk and all the mice died by escence (Supplementary Material, Fig. S2). P10 (Fig. 1B; Supplementary Video S1). Integrin-mediated signaling is decreased in Ptrh2 null Ptrh2 null mice present with muscle damage mice To determine the pathology underlying Ptrh2 loss in skeletal We previously reported that Ptrh2 provides a protective func- muscle, we examined H&E-stained cryosections of gastrocne- tion by activating a FAK-PI3K-AKT pro-survival pathway (6). mius muscle from Ptrh2 null mice and age-matched littermate Indeed, the a7b1 integrin relays a similar pro-survival effect by controls at P7. We detected severe myopathic changes typical of activating AKT (16). We analyzed gastrocnemius muscle from 209

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Figure 1. Ptrh2 null mice demonstrate progressive skeletal muscle pathology at P7. (A) Ptrh2 KO mice exhibit severe joint contractures at P7 compared to wild-type (WT) littermates. (B) The viability of ptrh2 KO mice (n ¼ 25) is severely reduced compared with wild-type (n ¼ 25). All Ptrh2 KO died at 7-10 days. (C) Quantitation of centrally- located nuclei in triceps, tibialis anterior and gastrocnemius skeletal muscle at P7 (n ¼ 3 per genotype and muscle type; 1,000 myofibers/animal; *P < 0.01; **P < 0.001). (D) Quantitation of creatine kinase levels in WT and Ptrh2 KO gastrocnemius muscle at P7 (n ¼ 4 per genotype, **P < 0.001). Creatine kinase detects severe muscle dam- age. (E) Myofibers 10 lm diameter were increased in Ptrh2 KO gastrocnemius muscle compared to WT (n ¼ 8 per genotype; 1000 myofibers/animal, ***P < 0.0001) indi- cating a significant increase in small regenerating fibers. (F) Increased endomysial fibrosis in gastrocnemius muscle (arrows) as determined by Trichrome staining in Ptrh2 KO mice compared to WT (longitudinal sections; bar 50 lm.) (G) Quantitation of Trichrome staining demonstrated a significant increase of fibrosis in the Ptrh2 KO mice compared to aged matched littermate controls at P7. (n ¼ 4 per genotype, *P < 0.01). (H,I) Ptrh2 KO mice had reduced sarcolemmal integrity as determined by the uptake of Evans blue dye (red stain; arrows) in gastrocnemius muscle compared to WT (n ¼ 4 per genotype; 1,500 myofibers/animal; *P < 0.01). Myofibers were counter- stained with Oregon Green-488-conjugated WGA (green; bar 50 lm). (I) Increased eMyHC expression in Ptrh2 KO mouse gastrocnemius muscle compared to age- matched WT littermates at P7 (n ¼ 4 per genotype; ***P < 0.0001).

Ptrh2 and integrin a7 null mice and found that pAKT/AKT levels dystrophin deficient mdx mice. Ptrh2 and pAKT/AKT expression were decreased in both null mouse models (Fig. 3C). Taken to- was decreased in both a7 deficient and laminin-a2 dyW nulls gether, these findings suggest that Ptrh2 may function, at least (Fig. 4A and B) but increased in mdx mice (Fig. 4B), suggesting in part, as a cytoplasmic effector of a7b1 integrin pro-survival that Ptrh2 may play an important role in these muscle signaling in muscle. pathologies.

Discussion Ptrh2 expression is altered in mouse models of muscular dystrophy Muscle disease occurs when the ECM-integrin-cytoskeleton complex is disrupted, thereby altering downstream pro-survival Because we observed that mutant PTRH2 causes distal muscle signals. Our data demonstrate that Ptrh2 associates in a com- pathology in patients and mice, Ptrh2 and the a7 integrin asso- plex with the a7b1 integrin at the sarcolemma, as determined ciate in a complex and loss of the a7 integrin promotes muscu- by co-localization studies and co-immunoprecipitation experi- lar dystrophy in humans and mice (3,4), we next examined the ments. Thus, in skeletal muscle, Ptrh2 is localized at sites of Ptrh2 expression in classical mouse models of muscular dystro- integrin signaling. PTRH2 is a regulator of myogenic differenti- phy including a7 integrin deficient, laminin-a2 dyW null and ation and loss of PTRH2 expression promotes early muscle 210

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Deletion of ILK, a known cytoplasmic effector of the a7integ- rin, induces a mild muscular dystrophy similar to the a7 integrin null phenotype (4), establishing a7 integrin-mediated signals as essential for normal muscle development and function. Our data points to a critical role for Ptrh2-mediated signaling in muscle cell development and function. The skeletal muscle phenotype of the Ptrh2 null mouse is more severe than that of the a7 integrin null mice and resembles, in severity, the dystrophin and a7integ- rin double knockout mice (mdx/a7-/-;) (18). These mice develop early-onset muscular dystrophy with fibrosis and die at 2–4 weeks of age (18) whereas the Ptrh2 null mice develop severe muscle pathology and die by day 10. The lack of Ptrh2-mediated signaling induces a more severe phenotype compared to a7 integ- rin nulls as detected by the presence of muscle regeneration, cen- tral nuclei, elevated creatine kinase activity and endomysial fibrosis at P7. Skeletal muscle from the a7 nulls present with ap- proximately 2% central nuclei. Ptrh2 nulls present with approxi- mately 15% central nuclei as examined in triceps, tibialis anterior and gastrocnemius skeletal muscle. This observed increase in Figure 2. Ptrh2 associates in a complex with the a7B integrin in skeletal muscle. (A) Ptrh2 co-localizes with the muscle-specific a7B integrin at the sarcolemma of central nuclei is similar to the mdx/a7 nulls, which demonstrate myofibrils in gastrocnemius muscle of WT mice as examined by immunofluor- approximately 15% central nuclei in the tibialis anterior muscle escence microscopy. Arrows indicate co-localization of Ptrh2 and a7 integrin (18). Moreover, we observe a higher number of eMyHC positive that is displayed in yellow in the merged image. Nuclei were stained with DAPI fibers from day 7 Ptrh2 null skeletal muscle compared with (blue). Immunofluorescence magnification 63x. (B) Ptrh2 and a7B integrin co- eMyHC positive fibers from 3 week old alpha 7 null skeletal immunoprecipitate in WT gastrocnemius muscle. Gastrocnemius muscle was muscle. eMyHC is expressed during normal muscle development collected, lysed and immunoprecipitated (IP) with anti-Ptrh2, anti-a7B integrin antibodies or pre-immune serum as a control. Control and immunoprecipitates in the embryo and transiently during muscle repair after birth. were analyzed by immunoblotting (IB) with either anti-a7B integrin (left) or anti- The observed increase in eMyHC may be due, in part, to the find- Ptrh2 antibody (right). Lower images are of 2% input lysate based on overall pro- ing that loss of PTRH2 expression promotes early skeletal muscle tein content immunoblotted with anti-a7B (left) or anti-Ptrh2 (right) antibodies. differentiation and that Ptrh2 null myoblasts prematurely express (C) Efficiency of antibodies used for IPs in gastrocnemius muscle that was col- muscle-specific proteins (10). Nevertheless, the eMyHC expres- lected, lysed and immunoprecipitated (IP) with anti-Ptrh2, anti-a7B integrin antibodies or pre-immune serum as a control. Control and immunoprecipitates sion in postnatal skeletal muscle is a gauge for active muscle re- were analyzed by immunoblotting (IB) with either anti-a7B integrin or anti-Ptrh2 generation and we observe increased eMyHC positive fibers in antibody. Lysate of 2% input based on overall protein content was immunoblot- the Ptrh2 null skeletal muscle. To address this further, we also ted with anti-a7B or anti-Ptrh2 antibodies. (D) Ptrh2 does not co-immunoprecipi- evaluated creatine kinase activity, which is a marker of muscle tate with a5orb1 integrin. Gastrocnemius muscle was collected, lysed and damage. Creatine kinase activity was significantly increased in immunoprecipitated (IP) with anti-Ptrh2, anti-a5, anti-b1 integrin antibodies or the Ptrh2 null mice at P7 further supporting the occurrence of pre-immune serum as a control. Control and immunoprecipitates were ana- lyzed by immunoblotting (IB) with either anti-a5 or anti-Ptrh2 antibodies. Lysate muscle damage at this early age. of 2% input based on overall protein content was immunoblotted with anti-a5 This data indicates loss of Ptrh2 expression in muscle alters integrin or anti-Ptrh2 antibodies. Data shown in A–D are representative of three the normal myofiber regenerative process. Interestingly, we independent experiments. found skeletal muscle from dystrophin deficient mdx mice up- regulate Ptrh2 levels, which correlate with elevated levels of the a7b1 integrin observed in mdx muscle and Duchenne muscular differentiation (10). In humans, mutations in the PTRH2 gene dystrophy (DMD) patients (19). Similar to the a7 integrin, Ptrh2 cause progressive congenital muscle weakness resulting in the expression was decreased in laminin-a2 dyW null gastrocne- most affected patients being wheelchair bound by age 15 (8). mius muscle. These findings point to a key role for PTRH2 in Ptrh2 is localized, in part, at the plasma membrane and associ- DMD and merosin deficient congenital muscular dystrophy ates in a complex with FAK to activate a PI3K-AKT-pro-survival (MDC1A) disease progression. signaling pathway in cancer and endothelial cells (6). Indeed, In humans, a mutation in the PTRH2 gene promotes a similar patient fibroblasts with a homozygous deletion in the PTRH2 phenotype as mutations in the a7 integrin gene both of which gene are sensitive to stress-induced apoptosis suggesting that result in progressive congenital muscle degeneration with PTRH2 expression is important in promoting cell survival during delayed motor milestones. Our data points to a new complex tissue development (8). whereby Ptrh2 associates with the a7 integrin and functions to The a7b1 integrin is involved in muscle cell survival and acti- regulate pro-survival signaling pathways in skeletal muscle. vates a pro-survival AKT pathway when re-expressed in dys- Further studies are required to delineate the precise role PTRH2 trophic muscle (16). Similar to earlier in vitro findings that plays in muscle disease. Identification of PTRH2 as a novel gene PTRH2 blocks staurosporine-induced apoptosis (6), a7 integrin in human muscle pathologies may provide a potential new therapeutic target for these fatal muscle diseases. expression prevents staurosporine-induced apoptosis in myo- blasts in vitro (17). We demonstrate here that pAKT signaling is reduced in skeletal muscle of Ptrh2 and a7 integrin null mice. We Materials and Methods further show that loss of Ptrh2 expression decreases a7 integrin PTRH2 null mice expression but not laminin. Taken together, these findings sug- gest Ptrh2 may function as an a7 cytoplasmic effector in skeletal All animals received care in compliance with the principles of muscle. laboratory animal care and use formulated by the Institutional 211

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Figure 3. a7 integrin expression is decreased in Ptrh2 null skeletal muscle. (A) Immunofluorescence and Western blot (B) of the a7 integrin showed reduced protein lev- els in gastrocnemius muscle in Pthr2 null (Ptrh2-/-) mice at P7 compared to WT littermate controls (n ¼ 3 per genotype for immunoblots and immunofluorescence; bar 10 lm). (C) Western blot analysis of the gastrocnemius muscle lysate indicated that Ptrh2 and pAKT/tAKT are decreased in Ptrh2-/- and a7-/-mice compared to WT (n ¼ 3 per genotype for immunoblots and immunofluorescence; **P<0.001). Data shown in A–C are representative of three independent experiments.

Figure 4. Ptrh2 is differentially expressed in mouse models of congenital muscular dystrophy. (A) Ptrh2 immunofluorescence was lower in a7 integrin and laminin-a2 dyW null gastrocnemius muscle compared to WT. The absence of Ptrh2 expression was confirmed in Ptrh2-/- mice (n ¼ 3 per genotype; bar 10 lm). (B) Western blot ana- lysis demonstrated that Ptrh2 and pAKT/tAKT levels were increased in mdx (right panel) and decreased in laminin-a2 dyW null (left panel) gastrocnemius muscle com- pared to WT (n ¼ 3 per genotype; **P<0.001, ***P<0.0001). Tubulin was used as a loading control. Data shown in A and B are representative of three independent experiments.

Animal Care & Use Committee (USA). The generation of PTRH2 St Louis, MO), rinsed and mounted with DePeX medium (electron null mice inbred in a black 6 pure strain genetic background Microscopy Sciences, Washington, PA). that die by P10 have been described previously (9). Centrally located nuclei from gastrocnemius skeletal muscle were counted at 63X magnification by bright-field microscopy. The number of central nuclei per muscle fiber was determined Hematoxylin and eosin staining by counting 1000 muscle fibers each for triceps, tibialis anterior and gastrocnemius muscle per animal. The percentage of cen- For histological studies paraformaldehyde fixed, paraffin tral nuclei was expressed as the number of central nuclei/total embedded sections were stained with Hematoxylin and Eosin number of fibers counted. At least three animals from each (H&E) as described (18). Briefly, gastrocnemius muscles were cyro- genotype were analyzed (wild type, Ptrh2-/-). sectioned and 10-lm sections were placed on Surgipath micro- scope slides. Tissue sections were fixed in ice-cold 95% ethanol followed by 70% ethanol and re-hydrated in water. Tissue sections Immunofluorescence were then stained with Gill’s hematoxylin (Fisher Scientific, Fair Gastrocnemius muscles were embedded in Tissue-TEK Optimal Lawn, NJ), rinsed in water and stained with eosin (Sigma-Aldrich, Cutting temperature compound (Sakura Finetek USA Inc., 212

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Torrance, CA) as described (18). Ten-lm sections were cut using PMSF and a 1:200 dilution of Protease Inhibitor Cocktail Set III a Leica MC1850 cryostat and placed onto Surgipath microscope (Calbiochem, EMD Biosciences, San Diego, CA). For the detection slides (Surgipath Medical Industries, Richmond, IL). Integrin a7 the a7 integrin: protein was quantified by Bradford assay and was detected with anti-CA5.5 rat monoclonal antibody (Sierra 80 lg of total protein were separated on 8%% SDS-PAGE gels Biosource, Morgan Hill, CA) followed by FITC-anti-rat secondary under non-reduced conditions, and transferred to nitrocellulose (see the Supplementary Appendix). PTRH2 was detected by rab- membranes. Membranes were blocked in Odyssey Blocking buf- bit polyclonal anti-Ptrh2 antibody (developed at Washington fer (LiCor Biosciences, Lincoln, NE) that was diluted 1:1 in Biotechnology Columbia, MD using the PTRH2 amino acid se- phosphate-buffered saline (PBS). The a7 integrin was detected quence GPADLIDKVTAGHLKL;(6). Laminin-a2 chain was de- with a rabbit anti-a7A (a2 345) polyclonal antibody and an Alexa tected with a rabbit anti-a2G polyclonal antibody. Slides were Fluor 680-coupled goat anti-rabbit IgG (Molecular Probes, mounted using Vectashield with DAPI (Vector Laboratories) and Eugene OR) to detect primary antibodies. visualized using a Zeiss Axioskop 2 Plus fluorescent microscope For the detection of PTRH2, pAKT, tAKT, tubulin: 30 lg total and images were captured using a Zeiss AxioCam HRc digital protein was separated on a 4–12% SDS-PAGE gel under reducing camera with Axiovision 4.1 software. conditions and transferred to nitrocellulose membranes. Central nuclei from tibialis anterior and triceps muscle were Membranes were blocked in the Odyssey Blocking buffer and determined from embedded tissue that was sectioned into 10 incubated with either anti-PTRH2 (developed at Washington micron thick sections using a cryosectioner. Sections were incu- Biotech), anti-AKT or anti-phospho-AKT (Cell Signaling) or anti- bated in 1X PBS for 5 mins to reconstitute tissue and then for 10 microtubulin (BioLegend) for 2 h. Membranes were washed and mins in 1:100 wheat germ agglutinin – alexa 647 conjugated (to incubated with Li-Cor secondary antibodies (ant-rabbit IR 800 delineate muscle fibers), washed 2 min three times with 1X PBS, CW and anti-mouse IR 680 CW; Li-Cor) for 1 h. Immunoblots mounted in Vectashield containing DAPI stain (stains nuclei) were analyzed via Odyssey software, provided with the Li-Cor and imaged using confocal microscope. Central nuclei were system. counted as nuclei located in the center of muscle fibers (more than one nuclei per fiber is only counted as one CLN fiber) and averaged to the total fiber number. The number of central nuclei Statistical analysis per muscle fiber was determined by counting 1000 muscle fibers All statistical analyses were performed using GraphPad Prism 6 each muscle type per animal. The percentage of central nuclei software. Averaged data are reported as the mean 6 the stand- was expressed as the number of central nuclei/total number of ard error of the mean. Individually reported data points were re- fibers counted. At least three animals from each genotype were ported as mean 6 standard deviation. Comparison of two analyzed (wild type, Ptrh2-/-). groups was performed using a Student’s t-test and between multiple groups using one-way analysis of variance with a Embryonic myosin heavy chain Bonferroni post-test with P < 0.05 considered statistically significant. Embryonic myosin heavy chain (eMyHC) was used in immuno- fluorescence experiments to measure muscle regeneration. Immunofluorescence was performed on 10-lm sections from Supplementary Material the gastrocnemius muscle from day 7 PTRH2 null mice and wild Supplementary Material is available at HMG online. type littermate controls with an anti-eMyHC antibody (FL.652, Developmental Studies Hybridoma Bank, University of Iowa IA). The primary antibody was detected with a FITC-conjugated Acknowledgements mouse secondary antibody. Myofibers were incubated with The authors thank the family members who participated in this rhodamine labeled WGA to outline the myofibers. Multiple adja- study and Anna Leychenko for immunoblotting and mouse hus- cent sections were analyzed with twenty random, non- bandry expertise. overlapping microscopic fields that were counted per animal at 630X magnification using a Zeiss Axioskop 2 Plus fluorescent Conflict of Interest statement. None declared. microscope and images were captured using a Zeiss AxioCam HRc digital camera with Axiovision 4.1 software. Data were re- ported as percent of eMYHC-positive fibers. Funding This work was supported by: National Institutes of Health (NCRR P20-RR016453 to M.L.M; RO1-AR064338 to D.J.B.), the Creatine kinase activity assay Ingeborg Foundation (M.L.M), the German Research Foundation -/- Gastrocnemius muscle from WT and Ptrh2 null mice was iso- (SFB665, DFG, A.M.K.), the Berlin Institute of Health (BIH, lated, washed with PBS and weighed (1 mg of muscle was lysed A.M.K.), the Sonnenfeld Stiftung (A.M.K), the Deutsche in 5ul 50mM potassium phosphate lysis buffer and sonicated). Gesellschaft fu¨ r Muskelkranke DGM (A.M.K.), the Max-Planck Creatine kinase activity was quantitated as per the kit protocol Society (H.H.), and the EU FP 7 project GENCODYS, #241995 (MAK 116; Sigma Aldrich, St Louis, MO). (H.H.).

Western blotting References The gastrocnemius muscle from day 7 after birth (P7) was 1. Carmignac, V. and Durbeej, M. (2012) Cell-matrix inter- ground in liquid nitrogen. Protein was extracted in 200 mM actions in muscle disease. J. Pathol., 226, 200–218. octyl-b-D-glucopyranoside (Sigma Aldrich, St Louis, MO), 50 mM 2. Hayashi, Y.K., Chou, F.L., Engvall, E., Ogawa, M., Matsuda, C.,

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Kaufman, S.J., et al. (1998) Mutations in the integrin alpha7 11. Grady, R.M., Teng, H., Nichol, M.C., Cunningham, J.C., gene cause congenital myopathy. Nat. Genet., 19, 94–97. Wilkinson, R.S. and Sanes, J.R. (1997) Skeletal and car- 3. Mayer, U., Saher, G., Fassler,€ R., Bornemann, A., diac myopathies in mice lacking utrophin and dystrophin: Echtermeyer, F., von der Mark, H., Miosge, N., Po¨ schl, E. and a model for Duchenne muscular dystrophy. Cell, 90, 729–738. von der Mark, K. (1997) Absence of integrin alpha 7 causes a 12. McGeachie, J.K., Grounds, M.D., Partridge, T.A. and Morgan, novel form of muscular dystrophy. Nat. Genet., 17, 318–323. J.E. (1993) Age-related changes in replication of myogenic 4. Gheyara, A.L., Vallejo-Illarramendi, A., Zang, K., Mei, L., Rene cells in mdx mice: quantitative autoradiographic St.-Arnaud, Dedhar, S. and Reichardt, L.F. (2007) Deletion of studies. J. Neurol. Sci., 119, 169–179. integrin-linked kinase from skeletal muscles of mice resem- 13. Webster, C., Silberstein, L., Hays, A.P. and Blau, H.M. (1988) bles muscular dystrophy due to alpha 7 beta 1-integrin defi- Fast muscle fibers are preferentially affected in Duchenne ciency. Am. J. Pathol., 171, 1966–1977. muscular dystrophy. Cell, 52, 503–513. 5. Jan, Y., Matter, M., Pai, J.T., Chen, Y.L., Pilch, J., Komatsu, M., 14. Burkin, D.J. and Kaufman, S.J. (1999) The alpha7beta1 integ- Ong, E., Fukuda, M. and Ruoslahti, E. (2004) A mitochondrial rin in muscle development and disease. Cell Tissue Res., 296, protein, Bit1, mediates apoptosis regulated by integrins and 183–190. Groucho/TLE corepressors 1. Cell, 116, 751–762. 15. Lopez, M.A., Mayer, U., Hwang, W., Taylor, T., Hashmi, M.A., 6. Griffiths, G.S., Grundl, M., Leychenko, A., Reiter, S., Young- Jannapureddy, S.R. and Boriek, A.M. (2005) Force transmis- Robbins, S.S., Sulzmaier, F.J., Caliva, M.J., Ramos, J.W. and sion, compliance, and viscoelasticity are altered in the Matter, M.L. (2011) Bit-1 Mediates Integrin-dependent Cell alpha7-integrin-null mouse diaphragm. Am. J. Physiol. Cell Survival through Activation of the NF{kappa}B Pathway. Physiol., 288, C282–C289. J. Biol. Chem., 286, 14713–14723. 16. Boppart, M.D., Burkin, D.J. and Kaufman, S.J. 7. Matter, M.L., Ginsberg, M.H. and Ramos, J.W. (2001) (2011) Activation of AKT signaling promotes cell growth Identification of cell signaling molecules by expression clon- and survival in alpha7beta1 integrin-mediated allevi- ing. Sci.STKE, 2001, L9. ation of muscular dystrophy. Biochim. Biophys. Acta, 1812, 8. Hu, H., Matter, M.L., Issa-Jahns, L., Jijiwa, M., Kraemer, N., 439–446. Musante, L., de la Vega, M., Ninnemann, O., Schindler, D., 17. Liu, J., Burkin, D.J. and Kaufman, S.J. (2008) Damatova, N., et al. (2014) Mutations in PTRH2 cause novel Increasing alpha 7 beta 1-integrin promotes muscle cell pro- infantile-onset multisystem disease with intellectual dis- liferation, adhesion, and resistance to apoptosis without ability, microcephaly, progressive ataxia, and muscle weak- changing gene expression. Am. J. Physiol. Cell Physiol., 294, ness. Ann. Clin. Transl.Neurol., 1, 1024–1035. C627–C640. 9. Kairouz-Wahbe, R., Biliran, H., Luo, X., Khor, I., Wankell, M., 18. Rooney, J.E., Welser, J.V., Dechert, M.A., Flintoff-Dye, N.L., Besch-Williford, C., Oshima J., Pascual R. and Ruoslahti, E. Kaufman, S.J. and Burkin, D.J. (2006) Severe muscular dystro- (2008) Anoikis effector Bit1 negatively regulates Erk activity phy in mice that lack dystrophin and alpha7 integrin. J. Cell 8. Proc. Natl. Acad. Sci. U.S.A, 105, 1528–1532. Sci., 119, 2185–2195. 10. Griffiths, G.S., Doe, J., Jijiwa, M., Van Ry, P., Cruz, V., de la 19. Hodges, B.L., Hayashi, Y.K., Nonaka, I., Wang, W., Arahata, K. Vega, M., Ramos, J.W., Burkin, D.J. and Matter, M.L. (2015) Bit- and Kaufman, S.J. (1997) Altered expression of the alpha7- 1 is an essential regulator of myogenic differentiation. J. Cell beta1 integrin in human and murine muscular dystrophies. Sci., 128, 1707–1717. J. Cell Sci., 110 (Pt 22), 2873–2881. 214

Journal of Pathology J Pathol 2015; 237: 282–284 INVITED COMMENTARY Published online 26 August 2015 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/path.4587 Mesothelioma cells breaking bad: loss of integrin 𝛂7 promotes cell motility and poor clinical outcomes in patients† Dean J Burkin* and Tatiana M Fontelonga

Department of Pharmacology, Center for Molecular Medicine, University of Nevada School of Medicine, 1664 N Virginia Avenue, Reno, NV 89557, USA

*Correspondence to: DJ Burkin, Department of Pharmacology, Center for Molecular Medicine, University of Nevada School of Medicine, 1664 N Virginia Avenue, Reno, NV 89557, USA. E-mail: [email protected] † Invited Commentary for Laszlo V, Hoda MA, Garay T, et al. Epigenetic down-regulation of integrin α7 increases migratory potential and confers poor prognosis in malignant pleural mesothelioma. J Pathol 2015; 237: 203–214.

Abstract Mesothelioma is a disease of pleural cells lining the lungs which is often caused by exposure to asbestos. The molecular mechanism(s) that regulate the transformation of pleural mesothelioma cells to a migratory and malignant phenotype are unclear. In recent work published in this journal, Laszlo et al performed a set of elegant experiments to identify a key molecular mechanism that may explain the aggressive nature of this disease. Using patient-derived mesothelioma cells with high versus low migratory activity, the authors conducted a genome-wide expression analysis. They identified a significant reduction in ITGA7 expression only in highly migratory malignant pleural mesothelioma cells and showed that loss of ITGA7 expression was associated with methylation of the promoter. Forced expression of integrin 𝛂7 reversed the migratory phenotype of these cells. Finally, the authors identified a strong correlation between ITGA7 expression and patient survival. Together, these results identify expression of integrin 𝛂7 as a molecular mechanism for the aggressive migratory transformation of mesothelioma and identify a potentially novel diagnostic and therapeutic target. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Keywords: integrin α7; mesothelioma; malignancy; epigenetics

Received 22 June 2015; Revised 6 July 2015; Accepted 10 July 2015

Conflict of interest statement: The University of Nevada, Reno has patents pending on the therapeutic use of integrin enhancing molecules, proteins and microRNAs, their derivatives and compositions. UNR has licensed this technology to StrykaGen Corp, founded and owned by Dr Dean J Burkin. Tatiana M Fontelonga has no conflicting interests to declare.

Introduction a major laminin receptor found in many tissues [2]. Mutations in the integrin α7 gene cause congenital Mesothelioma is a disease of the pleural cells lining the myopathy in patients and mice [3–5], and α7β1 inte- lung often caused by exposure to asbestos and treat- grin is a major modifier of disease progression in mouse ment usually involves surgical intervention. Malignant models and patients with muscular dystrophy [6–8]. pleural mesothelioma is often refractory to surgery, Although the majority of studies on integrin α7have due to the recurrence of tumour growth, with median focused on skeletal muscle, there is increasing evidence survival times of 9–12 months [1]. During the tran- that this integrin acts as a powerful tumour suppressor sition to the malignant form, pleural mesothelioma in many forms of cancer. This activity appears to reside cells become substrate-independent and highly migra- with the adhesive properties of α7β1 integrin, which tory. The molecular pathways that regulate this transi- serves as a ‘molecular glue’, reducing the metastatic tion are poorly understood. Laszlo et al [1] performed potential of tumour cells. Mutations in the ITGA7 gene genome-wide transcriptome analysis to identify changes that result in reduced levels of α7β1 integrin have been in gene expression associated with the migratory phe- identified in prostate cancers, hepatocellular carcino- notype of pleural mesothelioma cells. They identified mas, glioblastomas, and leiomyosarcomas [9]. Associ- 139 genes that were differentially expressed in highly ated with loss of integrin α7 are changes in cell sig- motile cells. Abundance of ITGA7 transcript, a known nalling pathways associated with malignancy including tumor suppressor, was greatly reduced in highly motile increased cell growth and motility. These observations malignant pleural mesothelioma cells [1]. indicate that integrin α7 can act to regulate cell mobility, tumour growth, and transition to metastasis, and as such, Integrin 𝛂7 in disease and as a tumour suppressor serves as a major tumour suppressor in many cancers. In recent work published in The Journal of Pathol- Integrin α7 chain (encoded by ITGA7) pairs with the β1 ogy, Laszlo et al [1] extend the role that integrin α7 integrin chain to form the α7β1 integrin heterodimer, plays in cancer to the malignant transformation of

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 282–284 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com 215

Mesothelioma cells breaking bad 283

Figure 1. Model of the role that integrin α7 plays in the transition of mesothelioma to pleural metastatic mesothelioma and potential targets for therapeutic intervention. pleural mesothelioma. Malignant pleural mesothelioma methylation with clinical outcomes. While normal plu- cells from patients were screened for their ability to ral cells exhibited varying levels of integrin α7, ITGA7 migrate in cell culture and grouped as low or high hypermethylation strongly correlated with poor prog- motility cells [1]. These cells were then subjected to nostic outcomes in patients [1]. genome-wide transcriptome analysis, which identi- fied differential expression of 139 genes between the two groups. The results showed significantly reduced Biomarker and therapy development ITGA7 transcript in cells from the high mobility group (Figure 1). for mesothelioma

The current treatment for malignant pleural mesothe- Epigenetic regulation of integrin 𝛂7 expression lioma is surgical intervention; however, this approach often results in recurrent tumour growth. Laszlo et al [1] identified a novel molecular mechanism by which Previous studies in prostate cancers, hepatocellular car- mesothelioma transitions to malignant pleural mesothe- cinomas, glioblastomas, and leiomyosarcomas revealed lioma that involves down-regulating expression of mutations in the ITGA7 gene [9]. In this study, the integrin α7. These cells are likely selected for their authors demonstrate hypermethylation of the ITGA7 ability to rapidly migrate and move from the primary promoter region in high mobility malignant pleural tumour site to distant locations (Figure 1). Increasing mesothelioma cells, indicating epigenetic suppression of integrin α7 in these cells is one potential therapeutic integrin α7 gene expression [1]. To substantiate that loss option; however, since the ITGA7 promoter is hyper- of integrin α7 expression was the primary cause of the methylated in malignant pleural mesothelioma cells, migratory phenotype, the authors forced expression of drugs that target ITGA7 transcription in these cells are the ITGA7 gene and were able to show that restoring unlikely to be effective. In addition, because integrin α7 expression inhibited migration of the cells. Thus, the α7 is found in many tissues and regulates complex cell findings [1] demonstrate a novel epigenetic mechanism signalling pathways, off-target drug activity in other of integrin α7 gene regulation that led to a highly motile tissues and cells in which the ITGA7 gene promoter is cell phenotype (Figure 1). not methylated may be problematic, although this might be overcome by restricting drug delivery. A second pharmacological option may be to utilize therapeutics Reduced ITGA7 expression correlates with a poor that inhibit the actions of methyltransferases and pre- clinical outcome vent hypermethylation of the integrin α7 promoter in mesothelioma cells to slow disease progression [10]. To determine if loss of ITGA7 expression was asso- The use of integrin α7 enhancing drugs in combina- ciated with disease progression in malignant pleural tion with therapies that inhibit methyltransferases or mesothelioma, the authors compared ITGA7 promoter demethylate the ITGA7 promoter may have efficacy

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 282–284 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com 216

284 DJ Burkin and TM Fontelonga in the treatment of malignant pleural mesothelioma 2. Burkin DJ, Kaufman SJ. The α7β1 integrin in muscle development (Figure 1). Further research will be required to develop and disease. Cell Tissue Res 1999; 296: 183–190. these approaches and reduce the potential of unwanted 3. Hayashi YK, Chou FL, Engvall E, et al. Mutations in the integrin α7 side effects. Finally, since the authors show a strong gene cause congenital myopathy. Nature Genet 1998; 19: 94–97. correlation between ITGA7 promoter hypermethylation 4. Welser JV, Lange ND, Flintoff-Dye N, et al. Placental defects in alpha7 integrin null mice. Placenta 2007; 28: 1219–1228. and clinical outcome [1], this study suggests a diagnos- 5. Guo C, Willem M, Werner A, et al. Absence of alpha 7 integrin in tic test may be developed that examines the methylation dystrophin-deficient mice causes a myopathy similar to Duchenne status of the ITGA7 promoter in biopsy samples that can muscular dystrophy. Hum Mol Genet 2006; 15: 989–998. be used to monitor disease progression in patients with 6. Burkin DJ, Wallace GQ, Nicol KJ, et al. Enhanced expression of mesothelioma. the alpha 7 beta 1 integrin reduces muscular dystrophy and restores viability in dystrophic mice. J Cell Biol 2001; 152: 1207–1218. 7. Doe JA, Wuebbles RD, Allred ET, et al. Transgenic overexpression of the α7 integrin reduces muscle pathology and improves viability Author contribution statement in the dy(W) mouse model of merosin-deficient congenital muscular dystrophy type 1A. J Cell Sci 2011; 124: 2287–2297. DJB and TMF wrote and edited the manuscript. 8. Rooney JE, Welser JV, Dechert MA, et al. Severe muscular dystro- phy in mice that lack dystrophin and alpha7 integrin. J Cell Sci 2006; 119: 2185–2195. 9. Ren B, Yu YP, Tseng GC, et al. Analysis of integrin alpha7 muta- References tions in prostate cancer, liver cancer, glioblastoma multiforme, and 1. Laszlo V, Hoda MA, Garay T, et al. Epigenetic down-regulation leiomyosarcoma. J Natl Cancer Inst 2007; 99: 868–880. of integrin α7 increases migratory potential and confers poor 10. Gray SG, Baird AM, O’Kelly F, et al. Gemcitabine reactivates epi- prognosis in malignant pleural mesothelioma. J Pathol 2015; 237: genetically silenced genes and functions as a DNA methyltransferase 203–214. inhibitor. IntJMolMed2012; 30: 1505–1511.

Copyright © 2015 Pathological Society of Great Britain and Ireland. J Pathol 2015; 237: 282–284 Published by John Wiley & Sons, Ltd. www.pathsoc.org.uk www.thejournalofpathology.com 217

Appendix B

Provisional Patent: SHP-2 Inhibitors for the Treatment of Muscular Dystrophy

218

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Appendix C

Copyright Releases for Use of Published Work

273

9/19/2018 Copyright and Permissions

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Copyright and Permissions

Reuse by Authors of Their Work Published by APS Reuse by Non-authors of APS Published Content

Reuse in APS Publications of non-APS Published Content

Reuse by Authors of Their Work Published by APS

The APS Journals are copyrighted for the protection of authors and the Society. The Mandatory Submission Form serves as the Society's ofcial copyright transfer form. Author’s rights to reuse their APS-published work are described below:

Republication Authors may republish parts of their nal-published work (e.g., gures, tables), in New without charge and without requesting permission, provided that full citation of the Works source is given in the new work.

Meeting Authors may use their work (in whole or in part) for presentations (e.g., at meetings Presentations and conferences). These presentations may be reproduced on any type of media in and materials arising from the meeting or conference such as the proceedings of a Conferences meeting or conference. A copyright fee will apply if there is a charge to the user or if the materials arising are directly or indirectly commercially supported1. Full citation is required.

Theses and Authors may reproduce whole published articles in dissertations and post to thesis Dissertations repositories without charge and without requesting permission. Full citation is required.

Open Authors may post articles, chapters or parts thereof to a public access courseware Courseware website. Permission must be requested from the APS1. A copyright fee will apply to a book chapter and during the rst 12 months of a journal article’s publication. Full citation is required.

Websites Authors may not post a PDF of the accepted or nal version of their published work to any website including social and research networking platforms; instead, links may be posted to the APS or publisher partner website where the work is published1 (see exception to authors’ own institution’s repository, as note below).

https://www.physiology.org/author-info.permissions 1/2 274

9/19/2018 Copyright and Permissions Institutional Authors may deposit their accepted, peer-reviewed journal manuscripts into an R epoJsiOtUoRriNAesLS  institutional repository providing: (non-theses) the APS retains copyright to the article1 a 12-month embargo period from the date of nal publication of the article is observed by the institutional repository and the author a link to the article published on the APS or publisher-partner website is prominently displayed alongside the article in the institutional repository the article is not used for commercial purposes self-archived articles posted to repositories are without warranty of any kind

1Unless it is published under the APS Open Access (AuthorChoice) option, which allows for immediate public access under a Creative Commons license (CC BY 4.0) (See also the APS Policy on Depositing Articles in PMC.)

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Please insert the author and article name, as well as the official publication name and send the document to [email protected].

The American Society of Gene Therapy (ASGCT) grants permission for ______Tatiana Fontelonga to use ______* (see below) figure/text in ______.Doctoral dissertation ASGCT reserves all rights to this information.

Please also fax or email a copy to [email protected] of the text where the citation is going to be placed.

Signed: ______Date:______

* SU9516 Increases a7b1 Integrin and Ameliorates Disease Progression in the mdx Mouse Model of Duchenne Muscular Dystrophy 276

Tatiana M Fontelonga

From: Susan Brettingen (ASGCT) Sent: Wednesday, September 19, 2018 1:49 PM To: Tatiana M Fontelonga Subject: FW: Molecular Therapy Permissions Attachments: ASGCT_Permissions_SU9516.pdf

Hello Tatiana,

Permission is granted for you to use the manuscript as you have requested.

All the best to you with your doctoral thesis.

Regards,

Sue Brettingen Journal Coordinator American Society of Gene & Cell Therapy 414.918.3070

From: Tatiana M Fontelonga [mailto:[email protected]] Sent: Wednesday, September 19, 2018 12:24 PM To: info (ASGCT) Subject: Molecular Therapy Permissions

Dear all,

Please find attached the request form to use said manuscript in my doctoral thesis to be printed at the University of Nevada, Reno’s repository with full acknowledgement of the original source.

Best regards,

‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐

Tatiana Fontelonga M.S. Ph.D. Candidate‐ Cell and Molecular Physiology and Pharmacology 1 277

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License Number 4451031372229 License date Oct 16, 2018 Licensed content publisher Oxford University Press Licensed content publication Human Molecular Genetics Licensed content title PTRH2 gene mutation causes progressive congenital skeletal muscle pathology Licensed content author Doe, Jinger; Kaindl, Angela M. Licensed content date Feb 8, 2017 Type of Use Thesis/Dissertation Institution name Title of your work Development of Small Molecule Therapies Targeting Regeneration for the Treatment of Duchenne Muscular Dystrophy Publisher of your work n/a Expected publication date Dec 2018 Permissions cost 0.00 USD Value added tax 0.00 USD Total 0.00 USD Title Development of Small Molecule Therapies Targeting Regeneration for the Treatment of Duchenne Muscular Dystrophy Institution name n/a Expected presentation date Dec 2018 Portions Entire text used as an appendix at the end of dissertation Requestor Location University of Nevada, Reno 1664 N. Virginia St. MS 573, Room CMM304

RENO, NV 89557 United States Attn: Tatiana Fontelonga Publisher Tax ID GB125506730

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STANDARD TERMS AND CONDITIONS FOR REPRODUCTION OF MATERIAL FROM AN OXFORD UNIVERSITY PRESS JOURNAL 1. Use of the material is restricted to the type of use specified in your order details. 2. This permission covers the use of the material in the English language in the following territory: world. If you have requested additional permission to translate this material, the terms and conditions of this reuse will be set out in clause 12. 3. This permission is limited to the particular use authorized in (1) above and does not allow you to sanction its use elsewhere in any other format other than specified above, nor does it apply to quotations, images, artistic works etc that have been reproduced from other sources which may be part of the material to be used. 4. No alteration, omission or addition is made to the material without our written consent. Permission must be re-cleared with Oxford University Press if/when you decide to reprint. 5. The following credit line appears wherever the material is used: author, title, journal, year, volume, issue number, pagination, by permission of Oxford University Press or the sponsoring society if the journal is a society journal. Where a journal is being published on behalf of a learned society, the details of that society must be included in the credit line. 6. For the reproduction of a full article from an Oxford University Press journal for whatever purpose, the corresponding author of the material concerned should be informed of the proposed use. Contact details for the corresponding authors of all Oxford University Press journal contact can be found alongside either the abstract or full text of the article concerned, accessible from www.oxfordjournals.org Should there be a problem clearing these rights, please contact [email protected] 7. If the credit line or acknowledgement in our publication indicates that any of the figures, images or photos was reproduced, drawn or modified from an earlier source it will be necessary for you to clear this permission with the original publisher as well. If this permission has not been obtained, please note that this material cannot be included in your publication/photocopies. 8. While you may exercise the rights licensed immediately upon issuance of the license at the end of the licensing process for the transaction, provided that you have disclosed complete and accurate details of your proposed use, no license is finally effective unless and until full payment is received from you (either by Oxford University Press or by Copyright Clearance Center (CCC)) as provided in CCC's Billing and Payment terms and conditions. If full payment is not received on a timely basis, then any license preliminarily granted shall be deemed automatically revoked and shall be void as if never granted. Further, in the event that you breach any of these terms and conditions or any of CCC's Billing and Payment terms and conditions, the license is automatically revoked and shall be void as if never granted. Use of materials as described in a revoked license, as well as any use of the materials beyond the scope of an unrevoked license, may constitute copyright infringement and Oxford University Press reserves the right to take any and all action to protect its copyright in the materials. 9. This license is personal to you and may not be sublicensed, assigned or transferred by you to any other person without Oxford University Press’s written permission. 10. Oxford University Press reserves all rights not specifically granted in the combination of (i) the license details provided by you and accepted in the course of this licensing transaction, (ii) these terms and conditions and (iii) CCC’s Billing and Payment terms and conditions. 11. You hereby indemnify and agree to hold harmless Oxford University Press and CCC, and their respective officers, directors, employs and agents, from and against any and all claims arising out of your use of the licensed material other than as specifically authorized pursuant to this license. 12. Other Terms and Conditions: v1.4 https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=0c2ba19d-dbff-4927-a371-0de59cafbc6a 2/3 279

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OXFORD UNIVERSITY PRESS LICENSE TERMS AND CONDITIONS Oct 17, 2018

This Agreement between University of Nevada, Reno -- Tatiana Fontelonga ("You") and Oxford University Press ("Oxford University Press") consists of your license details and the terms and conditions provided by Oxford University Press and Copyright Clearance Center.

License Number 4451030230992 License date Oct 16, 2018 Licensed content publisher Oxford University Press Licensed content publication Human Molecular Genetics Licensed content title Impaired fetal muscle development and JAK-STAT activation mark disease onset and progression in a mouse model for merosin- deficient congenital muscular dystrophy Licensed content author Nunes, Andreia M.; Wuebbles, Ryan D. Licensed content date Mar 7, 2017 Type of Use Thesis/Dissertation Institution name Title of your work Development of Small Molecule Therapies Targeting Regeneration for the Treatment of Duchenne Muscular Dystrophy Publisher of your work n/a Expected publication date Dec 2018 Permissions cost 0.00 USD Value added tax 0.00 USD Total 0.00 USD Title Development of Small Molecule Therapies Targeting Regeneration for the Treatment of Duchenne Muscular Dystrophy Institution name n/a Expected presentation date Dec 2018 Portions The entire text shown as published work at the end of dissertation Requestor Location University of Nevada, Reno 1664 N. Virginia St. MS 573, Room CMM304

RENO, NV 89557 United States Attn: Tatiana Fontelonga

Publisher Tax ID GB125506730 Billing Type Invoice

Billing Address University of Nevada, Reno 1664 N. Virginia St. MS 573, Room CMM304

RENO, NV 89557 United States Attn: Tatiana Fontelonga https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=68d30276-95e4-43e3-bae6-3d707e7288f9 1/3 280

10/17/2018 RightsLink Printable License Total 0.00 USD Terms and Conditions

STANDARD TERMS AND CONDITIONS FOR REPRODUCTION OF MATERIAL FROM AN OXFORD UNIVERSITY PRESS JOURNAL 1. Use of the material is restricted to the type of use specified in your order details. 2. This permission covers the use of the material in the English language in the following territory: world. If you have requested additional permission to translate this material, the terms and conditions of this reuse will be set out in clause 12. 3. This permission is limited to the particular use authorized in (1) above and does not allow you to sanction its use elsewhere in any other format other than specified above, nor does it apply to quotations, images, artistic works etc that have been reproduced from other sources which may be part of the material to be used. 4. No alteration, omission or addition is made to the material without our written consent. Permission must be re-cleared with Oxford University Press if/when you decide to reprint. 5. The following credit line appears wherever the material is used: author, title, journal, year, volume, issue number, pagination, by permission of Oxford University Press or the sponsoring society if the journal is a society journal. Where a journal is being published on behalf of a learned society, the details of that society must be included in the credit line. 6. For the reproduction of a full article from an Oxford University Press journal for whatever purpose, the corresponding author of the material concerned should be informed of the proposed use. Contact details for the corresponding authors of all Oxford University Press journal contact can be found alongside either the abstract or full text of the article concerned, accessible from www.oxfordjournals.org Should there be a problem clearing these rights, please contact [email protected] 7. If the credit line or acknowledgement in our publication indicates that any of the figures, images or photos was reproduced, drawn or modified from an earlier source it will be necessary for you to clear this permission with the original publisher as well. If this permission has not been obtained, please note that this material cannot be included in your publication/photocopies. 8. While you may exercise the rights licensed immediately upon issuance of the license at the end of the licensing process for the transaction, provided that you have disclosed complete and accurate details of your proposed use, no license is finally effective unless and until full payment is received from you (either by Oxford University Press or by Copyright Clearance Center (CCC)) as provided in CCC's Billing and Payment terms and conditions. If full payment is not received on a timely basis, then any license preliminarily granted shall be deemed automatically revoked and shall be void as if never granted. Further, in the event that you breach any of these terms and conditions or any of CCC's Billing and Payment terms and conditions, the license is automatically revoked and shall be void as if never granted. Use of materials as described in a revoked license, as well as any use of the materials beyond the scope of an unrevoked license, may constitute copyright infringement and Oxford University Press reserves the right to take any and all action to protect its copyright in the materials. 9. This license is personal to you and may not be sublicensed, assigned or transferred by you to any other person without Oxford University Press’s written permission. 10. Oxford University Press reserves all rights not specifically granted in the combination of (i) the license details provided by you and accepted in the course of this licensing transaction, (ii) these terms and conditions and (iii) CCC’s Billing and Payment terms and conditions. 11. You hereby indemnify and agree to hold harmless Oxford University Press and CCC, and their respective officers, directors, employs and agents, from and against any and all claims arising out of your use of the licensed material other than as specifically authorized pursuant to this license. https://s100.copyright.com/CustomerAdmin/PLF.jsp?ref=68d30276-95e4-43e3-bae6-3d707e7288f9 2/3 281

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References

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