A New Model for the Dystrophin Associated Protein Complex in Striated

Home , CSRP3, DTNA, Dysbindin, Dysferlin, Dystrobrevin, Dystroglycan

A new model for the dystrophin associated protein complex in striated

muscles.

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of

Philosophy in the Graduate School of The Ohio State University

Eric K. Johnson

Graduate Program, The Ohio State Biochemistry Program

The Ohio State University

2012

Dissertation Committee:

Federica Montanaro , PhD, Advisor

Denis C. Guttridge, PhD

Muthu Periasamy, PhD

Jill A. Rafael-Fortney, PhD

Eric K. Johnson

2012

Abstract

Dystrophin is a large, cytoskeletal protein localized to the intracellular side of the muscle membrane, and is the central organizer of a large protein complex known as the dystrophin associated protein complex (DAPC). Through the

DAPC, dystrophin links the actin cytoskeleton to the extracellular matrix and functions to stabilize the membrane from the forces generated by muscle contraction. However, further studies have also shown that dystrophin functions in intracellular signaling mediated through DAPC proteins. Absence of dystrophin destabilized the DAPC disrupting membrane integrity and muscle function ultimately leading to muscle damage and necrosis. Clinically, mutations in the dystrophin gene give rise a group of muscular dystrophies, termed the dystrophinopathies which represent the most common form of all muscular dystrophy.

The functions of dystrophin are generally believed to be similar for all striated muscles. However, it has become clear that loss of dystrophin does not affect all muscles types equally, particularly the heart. Clinically, studies have shown that there is no correlation in disease severity or age of onset between cardiac and skeletal muscles. Coupled with additional lines of evidence it appears that dystrophin has unique tissue specific functions yet to be elucidated.

Because the majority of dystrophin functions are facilitated though the DAPC, we

ii have hypothesized that the tissue specific functions of dystrophin are mediated by unique protein interactions.

In order to identify dystrophin associated proteins, we first developed a high throughput proteomics approach that combines dystrophin immunoprecipitation with downstream protein identification by shotgun mass spectrometry. Using this approach we identified major differences in the protein interactions of dystrophin between cardiac and skeletal muscle, including differences in the composition of known DAPC proteins. Furthermore, we identified novel cardiac-specific dystrophin associated proteins known to regulate cardiac contraction and to be involved in cardiac disease.

From our studies in the heart, we next extended our approach to the study of dystrophin associated proteins in the diaphragm. In the mdx mouse the diaphragm is the most affected muscle and is the only muscle that closely resembles pathology seen in humans. Because of this, the diaphragm has been intensely studied in an attempt to understand this unique pathology. However the reasons for the more dramatic phenotype are not fully understood. Using our immunoprecipitation approach we show that in the diaphragm, dystrophin has a unique set of protein interactions compared to limb muscles. A subset of these proteins are involved in membrane repair. Therefore, this finding suggests for the first time a possible molecular basis for the more severe phenotype observed in the diaphragm.

An important aspect of our approach is that it can easily be adapted to the study of different proteins. Specifically, we chose to study β-dystroglycan, the

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DAPC protein that anchors dystrophin to the membrane. Our current model of the DAPC is that in the absence of dystrophin, the DAPC is destabilized and lost from the sarcolemmal membrane in skeletal muscle. However, mutations affecting the functions of dystroglycan give rise to a much more severe pathology than loss of dystrophin, and in the absence of dystrophin, β-dystroglycan and some other DAPC proteins are still expressed at the membrane. Together this suggests that dystroglycan must have additional functions not mediated directly by the DAPC and may be mediated by yet unidentified protein complexes. Using a similar approach as to dystrophin we show that in skeletal muscle multiple complexes of β-dystroglycan exist and interact with a unique set of proteins compared to dystrophin. Importantly, although these complexes are distinct from the DAPC, a sub-set is disrupted upon loss of dystrophin. These findings suggest that three distinct β-dystroglycan complexes exist in skeletal muscle, and we propose a new model of β-dystroglycan function and organization in striated muscle.

The studies presented here provide clear evidence that the DAPC is a dynamic protein complex with unique differences between individual muscle tissues. These novel differences suggest tissue specific functions of dystrophin in the heart and diaphragm. Furthermore, our studies on β-dystroglycan add additional complexity our understanding of components of the DAPC and highlight new possible functions of both β-dystroglycan and dystrophin.

Importantly the technique described here overcomes a significant challenge in the field and offers a new method for studying dystrophin protein interactions.

The generation of the method and the novel findings described here will likely prove vital to our understanding of the mechanisms leading to muscle disease for patients with dystrophinopathies.

Acknowledgements

First and foremost I would like to thank my advisor, Dr. Federica

Montanaro for her support and guidance over the time I have been in her lab.

She has been an outstanding mentor and teacher, and has always provided me with an environment that promoted my academic achievements and scientific success.

I would also like to thank my former and current lab mates. Christopher

Penton has been a good friend and outstanding lab partner, and I thank him for all of his help and thoughtful discussion. Thanks to former lab members including

Jesse Gibbons for teaching me several techniques used throughout all of my studies, and Eva Partida for her assistance with the studies presented in chapter

III. I am also grateful for the positive experience and helpful discussions provided by other current and former lab members, Robert Orellana, Allison Macke, Dr.

Jennifer Thomas-Ahner, Nick Beastrom, Nelson Salgado, Stephanie Klatil, and

Dr. Rita Kaspar.

I would also like to personally thank my graduate committee members,

Drs. Denis Guttridge, Muthu Periasamy, and Jill Rafael-Fortney, for their guidance, suggestions, and time.

I am extremely grateful to have worked alongside several collaborators and thank them their help and thoughtful discussion including Dr. Paul Martin and

Dr. Jung Hae Yoon at the Research Institute at Nationwide Children’s Hospital,

Dr. Kari Green-Church and Liwen Zang at The Ohio State University Proteomics

Core, Dr. Stanley Froehner and Dr. Marvin Adams at the University of

Washington, Dr. Michael Freitas at the The Ohio State University, Dr. James

Ervasti at the Univeristy of Minnesota, Allistar Phillips at Cincinnati Children’s

Hospital, and Dr. Dongsheng Duan at the University of Missouri.

Finally I would like to thank my parents, Diane and Keith Johnson, my sister Kristen Johnson, and all of my family and friends for their love and support over the years.

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Vita

2007………………...………………………………………………..Bachelor of Arts Biochemistry/Molecular Biology Illinois State University

2007-Present……………………………………….Graduate Research Associate, OSBP Graduate Program The Ohio State University

Publications

Johnson, E.K., Zhang, L., Adams, M.E., Phillips, A., Freitas, M.A., Froehner, S.C., Green-Church, K.B., Montanaro, F. (2012) Proteomic analysis reveals differences in the cell signaling components of the dystrophin-associated protein complex between cardiac and skeletal muscle. PLOS ONE. 7(8): e43515.

Yoon, Jung; Johnson, Eric; Xu, Rui; Martin, Laura; Martin, Paul; Montanaro, Federica. (2012) Comparative proteomic profiling of dystroglycan-associated proteins in wild type, mdx and Galgt2 transgenic mouse skeletal muscle. J Proteome Res 11(9): 4413-442.

Beastrom, N., Lu, H., Macke, A., Canan, B. D., Johnson, E. K., Penton, C. M., Kaspar, B. K., Rodino-Klapac, L. R., Zhou, L., Janssen, P. M., and Montanaro, F. (2011) mdx(cv) mice manifest more severe muscle dysfunction and diaphragm force deficits than do mdx Mice. Am J Pathol 179, 2464-2474.

Lash, T. D., Lamm, T. R., Schaber, J. A., Chung, W. H., Johnson, E. K., and Jones, M. A. (2011) Normal and abnormal heme biosynthesis. Part 7. Synthesis and metabolism of coproporphyrinogen-III analogues with acetate or butyrate side chains on rings C and D. Development of a modified model for the active site of coproporphyrinogen oxidase. Bioorg Med Chem 19, 1492-1504.

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Field of Study

Major Field: Biochemistry

Table of Contents

Abstract ...... ii

Acknowledgements ...... vi

Vita ...... viii

Field of study ...... viiix

Publications ...... viii

Table of Contents ...... x

List of Tables ...... xvii

List of Figures ...... xviii

List of Abbreviations ...... xvii

Chapters

1. Introduction ...... 1

1.1. Dystrophinopathies ...... 2

1.1.1. Duchenne muscular dystrophy ...... 2

1.1.2. Becker muscular dystrophy ...... 4

1.1.3. Unique mutations ...... 7

1.1.4. X-Linked dilated cardiomyopathy ...... 8

1.1.4.1. Mutations affecting transcription and exon splicing of the DMD

gene ...... 8

1.1.4.2. Mutations that appear to disrupt cardiac specific functions of

dystrophin ...... 9

1.2. Animal models of DMD ...... 9

1.3. Dystrophin and the dystrophin-associated protein complex ...... 12

1.3.1. Dystrophin ...... 12

1.3.2. The dystrophin associated protein complex ...... 14

1.3.3. Dystroglycans ...... 16

1.3.3.1. β-Dystroglycan ...... 17

1.3.3.2. α-Dystroglycan ...... 19

1.3.4. Sarcoglycan-sarcospan complex ...... 20

1.3.4.1. Sarcoglycans ...... 20

1.3.4.2. Sarcospan ...... 22

1.3.5. Syntrophin-dystrobrevin complex ...... 23

1.3.5.1. Systrophins ...... 23

1.3.5.2. Dystrobrevins ...... 25

1.4. Mechanisms of Disease ...... 27

1.4.1. Membrane Destabilization ...... 28

1.4.2. Calcium Deregulation ...... 29

1.4.3. Disruption of Signaling Cascades ...... 30

1.5. Unsolved mysteries of the dystrophinopathies ...... 31

1.5.1. Heart ...... 32

1.5.2. Diaphragm ...... 35

1.5.3. Spared Muscles ...... 36

1.6. Summary ...... 37

1.6. Figures ...... 39

2. Proteomic analysis reveals new cardiac-specific dystrophin-associated proteins ...... 41

2.1. Abstract ...... 41

2.2. Introduction ...... 42

2.3. Results ...... 44

2.3.1. Dystrophin immunoprecipitation and identification of interacting

proteins by LC-MS/MS ...... 44

2.3.2. Identification of tissue-specific dystrophin protein interactions by multi-

factor spectral count analysis ...... 46

2.3.3. nNOS is not in a complex with full length cardiac dystrophin ...... 47

2.3.4.Cardiac and skeletal muscle DAPCs differ in syntrophin composition 48

2.3.5. Differential association of α-dystrobrevins with cardiac and skeletal

muscle DAPC ...... 49

2.3.6. New cardiac-specific protein associations of dystrophin ...... 51

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2.4. Discussion ...... 53

2.5. Methods ...... 56

2.5.1. Human ethics and cardiac biopsies...... 56

2.5.2. Animals ...... 57

2.5.3. Antibodies ...... 57

2.5.4. Immunoprecipitations ...... 58

2.5.5. LC-MS/MS ...... 59

2.5.6. Peptide sequence analysis ...... 61

2.5.7. Label-free quantitation ...... 62

2.5.8. Immunolabeling ...... 62

2.5.9. Immunoblots ...... 63

2.5.10. Densitometric analysis ...... 63

2.6. Tables ...... 65

2.7. Figures ...... 68

3. Identification of novel dystrophin-associated proteins links dystrophin to membrane repair in the diaphragm...... 77

3.1. Abstract ...... 77

3.2. Introduction ...... 78

3.3. Results ...... 81

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3.3.1. The diaphragm has unique protein interactions not present in

quadriceps muscle...... 81

3.3.2. Co-localization of cavin-1 and ahnak1 with dystrophin in quadriceps

and diaphragm...... 82

3.3.3. Localization of cavin-1 and ahnak1 is not disrupted in skeletal muscle

of patients with DMD...... 84

3.3.4. Loss of dystrophin leads to disruption in the localization of dysferlin. 84

3.4. Discussion ...... 85

3.5. Methods ...... 88

3.5.1. Human ethics and tissue biopsies ...... 88

3.5.2. Animals ...... 88

3.5.3. Antibodies ...... 88

3.5.4. Immunoprecipitations ...... 89

3.5.5. Immunoblots ...... 90

3.5.6. Immunohistochemistry ...... 90

3.6. Tables ...... 91

3.7. Figures ...... 92

4. Characterization of dystroglycan complexes in wild type and dystrophic skeletal muscles...... 97

4.1. Abstract ...... 97

4.2. Introduction ...... 98

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4.3. Results ...... 102

4.3.1. Dystroglycan is present at the membrane of myofibers in mdx and

mdx/utrn-/- mice...... 102

4.3.2. Core DAPC members are not major components of dystroglycan

complexes in the absence of dystrophin and utrophin...... 104

4.3.3. Only a small subset of dystroglycan complexes contain dystrophin in

wild type muscles...... 108

4.3.4. The majority of dystroglycan in muscle is expressed by muscle fibers.

...... 109

4.3.5. Novel protein interactions of dystroglycan complexes in muscle . Error!

Bookmark not defined.

4.4. Discussion ...... 114

4.5. Methods ...... 120

4.5.1. Human ethics and skeletal muscle biopsies ...... 120

4.5.2. Animals ...... 120

4.5.3. Antibodies ...... 120

4.5.4. Immunoprecipitations and WGA pull-downs ...... 121

4.5.5. LC-MS/MS...... 121

4.5.6. Peptide sequence analysis ...... 123

4.5.7. Immunohistochemistry ...... 124

4.5.8. Immunoblots...... 125

4.5.9. Molar Concentrations ...... 125

4.6. Tables ...... 126

4.7. Figures ...... 129

5. General discussion ...... 136

5.1. New Pathways for Dystrophin Function ...... 137

5.2. Novel functions of β-dystroglycan in skeletal muscle ...... 144

5.3. Plasticity of the Dystrophin Associated Protein Complex ...... 145

5.3. High Throughput Mutational Analysis ...... 147

5.4. Considerations of the Approach ...... 151

5.5. Figures ...... 153

References ...... 154

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List of Tables

2.1. Identification of DAPC members in DYS-IPs by LC-MS/MS...... 65

2.2. Comparison of proteins between cardiac and skeletal muscle IPs identified by multi-factor analysis of spectral counts ...... 67

3.1. Localization of novel proteins in wild type and mdx skeletal muscle ...... 91

4.1. Localization of DAPC proteins in wild type, mdx and mdx/utrn-/- mouse muscles ...... 126

4.2. Summary of protein concentrations for Laminin, α-dystrolgycan and dystrophin in mouse skeletal muscle extracts (SKM) and rabbit crude surface membrane preparations (CSM) ...... 127

4.3. Proteins identified by proteomics in β-dystroglycan but not dystrophin immunoprecipitations from wild type quadriceps muscles .. Error! Bookmark not defined.

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List of Figures

1.1. Schematic representation of the DMD gene and dystrophin protein...... 39

1.2. Schematic representation of the dystrophin associated protein complex. .. 40

2.1. MANDYS1 specifically immunoprecipitates dystrophin and associated

DAPC members...... 68

2.2. Immunoprecipitation strategy and background reduction ...... 69

2.3. nNOS does not associate with full-length dystrophin in cardiomyocytes. .... 70

2.4. Syntrophins differ between cardiac and skeletal muscle DAPC ...... 71

2.5. Immunolabeling for dystrophin, nNOS, syntrophins and dystrobrevins in wild type skeletal muscle ...... 72

2.6. Differences in -dystrobrevin splice variants between cardiac and skeletal muscle DAPC ...... 73

2.7. Novel cardiac-specific dystrophin-associated proteins ...... 74

2.8. Mass spectra for DAPC members identified by a single peptide in cardiac

DYS-IPs ...... 75

2.9. Mass spectra for DAPC members identified by a single peptide in skeletal muscle DYS-IPs ...... 76

3.1. Novel diaphragm dystrophin-associated proteins...... 92

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3.2. Localization of novel proteins are disrupted at the neuromuscular junction in mdx mice ...... 93

3.3. Localization of cavin-1 is disrupted specifically in the mdx diaphragm ...... 94

3.4. Localization of ahnak1 is disrupted specifically in the mdx diaphragm ...... 95

3.5. Localization of dysferlin is disrupted in the mdx diaphragm but not quadriceps ...... 96

4.1. β-dystroglycan is present at the membrane of myofibers in human DMD muscle biopsies and in mdx and mdx/utrn-/- mouse muscles ...... 129

4.2. Analysis of DAPC members that associate with β-dystroglycan in wild type, mdx and mdx/utrn-/- mouse muscles ...... 130

4.3. The localization of DAPC proteins show unique patterns of disruption in mdx and mdx/utr-/- skeletal muscle ...... 131

4.4. A significant pool of dystroglycan complexes are not bound to dystrophin in wild type muscle ...... 132

4.5. Non muscle cells within adult muscle do not significantly contribute to overall

β-dystroglycan expression...... 133

4.6. New dystroglycan-interacting proteins are differentially affected in mdx and mdx/utrn-/- muscles ...... 134

4.7. Proposed model for dystroglycan complexes in wild type and dystrophin- deficient muscle ...... 135

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5.1. Summary of the tissue specific differences in the DAPC and dystroglycan complexes in wild type muscles ...... 153

List of Abbreviations adbn -/-: α-dystrobrevin knockout mouse BMD: Becker muscular dystrophy BTX: bungarotoxin CRYAB: αB-crystallin DAPC: dystrophin associated protein complex DCM: dilated cardiomyopathy DKO: dystrophin/utrophin double knock out mouse DMD: Duchene muscular dystrophy DSHB: Developmental studies hybridoma bank DTNA: α-dystrobrevin DYS: dystrophin DYS-IP: dystrophin immunoprecipitation ECM: extracellular matric EOM: extraocular muscles IgG-IP: non-specific antibody control immunoprecipitation Kir: inwardly-rectifying potassium channel LC-MS/MS: liquid chromatography tandem mass spectrometry LGMD: Limb-girdle muscular dystrophy MDX: dystrophin deficient mouse Nav: voltage gated sodium channel NMJ: neuromuscular junction nNOS: neuronal nitric oxide synthase PMCA: plasma membrane calcium ATPase SYN: syntrophin TRPC: transient receptor potential channel UTR: utrophin WGA: wheat germ agglutinin XLDCM: X-linked dilated cardiomyopathy

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Chapter 1: Introduction

Muscular dystrophies are a heterogeneous group of inherited disorders characterized by progressive loss of skeletal muscle function and weakness ultimately leading to the loss of muscle tissue. Since the first clinical description of muscular dystrophy in the 1830s (Conte G 1836), many different forms have been identified, caused by mutations in an expanding list of genes. Among all muscular dystrophies, the dystrophinopathies are the most prevalent, and are caused by mutations in the DMD gene (Figure 1.1A). These include the most common type of all muscular dystrophies, Duchene muscular dystrophy (DMD) which affects 1:5000 male live births (Codd, Sugrue et al. 1989), and the less common Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy

(XLDCM). The dystrophinopathies are multisystem disorders affecting multiple organs, although skeletal and cardiac muscle are the primary affected tissues.

Accordingly mortality is associated with respiratory and /or cardiac failure.

1.1. Dystrophinopathies

1.1.1. Duchenne muscular dystrophy

DMD was originally described in the mid-1800s over a century before the causative gene was identified (Meryon 1852; Johnson 1977). Despite not knowing the cause of DMD, the clinical presentation and pathology of the disease was well defined. The first clinical symptoms of DMD present between the ages of 2-5 years and include impaired motor milestones including problems sitting, walking, and running. Additional symptoms include problems with balance where patients are often referred to as being clumsy before diagnosis of DMD.

This is subsequently followed by weakness of the proximal limb and lower limb muscles in addition to pseudohypertrophy, mainly of the calf muscle (Blake, Weir et al. 2002). Progression of muscle wasting leads to loss in ambulation resulting from severe muscle weakness, with patients becoming wheelchair dependent by the age of 12. Cognitive impairment is also often associated with DMD, but is unrelated to severity of muscle disease and it is non-progressive (Bresolin,

Castelli et al. 1994). Ultimately, patients succumb to the disease in their early twenties, where the leading cause of mortality is from respiratory or cardiac failure (Kaspar, Allen et al. 2009; Judge, Kass et al. 2011).

In addition to the relatively standard progression of disease, several well defined hallmarks of DMD have been described. This includes the Gowers maneuver named after W. Gowers who originally describe a method used by

DMD patients to move from a prone to standing position which involves using the

2 hands and arms to “walk” up their thighs (Gowers 1879). This is presumably due to weakness of the lower limb and trunk muscles. Most pathological changes in the muscle were not described until later in the in the 20th century. Histological studies on muscle biopsies showed variations in fiber size, degeneration and regeneration of muscle fibers, and accumulation of fibrotic and adipose tissue

(Bell and Conen 1968). This is further accompanied by extremely elevated levels of serum creatine kinase due to leaky muscle membranes (Okinaka, Kumagai et al. 1961). While predications could be inferred as to the cause of disease, without knowing the affected gene and its protein product research aimed at understanding the molecular mechanisms of DMD were limited and slow progressing.

It was not until over a decade and half after the first descriptions of

Duchenne muscular dystrophy that the DMD gene was identified and cloned, quickly followed by identification of its protein product dystrophin (Monaco, Neve et al. 1986; Hoffman, Brown et al. 1987; Koenig, Monaco et al. 1988). The DMD gene was identified by using positional cloning and was subsequently shown to be the largest known gene in the human genome spanning ~2.5 Mb, and was localized to the X-chromosome (Xp21) (Ahn and Kunkel 1993). The gene encompasses 79 exons (Figure 1.1A), multiple promoters, and tissue specific enhancers resulting in multiple isoforms of the dystrophin protein (Bastianutto,

De Visser et al. 2002; Muntoni, Torelli et al. 2003; Cohen and Muntoni 2004).

Three of these promoters are responsible for expression of the full length dystrophin, including the brain, muscle, and purkinje promoters (Muntoni, Torelli

3 et al. 2003). In striated muscle, the muscle promoter is responsible for full length dystrophin expression, although in skeletal muscle the purkinje promoter produces very low concentrations of full length dystrophin. At least four other promoters exist that drive expression of smaller dystrophin isoforms, named after their molecular weight. These include Dp260 (retinal), Dp140 (brain3), Dp116

(Schwann cells), and Dp71 (general) which are expressed in a tissue specific fashion (Muntoni, Torelli et al. 2003). In addition to alternative start sites, the

DMD gene transcript has further been shown to be alternatively spliced resulting in further variation of dystrophin protein products (Feener, Koenig et al. 1989;

Sadoulet-Puccio and Kunkel 1996).

1.1.2. Becker muscular dystrophy

As previously mentioned, the symptomatologies and pathological features of DMD are well defined and relatively static. However, in the mid-20th century, a new form of muscular dystrophy was described that while similar to DMD was milder and slower progressing (Becker and Kiener 1955). This type of muscular dystrophy would eventually be termed Becker muscular dystrophy (BMD) after its first description by P. Becker. Similar to DMD, BMD shows progressive weakness of the lower limb and proximal muscles. Patients with BMD also have pseudohypertrophy and elevated serum creatine kinase. While DMD and BMD show very similar phenotypes over time, clinically there are major distinguishing differences. Specifically, BMD has a later age of onset with the first symptoms becoming apparent around 11 years (DMD 2-5 years) (Emery and Skinner 1976).

The rate of progression is slower with loss of ambulation occurring at 20-30 years of age (DMD 12 years), and overall milder severity with average age of mortality in the 40s (DMD 20 years) (Emery and Skinner 1976). Although phenotypically similar to DMD, the aforementioned differences resulted in BMD being considered a separate and distinct X-linked disease. It was not until the discovery of dystrophin that DMD and BMD would be shown to be genetically linked and caused by mutations in the same gene. Because of the heterogeneity of BMD, a distinct classification of BMD was not always precise. It was not until the identification of the DMD gene that we gained the ability to distinguish BMD from other dystrophies including limb-girdle and Emery-Dreifuss muscular dystrophies.

The incidence of BMD was thus estimated to be 1:18,540 male births (Bushby,

Thambyayah et al. 1991).

From a scientific perspective, the study of BMD is particularly interesting although very challenging. DMD is caused by mutations that include relatively large deletions of one or more exons (60-70%), large duplications of one or more exons (10%), and point mutations (10-30%) (Darras, Miller et al. 1993).

Mutations that give rise to DMD tend to disrupt the reading frame and thus abolish expression of dystrophin. Because of this, the progression of the disease between individual patients does not significantly vary. The major molecular difference between DMD and BMD is that BMD is caused by mutations that do not completely prevent expression of dystrophin protein but allow for either reduced or altered protein products to be expressed. In turn, this results in disease progression and level of severity being highly variable between individual

5 patients. However, the types of mutations for BMD are not strikingly different than

DMD, including large deletions of one or more exons (80%), duplications(5%), and point mutations (10-15% )(Darras, Miller et al. 1993). Therefore, clinical presentation is often used to distinguish DMD from BMD. Additional tests including Western blotting and dystrophin staining of skeletal muscle biopsies may also be used to aid in diagnosis.

To further complicate our understanding of phenotype diversity for BMD, there is no correlation between size of deletion or duplication and severity of disease (Muntoni, Torelli et al. 2003). In an attempt to rationalize this observation, the reading frame “rule” was hypothesized (Monaco, Bertelson et al.

1988). The basis of this theory is that mutations that shift the open reading frame of transcription (out-of-frame) or result in pre-mature stop codons give rise to unstable mRNA and subsequent nonsense mediated decay. If this happens no dystrophin or nearly undetectable concentrations of truncated protein is produced and results in a clinical diagnosis of DMD. However, for mutations that give rise to BMD there is often a perseveration of the open reading frame (in-frame) and mutant but stable dystrophin protein products are expressed (Kaspar, Allen et al.

2009). Further testing of the reading frame theory has proven largely applicable to the majority of cases, where it holds true for 96% of DMD patients and 93% of

BMD patients (Ozawa 2010). However exceptions to this theory do exist including DMD patients with in-frame mutations and BMD patients with out-of- frame mutations (Muntoni, Torelli et al. 2003).

1.1.3. Unique mutations

In-frame and missense point mutations that give rise to DMD have been identified. These include mutations affecting critical domains of dystrophin that are important for its interactions with other proteins. These mutations disrupt critical functions of dystrophin and give rise to a DMD phenotype (Blake, Weir et al. 2002; Ozawa 2010).For some of these cases, dystrophin is still expressed and properly localized to the sarcolemma. Because of this it is important that both biochemical and genetic testing are used for an accurate diagnosis of DMD as opposed to BMD.

Some out-of-frame mutations that would be predicted to result in DMD actually give rise to a BMD clinical phenotype. In these cases, the milder phenotype is most often related to endogenous skipping of the affected exon leading to restoration of the reading frame (Muntoni, Torelli et al. 2003). These events almost always result in a truncated dystrophin product with an intact C- terminus. Examples of exon skipping can be seen by immunohistochemical analysis of muscle biopsies for dystrophin staining of revertant fibers. Additional out-of-frame mutations localized to the 5’ portion of the dystrophin gene have been shown to result in BMD due to an alternative start site in exon 8 which produces a truncated but semi-functional protein (Muntoni, Torelli et al. 2003).

These examples represent only a few of numerous mutations that have been identified.

1.1.4. X-linked dilated cardiomyopathy

Cardiac disease is a major component of all the dystrophinopathies with over 90% of DMD and BMD patients exhibiting cardiac involvement (Finsterer and Stollberger 2003). Because of this, heart failure accounts for about 30% and

50% of DMD and BMD mortality, respectively (Duboc, Meune et al. 2005). The third and most rare form of dystrophinopathy is X-linked dilated cardiomyopathy

(XLDCM). XLDCM is particularly interesting as symptomatically it is distinct from

DMD and BMD in that patients present with an almost exclusive cardiac involvement, mainly dilated cardiomyopathy (DCM) with no overt skeletal muscle disease. Although a subset of patients have elevated serum creatine kinase levels there is no signs of skeletal muscle weakness (Muntoni, Torelli et al.

2003). Mutations that give rise to XLDCM can generally be divided into two categories; 1) mutations affecting transcription and exon splicing of the DMD gene, and 2) mutations that appear to disrupt cardiac specific functions of dystrophin (Ferlini, Sewry et al. 1999).

1.1.4.1. Mutations affecting transcription and exon splicing of the DMD gene.

Most of these mutations are localized to the 5’ end of the dystrophin gene, and selectively disrupt the muscle specific promoter. In skeletal muscle the brain and purkinje promoters can compensate for the faulty muscle promoter

(Muntoni, Melis et al. 1995; Neri, Valli et al. 2012). However, in the heart this

8 compensatory mechanism not occur (Neri, Valli et al. 2012). Histologically this can be seen by dystrophin staining on skeletal and cardiac biopsy, and biochemically by Western blot analysis which shows a complete absence of dystrophin only in the heart. In rare instances skeletal muscle specific exon splicing has also been shown to rescue dystrophin expression in skeletal muscle but not the heart (Muntoni, Torelli et al. 2003). This indicates the existence of tissue-specific splicing regulation of dystrophin. Never the less, the major observation for this group is selective lack of dystrophin only in the heart.

1.1.4.2. Mutations that appear to disrupt cardiac specific functions of dystrophin.

Patients in this group show comparable levels of dystrophin expression in both cardiac and skeletal muscles; however only severe cardiac disease is present. Mutations in this group include nonsense and missense mutations and in-frame deletions (Towbin, Hejtmancik et al. 1993; Muntoni, Torelli et al. 2003).

Currently the reasons for the cardiac specific phenotype are not well understood.

However a reasonable explanation is that these mutations disrupt not yet identified cardio-specific functions of dystrophin.

1.2. Animal models of DMD

The dystrophin gene is relatively well conserved in metazoans (Greener and Roberts 2000; Pozzoli, Elgar et al. 2003; van der Plas, Pilgram et al. 2007).

Because of this several model organisms of DMD have either been identified or created from simple organisms such as Drosophila and C. Elegans to more complex higher organisms including Zebrafish, mice, cats, and dogs (Collins and

Morgan 2003). Interestingly, although all these models show some phenotype, the only one to show a phenotype similar to humans are canine models, specifically the golden retriever muscular dystrophy dog (Cooper, Winand et al.

1988).

Despite having a pathophysiological milder disease course than humans, the dystrophin deficient mdx mouse model is most commonly used laboratory model of DMD. The mdx mouse originated from a spontaneous nonsense mutation in exon 23 and was originally identified due to abnormally high plasma levels of creatine kinase (Bulfield, Siller et al. 1984). Since then additional mdx mouse models including mdx2cv, mdx3cv, mdx4cv, mdx5cv have been created by induced mutagenesis using ENU (Chapman, Miller et al. 1989; Im, Phelps et al.

1996). Pathologically, the mdx mouse undergoes traditional hallmarks of DMD including leaky membranes, fiber necrosis, regeneration, inflammation and accumulation of fibrotic tissue (Beastrom, Lu et al. 2011). Functional studies have further shown significant defects in force production and fatigue for both skeletal and cardiac muscle (Petrof, Shrager et al. 1993; Lynch, Hinkle et al.

2001; Beastrom, Lu et al. 2011; Xu, Delfin et al. 2011). Muscle pathology is the most pronounced between 2 and 8 weeks of age with a massive cycle of degeneration and regeneration between weeks 3 and 4 (Collins and Morgan

2003). The remainder of the mdx lifespan is marked by mild although present

10 myopathy evident by centrally located nuclei, a marker of regenerated fibers

(Collins and Morgan 2003). However, many features of DMD are not present until late age as mice display no prominent decrease in life span and remain ambulant. Similar to skeletal muscle, the heart in the mdx mouse is only mildly affected, although cardiac defects have been identified particularly with age and under stress conditions (Sapp, Bobet et al. 1996; Chu, Otero et al. 2002;

Quinlan, Hahn et al. 2004; Janssen, Hiranandani et al. 2005). Despite these caveats, the mdx mouse has still proven extremely useful for understandings the mechanisms of disease for DMD.

In an attempt to make a more physiologically accurate mouse model of

DMD, several groups have created double knockout mouse models which show a more severe myopathy. Most notable is the dystrophin/utrophin double knockout mouse (dko) (Deconinck, Rafael et al. 1997). Utrophin is a functional homolog of dystrophin, normally confined to the neuromuscular junction (NMJ) in adult wild type skeletal muscle. However, in embryonic and regenerating muscle fibers utrophin is highly expressed at the sarcolemma. Therefore, it is thought that utrophin can compensate for absence of dystrophin in the mdx mouse

(Rybakova, Patel et al. 2002) and overexpression of utrophin is able to rescue muscle pathology (Tinsley, Potter et al. 1996). Indeed, removal of both dystrophin and utrophin resulted in much more severe skeletal myopathy and cardiac disease which more accurately represents DMD (Deconinck, Rafael et al.

1997). Although the dko mouse more accurately represents the level of

11 pathology present in DMD, it is important to remember that this is no longer an exact genetic model.

Finally, a more recent mouse model for DMD has been generated by introducing an inactivating mutation in the CMAH gene, a gene involved in the biosynthesis of N-glycolylneuraminic acid onto the mdx background

(Chandrasekharan, Yoon et al. 2010). Humans do not have a functional CMAH gene and therefore the Cmah-/-mdx mouse more closely resembles DMD patients than mdx alone. Indeed, the Cmah-/-mdx mouse is characterized by a more severe phenotype and earlier onset of disease (Chandrasekharan, Yoon et al. 2010). Therefore, the Cmah-/-mdx mouse is more genetically similar to humans and represents yet another valuable model for the study of the disease mechanisms leading to DMD.

1.3. Dystrophin and the dystrophin-associated protein complex

1.3.1. Dystrophin

Dystrophin is the protein product of the DMD gene, that when disrupted is responsible for the dystrophinopathies (Figure 1.1A) (Hoffman, Brown et al.

1987). The full length dystrophin protein is a 427 kDa, 3685 amino acid rod shaped protein localized to the cytosolic face of the sarcolemmal membrane in striated muscle (Figure 1.1B). Dystrophin belongs to the spectrin family of proteins and has been shown to have features similar to α-actinin and β-spectrin including spectrin like repeats and actin binding domains (Broderick and Winder

2005). Based on sequence homology, dystrophin can be divided into four distinct domains; the N-terminal domain, the central rod domain, the cysteine-rich domain, and the C-terminal domain (Ervasti and Sonnemann 2008). The N- terminal domain contains a pair of calponin homology putative actin binding sequences that functionally comprise the actin binding domain thus linking dystrophin to the actin cytoskeleton (Rybakova, Amann et al. 1996). Adjacent to the N-terminus, the central rod domain represents the largest portion of dystrophin and is comprised of 24 spectrin like repeats interspersed with four proline rich hinge regions (Koenig and Kunkel 1990). Together, the spectrin-like repeats and hinge regions are believed to confer flexibility and give the protein a long rod shape. However, additional functions of the rod domain have been identified including a second actin binding site (Rybakova, Amann et al. 1996) and a nNOS binding site (Lai, Thomas et al. 2009). Following the rod domain, the cysteine-rich domain contains several protein interacting domains including two

EF hands, WW and ZZ modules (Ervasti 2007). All of these domains have been shown to be necessary for binding of β-dystroglycan (Ishikawa-Sakurai, Yoshida et al. 2004), a critical transmembrane protein involved in linking dystrophin to the extracellular matrix. Finally, the C-terminal domain is unique to dystrophin, although shares slight homology to α1-dystrobrevin and utrophin. The C-terminus mediates binding to several intracellular adapter proteins. It contains two coiled- coil domains, a well characterized protein binding motifs that is required for binding the dystrobrevins (Sadoulet-Puccio, Rajala et al. 1997). Finally the C- terminus also has two distinct syntrophin binding domains (Suzuki, Yoshida et al.

1995). Interestingly, the C-terminus of dystrophin including the regions involved in binding the syntrophins have been shown to be alternatively spliced and the inclusions of specific syntrophin isoforms may further be mediated by alternative splicing of dystrophin (Ahn and Kunkel 1995).

1.3.2. The dystrophin associated protein complex.

In striated muscles dystrophin is the central organizer of a large protein complex known as dystrophin associated protein complex (DAPC) (Figure 1.2).

The DAPC was identified in 1990 shortly after the identification of dystrophin

(Ervasti, Ohlendieck et al. 1990; Yoshida and Ozawa 1990; Ervasti, Kahl et al.

1991). Using wheat germ agglutinin (WGA), a plant lectin that binds N- acetylglucosamine, a sugar found on glycosylated proteins, large amounts of α- dystroglycan was purified from detergent solubilized skeletal muscle membranes.

In addition to α-dystroglycan, WGA chromatography purified several tightly bound proteins including dystrophin and associated proteins, now referred to as the

DAPC (Ervasti, Ohlendieck et al. 1990; Yoshida and Ozawa 1990; Ervasti, Kahl et al. 1991).

The DAPC is a large oligomeric protein complex (Figure 1.2) comprised of at least eleven core proteins including extracellular (α-dystroglycan), transmembrane (β-dystroglycan, α-, β-, γ-, δ-sarcoglycan, and sarcospan), and cytosolic (dystrophin, α1-, β1-, β2-syntrophin, and α-dystrobrevin) proteins. Early studies investigating the functions of the DAPC principally focused on its primarily role as a mechanical stabilizer of the sarcolemma (Ervasti and

Sonnemann 2008). By linking the extracellular matrix (ECM) with the actin cytoskeleton, the DAPC is believed to protect the membrane from the forces generated by muscle contraction and stretch. Loss of dystrophin destabilizes the

DAPC in turn weakening the sarcolemma (Ervasti and Sonnemann 2008). The fragile sarcolemma is thus more prone to contraction induced damage ultimately leading to loss of muscle fiber integrity. Unfortunately, the exact mechanisms leading to muscle damage and necrosis, including the distinction between primary and secondary disruptions are currently not fully understood.

Since the original identification of the DAPC, a growing body of evidence has identified a second function of the DAPC in cell signaling. Possibly the best characterized signaling function of the DAPC is through association with neuronal nitric oxide synthase (nNOS) in skeletal muscle (Abdelmoity, Padre et al. 2000). Association of nNOS is mediated through interaction with α1-syntrophin and dystrophin (Brenman, Chao et al. 1996) and localization at the sarcolemma has been shown to be disrupted in DMD and the mdx mouse (Brenman, Chao et al. 1995; Chang, Iannaccone et al. 1996). In skeletal muscle nNOS has been suggested to have several functions including: regulation of vasoconstriction, cytoprotection of free radicals, and cGMP-dependent cell survival pathways

(Rando 2001). Besides nNOS, other studies have also suggested additional signaling functions of the DAPC (described below) (Rando 2001).However, the exact cellular signaling pathways regulated by the DAPC are poorly defined, and more studies are needed to fully elucidate these functions and pathways.

Lack of dystrophin expression grossly disrupts the DAPC. More in depth analyses of individual components have highlighted the complexity of the DAPC.

Indeed, various muscular dystrophies have been identified that are caused by mutations in a variety of distinct DAPC genes. Therefore it is not surprising that mutations in the DMD gene have been suggested to be involved in various disease mechanisms.

1.3.3. Dystroglycans

Dystroglycan is the product of the Dag1 gene which is transcribed to produce a single mRNA sequence (Henry and Campbell 1999). The mRNA sequence is then translated into a single pre-mature polypeptide that is subsequently post-translationally cleaved into two distinct proteins, α- and β- dystroglycan (Holt, Crosbie et al. 2000). Dystroglycan was originally purified from rabbit skeletal muscle as part of the DAPC (Ibraghimov-Beskrovnaya, Ervasti et al. 1992). However, dystroglycan has since been shown to be expressed in almost every tissue in the human and mouse (Ibraghimov-Beskrovnaya,

Milatovich et al. 1993; Durbeej, Henry et al. 1998).Together α- and β- dystroglycan form a core component of the DAPC. By linking dystrophin inside the cell via transmembrane β-dystroglycan to the ECM through α-dystroglycan, the dystroglycan complex physically protects the muscle membrane. However, additional signaling functions of the dystroglycans have also been proposed

(discussed in detail below). Only recently has the first mutation in the Dag1 gene been identified (Hara, Balci-Hayta et al. 2011). This missense mutation affects

16 the glycosylation of α-dystroglycan and resulting in a clinical diagnosis of limb- girdle muscular dystrophy (Hara, Balci-Hayta et al. 2011). Because of the lack of mutations preventing dystroglycan expression, ablation of dystroglycan is believed to be embryonic lethal. This is further supported by knockout studies in the mouse where targeted knockout of dystroglycan is embryonic lethal

(Williamson, Henry et al. 1997). However, as discussed later, mutations affecting glycosylation of α-dystroglycan have been identified (Montanaro and Martin

2011). These mutations do not affect the expression but function of dystroglycan and give rise to a group of muscular dystrophies known as the dystroglycanopathies.

1.3.3.1. β-Dystroglycan

β-dystroglycan is a 43 kDa, single pass transmembrane protein with a intracellular proline rich c-terminal tail (Blake, Weir et al. 2002). The extracellular

N-terminus of β-dystroglycan non-covalently binds to α-dystroglycan (Henry and

Campbell 1999). Intracellularly, the last 15 amino acids bind tightly to the cysteine-rich domain of dystrophin. This interaction is primarily mediated though the WW motif of dystrophin which directly binds the PPXY consensus sequence of β-dystroglycan (Jung, Yang et al. 1995). However, it was subsequently shown that both the EF-hand, and ZZ moieties are further required for physiological binding of dystrophin (Chung and Campanelli 1999; Ishikawa-Sakurai, Yoshida et al. 2004). Besides dystrophin, β-dystroglycan has also been shown to bind several other intracellular proteins. Possibly the best studied protein interaction with respect to the DAPC is with the dystrophin homology utrophin. High

17 concentrations of utrophin are expressed at the membranes of immature muscle fibers bound to β-dystroglycan. In mature muscle, utrophin is restricted to the

NMJ where similar to immature muscle is bound β-dystroglycan. Binding of utrophin is similar to dystrophin, and presumably competes for the same PPXY consensus sequence (Chung and Campanelli 1999; Tommasi di Vignano, Di

Zenzo et al. 2000). Other examples of suggested binding partners of β- dystroglycan include grb2 (Yang, Jung et al. 1995), caveolin-3 (Sotgia, Lee et al.

2000),plectin 1f (Rezniczek, Konieczny et al. 2007) and vinexin (Thompson,

Moore et al. 2010) which interact with dystroglycan through the overlapping WW or SH3 domain binding motif. Through association with these proteins, β- dystroglycan could potentially be directly involved with Ras-MAP kinase signaling regulation cytoskeletal organization (Yang, Jung et al. 1995), stabilization and regulation of membrane activity (Sotgia, Lee et al. 2000), anchoring to the intermediate filaments (Rezniczek, Konieczny et al. 2007), and cell adhesion and spreading of myoblasts (Thompson, Moore et al. 2010), respectively.

Furthermore, β-dystroglycan has also been shown to bind rapsyn (Cartaud,

Coutant et al. 1998) at NMJ where it functions in acetylcholine receptor (AChRs) clustering (Gee, Montanaro et al. 1994; Jacobson, Montanaro et al. 1998;

Montanaro, Gee et al. 1998). The expansive list of β-dystroglycan interacting proteins suggests multiple signaling roles of dystroglycan and complicates our understanding of its functions in skeletal muscle. However, it is important to note that many of the β-dystroglycan interacting proteins all bind through similar or overlapping regions of the c-terminal tail of β-dystroglycan. Furthermore, some of

18 these proteins have been shown to directly compete with binding of dystrophin.

Finally many of these protein interactions were shown in vitro. Therefore, in vivo studies are needed to truly asses their biological association. The mechanisms of how these proteins all bind β-dystroglycan in striated muscles are not clear.

1.3.3.2. α-Dystroglycan

α-Dystroglycan is an extracellular, highly glycosylated protein that has an apparent molecular weight of ~156 kDa (not glycosylated 120 kDa) in muscle tissue (Blake, Weir et al. 2002). However, do to differential glycosylation, α- dystroglycan has varied molecular weight in non-muscle tissue. Sequence analysis and electron microscopy studies suggested a central mucin-like region flanked by two globular domains giving α-dystroglycan a dumbbell shape

(Brancaccio, Schulthess et al. 1995). Glycosylation of α-dystroglycan in turn strongly links the DAPC to the ECM though association with laminins

(Ibraghimov-Beskrovnaya, Ervasti et al. 1992). α-Dystroglycan has also binds agrin and perlecan in the ECM at the NMJ (Singhal and Martin 2011). Similar to

β-dystroglycan, α-dystroglycan is also involved in aggregation of AChRs through binding to agrin and laminins at the NMJ (Gee, Montanaro et al. 1994;

Montanaro, Gee et al. 1998). As previously mentioned only one missense mutation has been identified in the dystroglycan gene disrupting the proper glycosylation of α-dystroglycan. Although this is the only direct mutation of the

Dag1 gene, disruption of several proteins involved in the glycosylation of α- dystroglycan have been identified (Montanaro and Martin 2011). Biochemically,

19 these mutations result in hypo glycosylation of α-Dystroglycan. Clinically, these mutations are referred to as dystroglycanopathies, and represent an expanding group of muscular dystrophies. They encompasses various forms of muscular dystrophy including congenital muscular dystrophy 1C and 1D, muscle-eye-brain disease, Walker-Warburg syndrome, Fukuyama congenital muscular dystrophy, and limb girdle muscular dystrophy (LGMD) (Montanaro and Martin 2011).

1.3.4. Sarcoglycan-sarcospan complex

1.3.4.1. Sarcoglycans

The sarcoglycans form a homologous tetrameric sub-complex containing four main proteins (α-, β-, γ-, and δ-sarcoglycan) which are all encoded by separate genes. All of the sarcoglycans are single pass transmembrane type II

(C-terminal localized intracellularly) proteins with the exception of α-sarcoglycan which is type I (intracellular N-terminal) (Ervasti and Sonnemann 2008). Similar to α-dystroglycan, the sarcoglycans are glycosylated on their extracellular side

(Holt and Campbell 1998). Mutations in the sarcoglycan genes are termed sarcoglycanopathies and give rise to autosomal recessive LGMDs (Sandona and

Betto 2009). Beyond the original characterization of the four sarcoglycans, two additional sarcoglycans, ε- and δ-sarcoglycan are expressed in muscle. Although these are not components of the classical DAPC, they are suggested to compensate for loss of certain sarcoglycans. Specifically, ε-sarcoglycan is homologous to α-sarcoglycan, and studies have suggested that specific loss of

α-sarcoglycan results in replacement with ε-sarcoglycan (Liu and Engvall 1999;

Imamura, Mochizuki et al. 2005). Furthermore, δ-sarcoglycan shows homologies

20 to γ- and δ-sarcoglycan, and in vitro studies have suggest that it may compensate for absence of γ-sarcoglycan (Shiga, Yoshioka et al. 2006).

The functions of the sarcoglycan complex are not well defined. However, since their original identification with the DAPC (Ibraghimov-Beskrovnaya, Ervasti et al. 1992), two primary functions of the sarcoglycan complex have emerged.

Possibly the best studied and understood function is providing mechanical stability to the DAPC. Typically, disruption of one of the sarcoglycans results in absence or significant reduction of the remaining sarcoglycans (Blake, Weir et al.

2002). Mutations in β- or δ-sarcoglycan genes produce the greatest destabilization (Allikian and McNally 2007). However generation of individual knock-out mouse models for the sarcoglycans have identified exceptions where loss of particular sarcoglycans result in varied disruption of other DAPC members

(Blake, Weir et al. 2002). In all the sarcoglycan null mice, dystrophin is still properly localized to the sarcolemma (Lapidos, Kakkar et al. 2004). Never the less, these studies suggest to a possible structural roles of the sarcoglycan complex with the DAPC.

Depictions of the DAPC often reflect the sarcoglycans and forming a tightly associated sub-complex interacting with dystroglycan in the membrane.

Indeed studies have shown their tight association with dystroglycan, specifically

γ- and δ-sarcoglycan (Rando 2001). However, these depictions likely do not illustrate the complicated intertwinement of the sarcoglycan complex with other members of the DAPC. This includes association of α-dystroglycan and α- and γ- sarcoglycan with biglycan possibly forming a potential extracellular link between

21 the sarcoglycans and the dystroglycan sub-complex (Rafii, Hagiwara et al. 2006).

Furthermore, it has been suggested that the C-terminus of dystrophin may directly interact with β- and δ-sarcoglycan (Chen, Shi et al. 2006), and that the sarcoglycans, specifically β-sarcoglycan might bind the N-terminus of α- dystrobrevin (Yoshida, Hama et al. 2000). However, more in-depth studies, particularly performed in vivo are needed to fully understand the complex nature of the sarcoglycan interactions in the DAPC.

The second and less well defined function of the sarcoglycan complex is as a signaling component. Unfortunately, studies investigating the signaling functions of sarcoglycans are lacking. However, it has been reported that by associating with γ-filamin, the sarcoglycans may function in actin remodeling

(Dalkilic and Kunkel 2003).

1.3.4.2. Sarcospan

Sarcospan is a small tetraspan (4 transmembrane domains) protein that associates with the sarcoglycan complex (Crosbie, Lebakken et al. 1999). Little is known about the function of sarcospan and original studies on sarcospan knockout mice show no obvious phenotype (Lebakken, Venzke et al. 2000). However, more recent studies on the sarcospan null mouse suggest that sarcospan functions to stabilize the DAPC (Marshall, Chou et al. 2012). Furthermore, overexpression in the mdx mice results in upregulation of utrophin at the extrasynaptic membrane and stabilizes components of the DAPC (Peter,

Marshall et al. 2008).

1.3.5. Syntrophins-dystrobrevin complex

1.3.5.1. Syntrophin

The syntrophins were originally identified as a triplet of 59 kDa proteins

(Ervasti, Kahl et al. 1991) and comprise α1-, β1-, β2-syntrophin. The syntrophins all share a similar modular structure comprising one PDZ domain, two pleckstrin homology (PH) domains, and a unique C-terminus (Ervasti and Sonnemann

2008). Both the PDZ and PH domains are common protein-protein interacting domains, and thus the syntrophins are often referred to as adaptor proteins linking the DAPC to cell signaling pathways. Inclusion of the syntrophins in the

DAPC is mediated by both dystrophin and the dystrobrevins. Dystrophin has two binding site for the syntrophins in its c-terminus, and is capable of simultaneously binding two separate syntrophins via the syntrophin’s unique C-terminus and one of the PH domains (Ehmsen, Poon et al. 2002). Similar to dystrophin, the dystrobrevins are also capable of simultaneously binding two separate syntrophins. In striated muscle, α1-syntrophin is highly expressed at the membrane of muscle fibers and cardiomyocytes (Perry, Mori et al. 2001). Both

β1- and β2-syntrophin are expressed in skeletal and cardiac muscle (Johnson,

Zhang et al. 2012). However, in skeletal muscle β1-syntrophin is predominantly expressed in fast twitch type II muscle fibers (Peters, Adams et al. 1997). β2-

Syntrophin localized to the sarcolemma in humans (Jones, Compton et al. 2003) but in mice is restricted to the NMJ (Peters, Kramarcy et al. 1994).

As previously mentioned, the syntrophins are adapter proteins that link the

DAPC to cellular signaling pathways. The best characterized binding partner of the syntrophins is nNOS which is recruited to the membrane by binding the PDZ domain of α1-syntrophin (Brenman, Chao et al. 1995; Adams, Mueller et al.

2001). In α1-syntrophin knock-out mice the localization of nNOS at the sarcolemma is disrupted similar to mdx mice (Kameya, Miyagoe et al. 1999).

Interestingly no obvious phenotype arises from disruption of nNOS in the α1- syntrophin knock-out mouse, although recent studies have identified a point mutation that gives rise to long QT syndrome (Ueda, Valdivia et al. 2008; Wu, Ai et al. 2008). α1-Syntrophin has also been shown to bind and regulate the α1D- adrenergic receptor involved in regulating vascular tone (Tanoue, Nasa et al.

2002; Chen, Hague et al. 2006; Lyssand, Lee et al. 2011).

Other binding partners of the syntrophins include transmembrane channels. Specifically, α1-syntrophin is known to localize aquaporin-4 at the membrane of muscle fibers via the PDZ domain, and is disrupted in mdx mice

(Adams, Mueller et al. 2001). Little is known about the physiological role of aquaporin-4, but it has been suggested to functionally regulate water permeability during muscle contraction (Frigeri, Nicchia et al. 1998). A growing body of evidence has also linked the syntrophins to regulation of ion channels in both cardiac and skeletal muscle. Included are sodium (skeletal Nav1.4 and cardiac Nav1.5) (Gee, Madhavan et al. 1998; Gavillet, Rougier et al. 2006), potassium (Kir2.x and Kir4.1) (Leonoudakis, Conti et al. 2004), and more recently calcium (PMCA, TRPC1 and TRPC2) (Williams, Armesilla et al. 2006;

Vandebrouck, Sabourin et al. 2007; Sabourin, Lamiche et al. 2009) channels.

The ability of the syntrophins to regulate ion influx, particularly calcium is of great interest as deregulation of calcium homeostasis is hypothesized to be a major contributing factor to cardiac and skeletal muscle disease for the dystrophinopathies (Blake, Weir et al. 2002). Additional syntrophin binding proteins include microtubule-associated serine/threonine kinase (Lumeng,

Phelps et al. 1999), diacylglycerol kinase-δ (Abramovici, Hogan et al. 2003), grb2

(Oak, Russo et al. 2001), and ankyrin repeat-rich membrane spanning (Luo,

Chen et al. 2005) potentially linking dystrophin to the microtubule network, actin organization, intracellular signaling, and synaptic signaling respectively.

1.3.5.2. Dystrobrevins

Dystrobrevins are intracellular proteins that are distantly related to dystrophin and include α- and β-dystrobrevin. α-Dystrobrevin was originally identified as 87 kDa protein in the Torpedo electric organ post-synaptic membrane (Nakamori and Takahashi 2011). β-dystrobrevin does not associate with dystrophin in striated muscle and will not be discussed.

α-Dystrobrevin shares similarity to the cysteine-rich and C-terminus of dystrophin including two EF hands, a ZZ domain, and an α-helical coiled coil domain. The coiled-coil domain of both α-dystrobrevin and dystrophin is used for direct binding to each other (Sadoulet-Puccio, Rajala et al. 1997). In both human and mouse several splice variants of α-dystrobrevin exist, specifically α1-, α2-,

α3-dystrobrevin (Nakamori and Takahashi 2011). However, splicing is more

25 complex in humans where at least 5 distinct splice variants exist (α4- and α5- dystrobrevin, exclusive to the brain) (Bohm, Constantinou et al. 2009). In addition to the three major splice variants, alternative splicing of two variable regions

(three for α1-dystrobrevin) further add complexity to protein expression (Bohm,

Constantinou et al. 2009; Nakamori and Takahashi 2011). As previously mentioned the N-terminus of α-dystrobrevins binds the sarcoglycans, and α3- dystrobrevin which lacks the coiled-coil domain, is believed to associate with the

DAPC though the sarcoglycans (Yoshida, Hama et al. 2000). In addition to binding dystrophin and sarcoglycans, α1- and α2-dystrobrevin contain at least two independent syntrophin binding sites in tandem, although identification of a third was recently suggested (Bohm, Constantinou et al. 2009). The syntrophin binding sites are alternatively spliced and splicing may function to regulate not only stoichiometry of the syntrophins but isoform specificity (Bohm, Constantinou et al. 2009). The exact mechanisms controlling the association of the dystrobrevins with the syntrophins is not well understood.

Besides binding components of the DAPC, α-dystrobrevins interact with structural proteins including syncoilin, desmuslin (β-synemin), and dysbindin

(Benson, Newey et al. 2001; Mizuno, Thompson et al. 2001; Newey, Howman et al. 2001). Syncoilin and desmuslin are intermediate filaments and likely reinforce the link between the DAPC and intermediate filament network (Nakamori and

Takahashi 2011). The functions of dysbindin in muscle are not well defined, but it has been shown to associate with another protein myospryn and possibly protein

26 kinase A (Benson, Tinsley et al. 2004; Sarparanta 2008; Nakamori and

Takahashi 2011).

No mutations in the α-dystrobrevin gene have been identified that result in muscular dystrophy. Targeted deletion of α-dystrobrevin in the mouse (adbn−/−) has only a mild dystrophic phenotype, and the core DAPC proteins are unaffected (Grady, Grange et al. 1999). However, disruptions of the NMJ are apparent in these mice. Surprisingly, the adbn−/− mouse shows clear disruption of nNOS localization at the sarcolemma (Grady, Grange et al. 1999). More recent studies on the adbn−/− mouse have suggested that α-dystrobrevin helps to stabilize the interaction of dystrophin and β-dystroglycan (Bunnell, Jaeger et al.

2008). However, loss of this function does not appear to be critical for maintenance of the muscle fiber.

1.4. Mechanisms of disease

Dystrophin has been proposed to serve both structural and signaling functions primarily based on histological observations of the pattern of muscle damage and on the association of dystrophin with proteins involved in cell signaling. However while we know the upstream trigger (loos of dystrophin expression) and the ultimate outcome (myofiber necrosis), we currently do not have a clear grasp of the intervening steps. Similarly we do not know which events are a direct consequence of loss of dystrophin versus a secondary response to muscle injury. This section provides an overview of the observed

27 pathological changes in dystrophic muscle and the current theories of how they might be linked to dystrophin.

1.4.1. Membrane destabilization

The major function of dystrophin is believed to provide mechanical stability to the muscle membrane. In striated muscle dystrophin is enriched at the constameres, where it is believed to physically connect the contracting apparatus to the ECM. By doing so, dystrophin is believed to help to not only protect the membrane from the forces generated by muscle contraction, but also dissipate these forces into the ECM (Blake, Weir et al. 2002). In the late 1900s electron microscopy performed on DMD muscle identified disrupted sections of the sarcolemma overlaying triangular areas of abnormal cytoplasm referred to as delta-lesions (Mokri and Engel 1975; Blake, Weir et al. 2002). Disruptions in the sarcolemma membrane thus results in leaking of both intracellular proteins (e.g. creatine kinase (Okinaka, Kumagai et al. 1961)) out and extracellular proteins

(e.g. IgG and IgM (Straub, Rafael et al. 1997)) into the fibers. This has since further been shown by use of small impermeable dyes such as Evans blue which accumulate in the skeletal and cardiac muscle of mdx but not wild type muscle

(Straub, Rafael et al. 1997). The number of fibers that uptake Evans blue dye are further increased following mechanical stress such as exercise (Brussee, Tardif et al. 1997). Although the exact mechanisms leading to membrane permeability and consequence of this are unknown, membrane fragility resulting in increased leakiness of the mdx muscle fibers and cardiomyocytes are well established.

1.4.2. Calcium Deregulation

Ca2+ regulation is an important aspect of muscle function (Berchtold,

Brinkmeier et al. 2000). Several groups have reported increased intracellular concentrations of Ca2+ in DMD and mdx muscle fibers (Turner, Westwood et al.

1988; Turner, Fong et al. 1991; Hopf, Turner et al. 1996). Furthermore, uncontrollable Ca2+ sparks have also been identified in isolated mdx muscle fibers (Wang, Weisleder et al. 2005). The exact consequences of increased intracellular Ca2+ in DMD and the mdx mouse are not well defined, but several disease pathways have been hypothesized (Blake, Weir et al. 2002). Included is activation of Ca2+ dependent proteases, specifically calpains which have shown to be upregulated in both DMD and mdx mice (Bodensteiner and Engel 1978;

Spencer, Croall et al. 1995) and are involved in remodeling of cytoskeletal and membrane proteins (Tidball and Spencer 2000). Furthermore increased Ca2+ is linked to alterations in several signaling cascades, and disruption of mitochondrial membrane potentials (Goldstein and McNally 2010). Finally, high concentrations of Ca2+ have also been suggested to increase levels of reactive oxygen species (ROS) generated by the mitochondria (Whitehead, Yeung et al.

2006). ROS in turn has been suggested to increase proteolysis and inflammatory damage which could further worsen disease (Whitehead, Yeung et al. 2006).

The sources of increased Ca2+ in dystrophic muscle are currently not well understood, but several mechanisms have been proposed, mainly influx of extracellular Ca2+ due do membrane leaks. Based on the ability of impermeable dyes to accumulate in muscle cells, it is reasonable postulate that membrane

29 tears could be a potential source of extracellular Ca2+. Recent studies have also shown that dystrophin and the DAPC are directly associated with Transient receptor potential cation (TRPC) channels (Williams, Armesilla et al. 2006;

Vandebrouck, Sabourin et al. 2007; Sabourin, Lamiche et al. 2009), and calcium channel deregulation could represent an additional source of Ca2+. Indeed, abnormal calcium influx due to deregulation of TRPC channels has been shown in mdx muscle fibers (Franco and Lansman 1990; Vandebrouck, Duport et al.

2001) which was subsequently blocked by stretch-activated channel blockers

(Yeung, Whitehead et al. 2005). Additional studies have also suggested L-type

Ca2+ channels as a potential source of Ca2+ influx (Friedrich, von Wegner et al.

2008). While the L-type Ca2+ channels may contribute to increased Ca2+ influx, a direct association with the DAPC has not been shown and warrants further study.

1.4.3. Disruption of Signaling Cascades

Dystrophin and the DAPC have been suggested to be involved in several signaling cascades. Unfortunately, our understanding of the signaling functions of dystrophin are extremely limited, and therefore thorough analysis of their disruption has not been performed. However the majority of dystrophin’s signaling functions are likely mediated through the syntrophins that can bind to several proteins including enzymes (Brenman, Chao et al. 1995; Adams, Mueller et al. 2001), channels (Gee, Madhavan et al. 1998; Adams, Mueller et al. 2001;

Williams, Armesilla et al. 2006; Vandebrouck, Sabourin et al. 2007; Sabourin,

Lamiche et al. 2009), kinases (Lumeng, Phelps et al. 1999; Abramovici, Hogan et

30 al. 2003), and kinase substrates (Luo, Chen et al. 2005). Besides the syntrophins, β-dystroglycan has also been hypothesized to mediate signaling cascades though association with Grb2, potentially linking the DAPC to mitogen‐activated protein (MAP) kinase signaling pathway (Yang, Jung et al.

1995). Finally, β-dystroglycan has been suggested to directly bind MEK and ERK which are involved in downstream signaling cascades and transcriptional regulation (Spence, Dhillon et al. 2004). It is important to note that for some of these studies, predominantly those pertaining to β-dystroglycan were not performed in vivo and their association requires further studies.

1.5. Unsolved mysteries of the dystrophinopathies

The protein interactions of the DAPC, and in turn the functions of dystrophin are currently believed to be highly similar for all striated muscles including skeletal (limb and diaphragm) and cardiac. Despite this, it is clear from observations in the clinic and from animal models of DMD that not all mutations in the DMD gene affect all muscle groups equally. In particular there are subsets of muscle groups that appear to be differentially affected upon disruption of dystrophin.

1.5.1. Heart

Some mutations in the DMD gene exist and give rise to a predominant cardiac phenotype with relative sparing or complete absence of skeletal muscle disease including both nonsense and missense mutations and exon deletions

(Ferlini, Sewry et al. 1999; Kaspar, Allen et al. 2009). Possibly the most intriguing are missense mutations that do not completely abolish dystrophin expression and lie outside known protein interacting domains. One specific example is a point mutation that resulted in replacement of threonine at position 279 by an alanine affecting hinge 1 of the dystrophin protein. This mutation gave rise to early onset DCM in a large family where all patients harboring this mutation showed signs of cardiac involvement but no skeletal muscle weakness (Berko and Swift 1987; Towbin, Hejtmancik et al. 1993). Another example includes a nonsense mutation in exon 29. Due to alternative splicing, a dystrophin product lacking 50 amino acids in the rod domain was expressed in both cardiac and skeletal muscle (Franz, Muller et al. 2000). Surprisingly this disrupted the composition of DAPC including components of the sarcoglycan complex exclusively in the heart, and a clinical diagnosis of a XLDCM. For exon deletion mutations, the majority that predominantly affect the heart localize to mutational hot spots of the rod domain, primarily exons 45-55 (Muntoni, Di Lenarda et al.

1997; Kaspar, Allen et al. 2009). Analysis of these mutations identified two sub- classes; mutations that disrupt the phasing of the spectrin repeats and mutations preserving phasing (Kaspar, Allen et al. 2009). An early onset and more sever phenotype correlates with out of phase as opposed to conserved phasing.

Despite the difference in severity between the two sub-classes, the reasons for the predominant cardiac phenotype with sparing of skeletal muscle are not known. For all of these mutations no significant difference for dystrophin expression was seen between cardiac and skeletal muscle (Kaspar, Allen et al.

2009).

In addition to mutational analysis, molecular and biochemical studies have identified key differences between skeletal and cardiac muscle dystrophin.

Dystrophin has unique localization in the heart including expression at the T- tubules and cytosol, distinct from the localization at the sarcolemma (Peri,

Ajdukovic et al. 1994; Meng, Leddy et al. 1996). In these pools, dystrophin is suggested to interact with T-tubule and microfilament network only in cardiomyocytes (Peri, Ajdukovic et al. 1994; Meng, Leddy et al. 1996). Further iIsolation of the DAPC by WGA pull-downs depletes dystrophin in skeletal but not cardiac muscle, further suggesting that a sub-population of dystrophin exists that does not interact with glycosylated DAPC proteins (Peri, Ajdukovic et al. 1994).

The functions and binding partners of dystrophin in these separate pools are currently unknown.

Investigations into cardiac dystrophin associated proteins recently identified the cardiac specific sodium channel Nav1.5 as being a component of the cardiac DAPC (Gavillet, Rougier et al. 2006). Nav1.5 plays a key role in cardiac excitability and conduction, and decreases in the sodium current and alterations in cardiac conduction are evident in isolated mdx cardiomyocytes

(Gavillet, Rougier et al. 2006). As previously mentioned, dystrophin has been

33 suggested to regulate skeletal muscle Nav1.4 (Gee, Madhavan et al. 1998).

However, a direct association of Nav1.4 with the DAPC has not been shown, and alterations in Nav1.4 activity due to secondary affects cannot be ruled out.

Finally, gene therapy strategies utilizing truncated dystrophin constructs hold great promise for rescuing skeletal muscle integrity and function. A number of mini- and micro-dystrophin constructs have been generated to date, and where originally modeled after large exon deletions identified in mild patients with

BMD. Among these, a mini-dystrophin construct modeled after a very mild BMD patient (ΔH2-R19) which lacks the majority of the rod domain has been shown to completely rescue skeletal muscle of mdx mice to wild type levels (Davies, Smith et al. 1988; England, Nicholson et al. 1990; Harper, Hauser et al. 2002). When tested for its ability to restore muscle function, mini-dystrophin was able to correct all histological parameters of mdx skeletal muscle (Bostick, Yue et al.

2008). However, mini-dystrophin was incapable of completely normalizing the heart, specifically certain electrocardiogram and hemodynamic parameters

(Bostick, Yue et al. 2008). Unfortunately, there was no mention if any cardiac abnormalities were apparent in the patient used for the creation of mini- dystrophin (Davies, Smith et al. 1988). While the mini-dystrophin is a promising therapeutic construct, it is important to remember that it is still modeled after a mutation that gave rise to BMD, although very mild (England, Nicholson et al.

1990).

The functions of dystrophin and the DAPC are generally believed to be highly similar for skeletal muscle and the heart. However, as just discussed

34 genetic analysis coupled with building molecular and biochemical evidence suggests possible cardio specific functions of dystrophin.

1.5.2. Diaphragm

Respiratory failure is one of the leading causes of mortality for patients with DMD (Simonds 2002). Similarly the mdx mouse diaphragm exhibits a dystrophic phenotype including necrosis, fibrosis, and loss of function, although disruption does not lead to early mortality in the mouse as it does in humans

(Stedman, Sweeney et al. 1991). Compared to all other muscles, the mdx diaphragm is the most affected and the only muscle that closely mimics pathology in humans. Several hypotheses for this dramatic difference in severity have been proposed and tested including increased inflammation (Demoule,

Divangahi et al. 2005), increased work load (Stedman, Sweeney et al. 1991;

Dupont-Versteegden, McCarter et al. 1994), and reduced myogenic regeneration

(Anderson, Garrett et al. 1998; Matecki, Guibinga et al. 2004). While the evidence for these hypotheses is in support of potential pathways leading to disease, subsequent studies have questioned the validity of the majority of these hypotheses (Dupont-Versteegden, McCarter et al. 1994; Anderson, Garrett et al.

1998; Krupnick, Zhu et al. 2003; Matecki, Guibinga et al. 2004). At this time, no clear mechanism for the severe phenotype observed in the mdx diaphragm has been delineated.

1.5.3. Spared Muscles

Despite dystrophin being absent from all striated muscle fiber types in

DMD, a small subset of muscles appear unaffected by loss of dystrophin.

Included are extraocular (EOMs) (Khurana, Prendergast et al. 1995) and laryngeal muscles (Marques, Ferretti et al. 2007; Thomas, Joseph et al. 2008).

The most extensively studied of these are the EOMs. Surprisingly in both DMD patients and the mdx mouse no centrally located nuclei, a hallmark of regenerated fibers are apparent in the EOMs (Kaminski, al-Hakim et al. 1992;

Khurana, Prendergast et al. 1995; Porter, Rafael et al. 1998). Furthermore, the

EOMs have no other hallmarks of dystrophic muscle including both necrosis and fibrosis suggesting these muscle are not undergoing any damage or pathology

(Kaminski, al-Hakim et al. 1992; Khurana, Prendergast et al. 1995; Porter, Rafael et al. 1998).

Because of the preservation of EOMs in DMD, considerable attention has been given to understanding the mechanisms used for protection of muscle fibers. Indeed, several studies have investigated potential mechanisms for maintaining muscle integrity. Specifically, utrophin, a functional homolog of dystrophin has been shown to be considerably upregulated in mdx EOMs compared to that of limb muscle (Porter, Rafael et al. 1998). Utilizing a dko mouse, it was shown that absence of both proteins results in loss of protection of the muscle fiber and a severe dystrophic phenotype (Porter, Rafael et al. 1998).

Separate studies have investigated the ability of the EOMs to sequester influx of high cytosolic Ca2+ concentrations (Zeiger, Mitchell et al. 2010). EOMs have

36 been reported to have higher levels of Ca2+ handling proteins including sarco/endoplasmic reticulum Ca2+-ATPase, calsequestrin, and phospholamban

(Zeiger, Mitchell et al. 2010). It has been suggested that the increased buffering capacity of the EOMs helps to protect the muscle fiber by reducing the damage caused by increased intracellular Ca2+ (e.g. activation of calpains) (Zeiger,

Mitchell et al. 2010). Understanding the mechanisms involved in protecting the

EOMs and laryngeal muscles will not only help in delineating the mechanisms leading to disease but give insight into generation of future therapeutic strategies.

1.6. Summary

Since the original identification of dystrophin and the dystrophin associated protein complex nearly 30 years ago, great strides have been made towards our understanding of the mechanisms leading to the disease. Despite this tremendous effort, the functions of dystrophin have not been fully delineated and much more work is needed to fully grasp the roles of dystrophin. Never the less, dystrophin has been well defined as a structural protein. By connecting the actin cytoskeleton to the extracellular matrix through the DAPC, dystrophin protects the sarcolemma from the forces generated by muscle contraction. Loss of dystrophin in turn results in membrane fragility ultimately leading to loss of muscle function and mass.

Disruption of dystrophin clearly affects the organization of the DAPC and lead to multisystem disorders. In skeletal and cardiac muscle, loss of dystrophin

37 appears to disrupt several vital roles needed for proper muscle function.

Interestingly, mutations affecting individual components of the DAPC give rise to several forms of muscular dystrophy distinct from the dystrophinopathies. While the mechanisms leading to disease are not fully known, it can be inferred that the functions of dystrophin are likely more complex than a purely structural role.

Indeed, a second function of dystrophin as a mediator of cellular signaling has been shown by several independent groups. Based on these studies, the signaling functions of dystrophin are likely regulated through a complex network of protein interactions mediated by numerous members of the DAPC.

It appears that many of dystrophin functions are mediated through protein- protein interactions, including tissue specific associations. Unfortunately the identification and study of dystrophin associated proteins in vivo have been hampered by the complex nature of the DAPC. In order to fully understand the mechanisms of muscle disease for the dystophinopathies, there is a critical need for the generation of techniques directed at delineating the exact functions of dystrophin in vivo including identification of dystrophin associated proteins.

1.7. Figures

Actin- Membrane Flexible Rod domain (24 spectrin repeats + 4 hinges) Unique binding protein binding C-terminus 39 domain

Figure 1.1: Schematic representation of the DMD gene and dystrophin protein. A. The DMD gene is comprised of 79 exons encoding for the dystrophin protein. B. Dystrophin is a rod shaped protein comprised of four domains: N-terminal actin binding domain, flexible rod domain, cysteine-rich domain, and a unique C-terminal domain

Figure 1.2: Schematic representation of the dystrophin associated protein complex.

Chapter 2: Proteomic analysis reveals new cardiac-specific dystrophin- associated proteins.

2.1. Abstract

Mutations affecting the expression of dystrophin result in progressive loss of skeletal muscle function and cardiomyopathy leading to early mortality.

Interestingly, clinical studies revealed no correlation in disease severity or age of onset between cardiac and skeletal muscles, suggesting that dystrophin may play overlapping yet different roles in these two striated muscles. Since dystrophin serves as a structural and signaling scaffold, functional differences likely arise from tissue-specific protein interactions. To test this, we optimized a proteomics-based approach to purify, identify and compare the interactome of dystrophin between cardiac and skeletal muscles from as little as 50 mg of starting material. We found selective tissue-specific differences in the protein associations of cardiac and skeletal muscle full length dystrophin to syntrophins and dystrobrevins that couple dystrophin to signaling pathways. Importantly, we identified novel cardiac specific interactions of dystrophin with proteins known to regulate cardiac contraction and to be involved in cardiac disease. Our approach overcomes a major challenge in the muscular dystrophy field of rapidly and consistently identifying bona fide dystrophin-interacting proteins in tissues. In

41 addition, our findings support the existence of cardiac-specific functions of dystrophin and may guide studies into early triggers of cardiac disease in

Duchenne and Becker muscular dystrophies.

2.2. Introduction

Dystrophin is a large (427 kDa) sub-membrane protein that links the actin cytoskeleton to the extracellular matrix via the dystrophin-associated protein complex (DAPC; Figure 2.1A) (Ervasti and Sonnemann 2008). In skeletal muscle, the DAPC has a structural role important for membrane integrity and a signaling role mediated by its intracellular members, syntrophins and dystrobrevins (Albrecht and Froehner 2002). Mutations in dystrophin give rise to dystrophinopathies, a term that includes Duchenne muscular dystrophy (DMD),

Becker muscular dystrophy (BMD) and X-linked dilated cardiomyopathy

(XLDCM). DMD and BMD are characterized by both progressive skeletal muscle degeneration and cardiac involvement, contributing to early mortality by respiratory or cardiac failure (Kaspar, Allen et al. 2009; Judge, Kass et al. 2011).

By contrast, XLDCM patients show a selective severe cardiac involvement leading to heart failure (Cohen and Muntoni 2004). Although the functions of dystrophin and composition of the DAPC are generally thought to be similar between cardiac and skeletal muscle, clinical studies in dystrophinopathy patients show no correlation between cardiac and skeletal muscle disease with respect to severity or age of onset (Nigro, Politano et al. 1994; Cohen and

Muntoni 2004; Kaspar, Allen et al. 2009). In addition, mini- and micro-dystrophin constructs developed for gene-replacement therapy of DMD show differences in their ability to functionally rescue cardiac versus skeletal muscle (Townsend,

Blankinship et al. 2007; Bostick, Yue et al. 2008). These results suggest that dystrophin may have cardiac-specific functions that remain to be elucidated.

Since protein interactions mediate many of the structural and signaling functions of dystrophin, we hypothesized that dystrophin may associate with different proteins in cardiac and skeletal muscle.

Mass spectrometry based proteomic approaches are well positioned for the identification of large numbers of proteins within a complex sample and could provide a comprehensive view of the dystrophin interactome. To date, proteomic analysis of muscle membrane fractions enriched for dystrophin and the DAPC has proven challenging, achieving only a 2% coverage of the large dystrophin protein and incomplete detection of known dystrophin-interacting proteins(Lewis and Ohlendieck 2010). However, optimization of this approach is a worthwhile endeavor because it has the potential to reveal new tissue-specific dystrophin- binding proteins relevant to normal function and disease.

We describe here the successful combination of DAPC immunoprecipitation with shotgun proteomics (LC-MS/MS) to rapidly and consistently identify dystrophin-associated proteins from as little as 50 mg of tissue, allowing studies in individual mice and eventually biopsy material.

Furthermore, LC-MS/MS yielded higher sensitivity and protein coverage than previous gel-based approaches (Lewis and Ohlendieck 2010), allowing robust

43 detection of all known DAPC members with high protein sequence coverage. We further describe a spectral count analysis for subtraction of tissue-specific background and direct comparison of dystrophin’s interactome between cardiac and skeletal muscle. This analysis brought to the forefront tissue-specific differences in DAPC composition and revealed new dystrophin interacting proteins that are relevant to cardiac function and disease.

2.3. Results

2.3.1. Dystrophin immunoprecipitation and identification of interacting proteins by LC-MS/MS.

To identify proteins that selectively associate with dystrophin, we opted for the high specificity of antibody-based immunoprecipitation using the MANDYS1 monoclonal antibody to dystrophin. MANDYS1 recognizes a domain not involved in interactions with known DAPC members that is absent in shorter dystrophin isoforms expressed in non-muscle cells (Nguyen and Morris 1993; Muntoni,

Torelli et al. 2003). Therefore we are isolating proteins associated with full-length dystrophin expressed in muscle cells. Furthermore, MANDYS1 does not cross- react with utrophin (Figure 2.1B), a homolog of dystrophin (Tinsley, Blake et al.

1992). Sample contamination by immunoglobulins was minimized by cross- linking the MANDYS1 antibody to the support matrix (Figure 2.2A, B). Western blot analysis confirmed that MANDYS1 reliably immunoprecipitated large amounts of dystrophin and co-purified transmembrane ( -dystroglycan),

44 extracellular ( -dystroglycan) and intracellular (syntrophins) DAPC members

(Figure 2.1C, D). LC-MS/MS analysis was performed on dystrophin immunoprecipitations from 50mg of cardiac or skeletal muscle (quadriceps) from wild type mice (DYS-IPs). Two controls for non-specific protein binding to immunoglobulins or to MANDYS1 were performed using an irrelevant isotype- matched antibody on wild type samples (IgG-IP) or the MANDYS1 antibody on samples from dystrophin-deficient mdx mice (Bulfield, Siller et al. 1984) (MDX-IP;

Figure 2.2C). Dystrophin and DAPC proteins were not detected in IgG- or MDX-

IPs with the exception of one skeletal muscle IgG-IP that was cross- contaminated with a small amount of a cardiac DYS-IP sample resulting in low level detection of cardiac proteins and DAPC members. Comparison of IgG- and

MDX-IPs revealed that they were equivalent in identifying contaminating proteins in DYS-IPs. However, different background proteins were identified in cardiac and skeletal muscle control immunoprecipitations. After tissue-specific background subtraction, dystrophin and the core DAPC members (with the exception of the more distantly associated sarcospan) were within the top 15 proteins specifically detected in all DYS-IPs with high confidence scores typically above 100, multiple unique peptide matches, and high protein sequence coverage (Table 2.1). This included DAPC members known to be at least one interaction removed from dystrophin, such as the sarcoglycans. Furthermore, we consistently identified and obtained good peptide coverage for transmembrane proteins ( -dystroglycan and all sarcoglycans) whose hydrophobicity renders identification by mass spectrometry challenging. Of note, two DAPC members,

45 neuronal nitric oxide synthase (nNOS) and 2-syntrophin were consistently differentially detected between cardiac and skeletal muscle. Overall these results indicate that our approach allows for the reliable and rapid one-shot identification of proteins that interact directly or indirectly with dystrophin from as little as 50mg of muscle tissue.

2.3.2. Identification of tissue-specific dystrophin protein interactions by multi-factor spectral count analysis.

To identify in an unbiased fashion both known and novel proteins that interact with dystrophin in skeletal and cardiac muscle, we adapted a multi-factor sample analysis commonly used in genomics (Robinson, McCarthy et al. 2010) to the analysis of protein spectral counts. First, a multi-factor significance analysis test was used to account for variability between replicates and to generate a fold change and associated p-value between each DYS-IP and its corresponding IgG-IP. Negative fold changes indicated enrichment in the IgG-IP allowing easy removal of contaminating proteins from analysis. Second, proteins were sorted based on their p value to identify differences in composition and protein abundance between cardiac and skeletal muscle DYS-IPs (Table 2.2). In agreement with our results based on total protein scores (Table 2.1), the spectral count analysis identified tissue-specific differences in the associations of nNOS and 2-syntrophin with cardiac and skeletal muscle dystrophin (Table 2.2). In addition, comparison of p values suggested a lower abundance of 1-syntrophin in cardiac compared to skeletal muscle DYS-IPs (Table 2.2). Importantly, this

46 analysis highlighted new potential dystrophin-associated proteins that were specific to cardiac DYS-IPs and involved proteins with known associations to cardiac disease in humans: Cavin-1 (PTRF) (Rajab, Straub et al. 2010; Shastry,

Delgado et al. 2010), Ahnak1 (desmoyokin) (Haase, Alvarez et al. 2005), Cypher

(LDB3, ZASP) (Vatta, Mohapatra et al. 2003; Hershberger, Parks et al. 2008;

Arimura, Inagaki et al. 2009), and Crystallin alpha B (CRYAB) (Inagaki, Hayashi et al. 2006; Sacconi, Feasson et al. 2011). Therefore, this multi-factor analysis of spectral counts can be used to rapidly compare the composition of purified dystrophin complexes between tissues and to bring to the forefront potential candidate proteins for tissue-specific association to dystrophin.

2.3.3. nNOS is not in a complex with full length cardiac dystrophin.

To test for tissue-specific differences in nNOS association with the DAPC, we first tested for the presence of nNOS in DYS-IPs by Western blot. As shown in Figure 2.3A, nNOS is readily detected in skeletal muscle dystrophin IPs, but not in cardiac DYS-IPs even when the entire DYS-IP eluate is loaded in a single lane and the film exposure is saturated. Furthermore, immunostaining of isolated cardiomyocytes showed a lack of co-localization of nNOS with dystrophin at lateral membranes (Figure 2.3B). Instead, nNOS localized to internal membranes consistent with its reported association with the sarcoplasmic reticulum (Barouch,

Harrison et al. 2002). These results support our proteomic analysis and indicate that nNOS does not interact with full length dystrophin in cardiomyocytes.

Recruitment of nNOS to the DAPC occurs via 1-syntrophin (Brenman, Chao et

47 al. 1996) and spectrin repeats 16 and 17 in the dystrophin rod domain (Lai,

Thomas et al. 2009). 1-syntrophin was readily detected in cardiac DYS-IPs by both LC-MS/MS (Table 2.1) and Western blot (Figure 2.3A). Analysis of dystrophin peptides detected by LC-MS/MS indicated that spectrin repeats 16 and 17 were present in cardiac dystrophin (Figure 2.3C). These results indicate that lack of association of nNOS with cardiac dystrophin is not due to loss of 1- syntrophin or to tissue-specific alternative splicing of the rod domain of dystrophin.

2.3.4. Cardiac and skeletal muscle DAPCs differ in syntrophin composition.

LC-MS/MS analysis suggested a selective association of 2-syntrophin with cardiac dystrophin (Tables 2.1 and 2.2). Western Blot analysis confirmed co- purification of 2-syntrophin in mouse cardiac but not skeletal muscle DYS-IPs

(Figure 2.4A). In addition, 2-syntrophin also co-purified with cardiac dystrophin in human cardiac biopsy samples (Figure 2.4B). Co-localization of dystrophin and

2-syntrophin by immunolabeling could not be established due to weak binding of the only available antibody to 2-syntrophin. Our spectral count analysis also indicated that 1-syntrophin is present in similar amounts in cardiac and skeletal muscle DYS-IPs while 1-syntrophin is less abundant in cardiac DYS-IPs (Table

2.2). Western blot analysis followed by densitometry confirmed a similar abundance of 1-syntrophin relative to dystrophin between skeletal and cardiac muscle DYS-IPs (Figure 2.4A). This agreed with immunohistochemical analysis

48 of cardiac and skeletal muscle tissue sections where 1-syntrophin was strongly expressed at the membrane of all cardiomyocytes (Figure 2.4C) and all myofibers (Figure 2.5) where it co-localized with dystrophin. By contrast, 4-fold less 1-syntrophin was associated with dystrophin in cardiac muscle compared to skeletal muscle DYS-IPs (Figure 2.4A). This was supported by immunohistochemistry where low levels of 1-syntrophin expression were seen at the membrane of a subset of cardiomyocytes (Figure 2.4C), while in skeletal muscle, 1-syntrophin was expressed at high levels in a subset of muscle fibers

(type IIB), as previously reported (Peters, Adams et al. 1997) (Figure 2.5).

Overall these results confirm that cardiac and skeletal muscle DAPCs differ in the presence of 2-syntrophin and in the relative abundance of 1-syntrophin. They also validate the use of p values derived from the spectral count analysis as good indicators of the relative abundance of a given protein between samples.

2.3.5. Differential association of -dystrobrevins with cardiac and skeletal muscle DAPC.

The stoichiometry of syntrophins is affected by alternative splicing of - dystrobrevin (Newey, Benson et al. 2000). We therefore tested for tissue-specific association of -dystrobrevin splice variants ( 1-, 2-, and 3-) with cardiac and skeletal muscle dystrophin. Because of their high sequence identity, - dystrobrevin splice variants could not be conclusively distinguished by LC-

MS/MS. However, Western blot analysis with a pan -dystrobrevin antibody

49 allowed distinction of -dystrobrevin variants based on molecular weight. Both

1- and 2-dystrobrevins were present in mouse cardiac and skeletal muscle

DYS-IPs but they differed in their relative stoichiometry to dystrophin (Figure

2.6A). The stoichiometry of 2-dystrobrevin to dystrophin was similar between skeletal and cardiac muscle DYS-IPs (Figure 2.6A) in agreement with strong 2- dystrobrevin immunolabeling at the membrane of all cardiomyocytes (Figure

2.6B) and skeletal muscle fibers (Figure 2.5) where dystrophin is also expressed.

By contrast, almost 5-fold less 1-dystrobrevin was associated with cardiac DYS-

IPs compared to skeletal muscle (Figure 2.6A). Accordingly, 1-dystrobrevin was expressed at the sarcolemma of all myofibers in skeletal muscle (Figure 2.5) but showed only weak discontinuous membrane staining of a subset of cardiomyocytes (Figure 2.6B).

Western blot analysis also revealed that 3-dystrobrevin, while expressed in both mouse skeletal and cardiac muscle, strongly associates with dystrophin in the heart (Figure 2.6A). In skeletal muscle DYS-IPs, a faint but specific 3- dystrobrevin band could only be detected after long exposures suggesting that only a small pool of 3-dystrobrevin associates with full length skeletal muscle dystrophin. Interestingly, 3-dystrobrevin is the only splice variant that lacks the known dystrophin and syntrophin binding domains (Blake, Nawrotzki et al. 1996;

Peters, Sadoulet-Puccio et al. 1998) and associates with the DAPC via direct binding to the intracellular domain of -sarcoglycan (Yoshida, Hama et al. 2000).

A prior study (Townsend, Blankinship et al. 2007) and our own data (Figure 2.6C) indicate that -sarcoglycan is preserved at the membrane of dystrophin-deficient

50 cardiomyocytes. We therefore predicted that 3-dystrobrevin would be similarly unaffected in the mdx heart. Surprisingly, loss of dystrophin in mdx mice is accompanied by an almost complete loss of 3-dystrobrevin expression in the heart (Figure 2.6D). Among all three -dystrobrevin splice variants, 3- dystrobrevin showed the most drastic decrease in expression in the mdx heart.

These results confirm that 3-dystrobrevin is a member of the cardiac DAPC and further indicate that its expression in cardiac muscle is tightly dependent upon dystrophin, even in the presence of -sarcoglycan at the membrane of mdx cardiomyocytes.

Finally, we confirmed by Western blot analysis that all three -dystrobrevin isoforms interact with dystrophin in the human heart (Figure 2.6E). Interestingly, the antibody to -dystrobrevin recognized additional bands in the human cardiac dystrophin IPs. Based on molecular weight, these additional bands likely correspond to the previously described human-specific -dystrobrevin splice variants (Nakamori, Kimura et al. 2008). These results suggest that while the human cardiac DAPC is similar to the mouse, it is also more complex in its - dystrobrevin composition.

2.3.6. New cardiac-specific protein associations of dystrophin.

Our comparative LC-MS/MS analysis suggested novel cardiac-specific interactions of dystrophin with Cavin-1, Ahnak1, Cypher and CRYAB (Table 2.2) and these proteins were specifically and consistently detected by Western blot

51 analysis in mouse cardiac but not skeletal muscle DYS-IPs (Figure 2.7A).

Although these proteins are abundant in mouse muscle, they were not detected in IgG- or MDX-IPs by LC-MS/MS (Table 2.2) or Western blots (Figure 2.7A), with the exception of low amounts of CRYAB detected by Western blot after long exposure. Of note, only a fraction of the total pool of CRYAB and Cypher co- purified with dystrophin suggesting that these interactions are either weak or rare. The interactions of these cardiac proteins with dystrophin was further confirmed by reverse immunoprecipitation for each individual protein on cardiac mouse tissue (Figure 2.7B). No dystrophin was detected in the IgG matched controls except for trace amounts in the cypher immunoprecipitation.

Strong immunoreactive bands corresponding to Cavin-1 and Ahnak1 were consistently detected in cardiac DYS-IPs from the mouse (Figure 2.7A). We therefore asked whether these interactions were conserved in the human heart.

Similar to the mouse, strong immunoreactive bands were detected in DYS-IPs from a human cardiac biopsy (Figure 2.7C). Densitometric analysis of the expression of Cavin-1 and Ahnak1 in mdx versus wild type mouse cardiac muscle did not reveal any significant change in overall expression. We therefore tested whether dystrophin was required for their association with the cardiac

DAPC. We immunoprecipitated the DAPC from wild type and mdx mouse heart using the MANDAG2 antibody to -dystroglycan, that is preserved at the membrane of mdx cardiomyocytes. Although MANDAG2 recognizes a region of

-dystroglycan involved in binding to dystrophin, it co-purified large amounts of dystrophin as well as Ahnak1 and Cavin-1 in wild type heart (Figure 2.7D). By

52 contrast, Ahnak1 and Cavin-1 were absent from MANDAG2-IPs from mdx mouse heart. Therefore, Ahnak1 and Cavin-1 are part of the cardiac DAPC and this association is affected in the dystrophin-deficient heart.

2.4. Discussion

The study of the interactome of dystrophin has been hampered by the large size of dystrophin and by the complexity of its direct and indirect interactions with extracellular, transmembrane, and intracellular proteins. Yet the identification of dystrophin-associated proteins is fundamental to our knowledge of the functions of dystrophin in healthy striated muscles and, by correlate, to our understanding of the molecular events that trigger cardiac and skeletal muscle disease in dystrophinopathies.

This study describes a rapid and simple protocol that allows for the high confidence and robust identification of proteins that interact either directly or indirectly with dystrophin within muscle tissues. Compared to previous studies

(Yoshida and Ozawa 1990; Lewis and Ohlendieck 2010), this approach offers the advantage of specifically purifying dystrophin-containing complexes with the

MANDYS1 antibody rather than using lectins that bind to multiple glycosylated proteins. We are also directly interrogating the entire protein eluate with minimal losses in sensitivity associated with gel separation of proteins. This resulted in impressive protein coverage, high confidence scores and reliable detection of all core DAPC members. This high level of sensitivity likely played a key role in our

53 ability to identify with high confidence Cypher, Ahnak1, Cavin-1, and CRYAB as new cardiac-specific dystrophin-associated proteins. Indeed, our Western blot results indicate that these proteins are not abundant in dystrophin immunoprecipitations, possibly explaining why they have not been previously detected. Finally, we downsized the amount of starting material required to 50 mg of tissue. The ability to study the interactome of dystrophin from small amounts of tissue is not of trivial importance. It enables the study of DAPC composition by proteomics in the mouse, an animal model for many human muscle disorders (Allamand and Campbell 2000; Vainzof, Ayub-Guerrieri et al.

2008). In combination with the spectral count analysis described here, this opens the door to future comparative studies in mouse models of muscular dystrophies aimed at understanding how disease-causing mutations may affect the composition of the DAPC. In addition, 50 mg is within the size range of a human tissue biopsy, suggesting that our protocol may be adapted in the future to directly study the DAPC in human muscles. Analysis of human cardiac samples may be particularly informative since our results with -dystrobrevin suggest a higher complexity of the human cardiac DAPC compared to the mouse.

The successful comparison of affinity purified dystrophin protein complexes between cardiac and skeletal muscles required a tissue-specific background strategy and was further made possible by a multi-factor quantitative bio-informatics approach applied to spectral counts. From a biological standpoint, our approach provided a global yet detailed view of the DAPC in cardiac and skeletal muscle that clearly revealed differences specifically affecting the

54 syntrophin-dystrobrevin sub-complex and its interaction with nNOS. The finding that nNOS is not part of the cardiac DAPC suggests that dystrophin is not involved in nNOS membrane localization in cardiomyocytes as it is in skeletal muscle fibers (Adams, Mueller et al. 2001; Lai, Thomas et al. 2009). This agrees with the reported interaction of nNOS with the ryanodine receptor in the heart and its spatial confinement to the sarcoplasmic reticulum (Barouch, Harrison et al. 2002) where dystrophin is absent. A link between cardiac dystrophin and nNOS has been postulated based on decreased nNOS activity in mdx hearts

(Bia, Cassidy et al. 1999; Chu, Otero et al. 2002) and on improved cardiac histopathology upon nNOS over-expression (Wehling-Henricks, Jordan et al.

2005). Our data favors a model where perturbations in cardiac nNOS activity are secondary to the loss of dystrophin, while in skeletal muscle deregulated nNOS function is a direct consequence of lack of dystrophin at the myofiber membrane

(Brenman, Chao et al. 1995; Percival, Anderson et al. 2010).

Our findings also raise the question of the identity of the proteins that interact with the cardiac syntrophin/dystrobrevin complex and the nature of the intracellular functions they may mediate. Given a general paucity of information on binding partners for syntrophins and dystrobrevins, we cannot at this time address the functional significance of the observed preferential inclusion of 2- syntrophin and 3-dystrobrevin in the cardiac DAPC. Interestingly, mice genetically engineered to lack 1-, 2- and 3-dystrobrevins show cardiomyocyte degeneration, inflammation and fibrosis indicating that - dystrobrevins are important for cardiac integrity (Grady, Grange et al. 1999). In

55 addition, in vitro studies have indicated that all three syntrophins can interact with and may regulate the activity of the cardiac sodium channel Nav1.5 which is disrupted in the dystrophin-deficient heart (Gavillet, Rougier et al. 2006).

Finally, our findings open the door to studies into the functional significance of the novel cardiac-specific association of dystrophin with Cavin-1,

Ahnak1, Cypher and CRYAB. These novel associations suggest a direct link between dystrophin and two major cardiac systems that are disrupted in the dystrophin-deficient heart: the regulation of ion channels that initiate and pace contraction, and the sarcomeric apparatus that ultimately mediates cardiac contraction. Indeed, the majority of dystrophinopathy patients have arrhythmias, long QT syndrome and contractile dysfunction (Kaspar, Allen et al. 2009), and therefore overlap in phenotype with patients with mutations in cypher, CRYAB,

Ahnak1, or Cavin-1 (Vatta, Mohapatra et al. 2003; Inagaki, Hayashi et al. 2006;

Reilich, Schoser et al. 2010; Shastry, Delgado et al. 2010; Sacconi, Feasson et al. 2011). Therefore, our proteomics findings suggest a novel molecular link between these different cardiac diseases that warrants further investigation.

2.5. Methods

2.5.1. Human ethics and cardiac biopsies.

Human cardiac biopsy material was obtained with informed written consent from guardians on behalf of the minor participants involved in the study under our approved IRB protocol (IRB07-00225). Surgeries were performed at

Nationwide Children’s Hospital. Discarded ventricular tissue from corrective cardiac surgery in infants (1 to 3 months old) diagnosed with Tetralogy of Fallot was used. Tetralogy of Fallot is a congenital heart defect that does not involve mutations in DAPC members and leads to an outgrowth of histologically normal ventricular tissue. Biopsies from two unrelated patients were analyzed in this study. For each patient, 100 mg of biopsy tissue were processed for one

MANDYS1 IP and one Control IP.

2.5.2. Animals.

C57BL/6J, dystrophin-deficient mdx5cv (referred to as mdx), nNOS knockout (KN1) (Huang, Dawson et al. 1993) and dystrobrevin knock-out (Grady,

Grange et al. 1999) mice between 12 and 20 weeks of age were used. Mice were provided with full access to food and water. Animal procedures were approved by the Institutional Animal Care and Use Committees at Nationwide Children’s

Hospital or the University of Washington.

2.5.3. Antibodies.

Anti-dystrophin (MANDYS1), isotype-matched control (MW8), and anti-β- dystroglycan (MANDAG2) antibodies were produced in-house from hybridoma cell lines (DSHB; University of Iowa) and concentrated using the Amicon ultra- filtration cell (Millipore). Antibodies to DAPC members are: isoform specific anti-

α1-, β1- or β2-syntrophin, and anti- α1- or α2-dystrobrevin antibodies (Peters,

Kramarcy et al. 1994; Peters, Adams et al. 1997; Peters, Sadoulet-Puccio et al.

1998); pan anti-syntrophin (ab11425, Abcam); Manex1011B to dystrophin and

MANDAG2 to β-dystroglycan (DSHB); clone IIH6C4 to α-dystroglycan (Upstate); anti-nNOS (#610308) and anti-α-dystrobrevin (#610766, BD Bioscience); anti-β- sarcoglycan (clone 5B1, Leica Microsystems).

2.5.4. Immunoprecipitations.

For mice, one quadriceps muscle and left and right cardiac ventricles were dissected per mouse, weighed and 100 mg of tissue were homogenized for protein extraction. This amount of tissue provided enough material for one experimental IP and one control IP from the same protein homogenate.

Quadriceps muscles were chosen for analysis because of their mixed fiber type composition. For human patients, cardiac biopsies of ventricular tissue were obtained from 2 patients and 100 mg of each were homogenized. Tissues from different mice or patients were not pooled. Tissues were homogenized 1:10 w/v in ice cold Buffer A (1% digitonin, 0.05% NP-40, NaCl 150mM, Tris 50mM, pH

7.4) with Complete Protease Inhibitors and PhosSTOP (Roche Diagnostics) using a polytron homogenizer (Power Gen 700, Fisher Scientific). Proteins were extracted on ice for 1 hr, centrifuged at 80,000 x g for 30 min and supernatant was pre-cleared with protein G agarose beads (Invitrogen). Protein concentration was determined with the Dc Protein Assay (Bio-Rad). MANDYS1, MANDAG2, or

MW8 control antibodies were incubated with Dynal protein G magnetic beads

(Invitrogen; 1.2 g antibody per 1 l beads) in 100 mM sodium phosphate (pH

5.0) overnight at 4 °C. Antibodies were cross-linked to the beads by incubation in

0.2 M Triethanolamine containing 20 mM dimethyl pimelimidate for 30 min at 20

°C. Antibody conjugated beads were incubated with 2-5 mg protein at 4 °C for 3 hr, washed in ice cold Buffer A without digitonin, and proteins were eluted in

Laemmli Reducing Sample Buffer for Western blot analysis or in 2% SDS, 100 mM DTT for LC-MS/MS analysis.

For reverse immunoprecipitation, samples were prepared as above.

Antibodies used were anti-Cavin-1 (ab48824, Abcam), anti-Ahnak1 (EM-09,

Cedarlane), anti-CRYAB (ab13496, Abcam), and anti-Cypher (ab40840, Abcam).

The controls for Ahnak and CRYAB immunoprecipitations used equal amounts of species matched MW8 antibody as previously mentioned. For Cavin-1 and

Cypher immunoprecipitations, species matched, non-specific rabbit (#305-005-

003, Jackson ImmunoResearch) and goat (#111-005-003, Jackson

ImmunoResearch) antibodies were used, respectively.

2.5.5. LC-MS/MS.

Proteomic analysis was performed on 2 MDX-IPs (2 biological replicates), and 3 DYS-IPs and their corresponding 3 IgG-IPs (3 biological replicates) from quadriceps and cardiac muscle samples. Eluted proteins were chloroform/methanol precipitated, resuspended in 5X Invitrosol protein solubilizer

(Invitrogen), diluted with 25 mM ammonium bicarbonate to a final volume of 1X

Invitrisol. The proteins were then reduced with 10 µL DTT (5 mg/mL solution in

100 mM ammonium bicarbonate) and carbamidomethylated with 10 µL

Iodoacetimide solution (15 mg/mL in 100 mM ammonium bicarbonate). Trypsin

(in 50 mM ammonium bicarbonate) was added to the protein solution with an enzyme to substrate ratio of 1:25 (w/w). Samples were incubated for 2 hr at 37

°C before quenching by acidification. Capillary-liquid chromatography-nanospray tandem mass spectrometry was performed on a Thermo Finnigan LTQ orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose CA) equipped with a microspray source (Michrom Bioresources Inc, Auburn, CA) operated in positive ion mode. Samples were loaded onto a precolumn Cartridge (Dionex, Sunnyvale,

CA) and desalted with 50 mM acetic acid for 10 minutes, then separated on the capillary column (0.2X150mm Magic C18AQ 3µ 200A, Michrom Bioresources

Inc, Auburn, CA) using an UltiMate™ 3000 HPLC system from LC-Packings A

Dionex Co (Sunnyvale, CA). Mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Flow rate was 2 µl/min.

Mobile phase B was increased from 2% to 50% in 250 min, then from 50% to

90% in 5 min, then kept at 90% for another 5 min The column was equilibrated at

2% of mobile phase B (or 98% A) for 30 min before the next sample injection.

MS/MS data was acquired with a spray voltage of 2 KV and a capillary temperature of 175 °C. The scan sequence of the mass spectrometer was based on the TopTen™ method. The full scan mass resolving power was set at 30,000 to achieve high mass accuracy MS determination. The CID fragmentation energy was set to 35%. Dynamic exclusion is enabled with a repeat count of 3 within 30 s, a mass list size limit of 200, exclusion duration of 350 s and a low mass width of 0.50 and high mass width of 1.50 Da.

2.5.6. Peptide sequence analysis.

The RAW data files collected on the mass spectrometer were converted to mzXML and MGF files by use of MassMatrix data conversion tools (version 1.3, http://www.massmatrix.net/download). Isotope distributions for the precursor ions of the MS/MS spectra were deconvoluted to obtain the charge states and monoisotopic m/z values of the precursor ions during the data conversion.

Resulting .mgf files were searched using Mascot Daemon (version 2.3.2, Matrix

Science, Boston, MA) against UniprotKBSwiss mouse database (version072711,

55,744 protein sequences). Trypsin was used as the enzyme and three missed cleavages were permitted. Considered variable modifications were oxidation

(Met) and carbamidomethylation (Cys). The mass accuracy of the precursor ions was set to 10ppm and the fragment mass tolerance to 0.5 Da. Accidental picking of one 13C peak was included into the search. The significance identity threshold was set at p<0.05 for valid protein identification. False discovery rates (FDR) for peptide matches were estimated using the target-decoy search strategy (Elias and Gygi 2007; Elias and Gygi 2010). All reported results are for peptides with less than 5% FDR except for one (heart control 2, which was 8.1%). Proteins with a Mascot mowse score of 25 or higher containing a minimum of one unique peptide with a -b or -y ion sequence tag of five residues or better were accepted.

However, identifications of proteins with one unique peptide were considered to be true positives only if: 1) the precursor ion has correct charge status and the mass accuracy is <10ppm. 2) b and -y ion sequential tag of five or more residues were present following manual validation of MS/MS spectra.

2.5.7. Label-free quantitation.

Label free quantitation was performed using the spectral count approach

(Liu, Sadygov et al. 2004; Colinge, Chiappe et al. 2005) in which the relative protein quantitation is measured by comparing the number of MS/MS spectra identified from the same protein in each of the multiple LC/MSMS datasets. To evaluate statistically significant differences in protein abundances, the MS/MS data for each treatment and its technical replicates were combined using an in- house developed application into parsimonious protein lists. Differential expression of protein between the IgG-IP and DYS-IP were determined by analysis of the spectral count data using the edgeR bioconductor package

(Robinson, McCarthy et al. 2010). Peptide spectral counts were modeled as an overdispersed Poisson/negative binomial distribution in which an empirical Bayes procedure was used to moderate overdispersion across each protein. An exact text for overdispersed data was then used to assess difference in protein abundance (Robinson and Smyth 2008).

2.5.8. Immunolabeling.

Heart and quadriceps were flash frozen in isopentane cooled in liquid nitrogen and 4-8 μm serial sections were cut. Cardiac ventricular myocytes were isolated from 12-16 week old mice (Santana, Kranias et al. 1997) and fixed with

0.5% paraformaldehyde in PBS. Immunofluorescence labeling of tissue sections

62 and cardiomyocytes was performed as described (Peters, Adams et al. 1997).

Images were captured using an Olympus BX61 microscope or a Leica TCS-NT confocal microscope.

2.5.9. Immunoblots.

Proteins separated on 4-12% gradient SDS-PAGE gels (Invitrogen) were transferred to nitrocellulose membranes (Whatman), blocked with 5% skim milk in 0.1% Tween 20/Tris-buffered saline and incubated with primary antibodies.

Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and enhanced chemiluminescence reagents (Pierce). Signal was detected on X-Ray film (RPI).

2.5.10. Densitometric analysis.

Protein band intensities from multiple non-saturated film exposures were quantified using ImageJ (NIH). Values in the linear range of pixel intensities were selected for quantifications. Samples to be compared (cardiac versus skeletal muscle, wild type versus dystrophin-deficient) were run side by side on the same gel. For immunoprecipitations, the membrane was cut at the 200 kDa marker to simultaneously probe for dystrophin (top portion) and syntrophins or dystrobrevins (bottom portion). Signal intensities were normalized to the dystrophin signal. For total protein lysates, membranes were probed for the protein(s) of interest then stripped and re-probed for GAPDH. Band intensities

63 were normalized to the GAPDH signal. Blots from 3 biological replicates were analyzed by densitometry for each protein.

2.6. Tables

Skeletal Muscle Heart

UniProt Protein Expt. Score % Pep. Score % Pep. ID Cov. # Cov. #

Dystrophin P11531 1 19064 51.9 167 35126 63.4 207 2 13864 42.2 125 17334 49.9 194

3 22567 52.5 158 16027 45 149

*Dystroglycan Q544G5 1 477 5.7 5 285 10.2 7 2 55 3.7 2 37 2 1

3 584 11.5 8 39 0.9 1

-Dystrobrevin E9QJX4 1 1388 27.7 14 2333 45.8 20 2 926 25.5 10 572 21.1 11

3 1674 30.9 16 522 17.6 10

Q9Z0J4- nNOS 1 11.3 12 - - - 5 2 81 2.7 2 - - -

3 86 1.5 2 - - -

-Syntrophin A2AKD7 1 1610 53.5 16 2134 47.1 19 2 1587 42.3 12 1328 62 19

3 2429 52.9 18 1698 43.8 17

-Syntrophin Q99L88 1 194 17.1 8 - - - 2 - - - 134 10.2 4

3 178 11.6 6 418 21 10

-Syntrophin Q542S9 1 - - - 228 13.1 4 2 - - - 356 12.3 5

3 - - - 469 13.3 7

* -Sarcoglycan Q5SWB2 1 715 26.4 8 963 32.3 10 2 28 3.8 1 311 17.3 5

3 328 25.7 8 467 20.1 7

* -Sarcoglycan P82349 1 779 40.4 9 1074 40.4 8 2 35 6.9 1 263 22.4 4

3 396 23.4 5 619 24.9 5

-Sarcoglycan P82347 1 548 27.1 7 1412 33.6 8 2 559 23 6 337 19.9 6

3 559 23 6 614 21.9 6

-Sarcoglycan P82348 1 428 19.2 5 366 16.8 4 2 201 11.3 2 310 14.4 4

3 113 12 3 194 12 3

*Sarcospan E9Q8Y7 1 59 6.3 1 93 6.3 1 2 - - - 49 7.7 1

3 68 6.3 1 133 14 2

Table 2.1: Identification of DAPC members in DYS-IPs by LC-MS/MS. Results from three independent experiments (biological replicates) are shown for each protein. UniProt ID: UniProt protein identifier; Score: Mascot protein score; % Cov: Percent protein sequence coverage; Pep. #: Number of unique peptides with individual peptide score >30. * For additional information for proteins identified by one unique peptide see Figure 2.8, Figure 2.9.

Heart spectral counts Skm spectral counts IgG-IPs DYS-IPs IgG-IPs DYS-IPs Uniprot ID Protein Name 1 2 3 1 2 3 p value 1 2 3 1 2 3 p value P11531* Dystrophin 0 0 0 1163 642 501 7.66E-08 3 0 0 621 490 917 1.09E-07 A2AKD7* -Syntrophin 0 0 0 79 51 59 4.00E-05 0 0 0 55 45 105 4.38E-06 E9QJX4* -Dystrobrevin 0 0 0 80 25 24 1.63E-04 0 0 0 49 45 73 7.21E-06 P82347* -Sarcoglycan 0 0 0 39 14 16 6.40E-04 1 0 0 18 11 36 1.45E-03 Q5SWB2* -Sarcoglycan 0 0 0 34 9 14 1.06E-03 0 0 0 22 2 27 6.40E-04 Q9Z0J4-5* nNOS 0 0 0 28 5 9 8.01E-04 O54724 Cavin-1 0 0 0 14 8 35 1.01E-03

67 P82349* -Sarcoglycan 0 0 0 27 8 17 1.17E-03 0 0 0 24 2 15 1.05E-03

P82348* -Sarcoglycan 0 0 0 15 7 10 3.16E-03 0 0 0 15 6 8 1.45E-03 Q99L88* -Syntrophin 0 0 0 0 5 23 1.33E-02 0 0 0 17 0 15 2.02E-03 Q542S9* -Syntrophin 0 0 0 7 13 14 2.91E-03 Q52L78 CRYAB 0 0 0 7 12 8 4.85E-03 Q9JKS4-3 Isoform 3 of Cypher 0 0 0 3 3 13 1.13E-02

F7BRM2 AHNAK1 0 0 0 2 2 10 1.98E-02 Q544G5* Dystroglycan 0 0 0 14 1 1 2.99E-02 3 0 0 13 4 28 2.09E-02

Table 2.2: Comparison of proteins between cardiac and skeletal muscle IPs identified by multi-factor analysis of spectral counts. Spectral counts are shown for three independent biological replicates for DYS-IP and corresponding control IgG-IPs from cardiac and skeletal muscles. Proteins listed were enriched in DYS-IP with a p value ≤ 0.03. *Known DAPC members.

2.7. Figures

Figure 2.1: MANDYS1 specifically immunoprecipitates dystrophin and associated DAPC members. A. Schematic representation of the core DAPC in skeletal muscle. B. MANDYS1 does not recognize utrophin (Utr). Western blot of lysates and DYS-IPs from cardiac (C) and skeletal (S) muscle probed for utrophin, then stripped and re-probed for dystrophin (Dys). C. - and - dystroglycan ( -DG, -DG) are detected in DYS-IPs but not control IgG-IPs. D. Syntrophins (Syn) and -dystroglycan in DYS-IP from wild type but not mdx skeletal muscle or in IgG-IPs. Dystrophin is depleted in Post-IP lysates from WT muscle.

Figure 2.2: Immunoprecipitation strategy and background reduction. A. Effect of antibody cross-linking to beads on the contamination of immunoprecipitation samples by immunoglobulins. Western blot analysis of indicated DAPC members and immunoglobulins present in dystrophin (DYS) immunoprecipitations using MANDYS1-conjugated beads that were pre-treated (+) or not (-) with a cross-linking agent. Antibody bands (IgG) obscure syntrophin (Syn) and -dystroglycan ( -DG) detection in the absence of cross-linker. B. Antibody cross-linking eliminates contamination of immunoprecipitated proteins by immunoglobulins. No large IgG bands are seen at 50kDa and 25kDa after Deep Purple total protein dye staining of SDS-Page gel. IP: proteins eluted following skeletal muscle dystrophin immunoprecipitation. L: Molecular weight ladder. C. Experimental design. Proteins were extracted from quadriceps muscle from wild type (WT) and dystrophin-deficient mdx mice. The MANDYS1 antibody to dystrophin and an isotype matched control antibody (IgG Control) were cross- linked to magnetic G-protein beads for immunoprecipitations. As an additional control for non-specific protein binding, MANDYS1 immunoprecipitations were also carried out on muscle tissue extracts from mdx mice. Bound proteins were eluted and processed for LC-MS/MS or immunoblots.

Figure 2.3: nNOS does not associate with full-length dystrophin in cardiomyocytes. A. Western blot analysis of DYS-IPs and IgG-IPs from wild type cardiac (C) and skeletal (S) muscle showing lack of nNOS detection in cardiac DYS-IP but presence of 1-syntrophin ( 1-syn). B. Immunolabeling of wild type (WT) and nNOS knock-out (nNOS-/-) cardiomyocytes for nNOS (green) and dystrophin (red) shows lack of co-localization. Arrows indicate non-specific labeling. C. Peptide coverage (blue amino acids) by LC-MS/MS of spectrin repeats 16 and 17 of cardiac dystrophin.

Figure 2.4: Syntrophins differ between cardiac and skeletal muscle DAPC. A. Western blot analysis of syntrophins in mouse cardiac (C) and skeletal (S) muscle protein lysates, DYS-IPs and IgG-IPs. 2-syntrophin ( 2-syn) associates with dystrophin only in the heart. Fold differences in syntrophin abundance in cardiac vs. skeletal muscle DYS-IPs relative to dystrophin are shown (averages ± SD, N=3 biological replicates). B. Western blot analysis of DYS-IPs from human (H) and mouse (M) cardiac samples showing association of 2-syntrophin with dystrophin in the human heart. C. Immunolabeling of cardiac sections from wild type mice for indicated proteins. Scale bar: 50 m. Arrows indicate blood vessels.

Figure 2.5: Immunolabeling for dystrophin, nNOS, syntrophins and dystrobrevins in wild type skeletal muscle. Immunolabeling of quadriceps muscle sections for dystrophin (DYS), 1-syntrophin ( 1-Syn), 1-syntrophin ( 1- Syn), nNOS, 1 and 2-dystrobrevin (DTNA1, DTNA2) (red) and DAPI (blue). Scale bar= 50 mm.

Figure 2.6: Differences in -dystrobrevin splice variants between cardiac and skeletal muscle DAPC. A. Western blot analysis of -dystrobrevins (DTNA) in mouse cardiac (C) and skeletal (S) muscle total protein lysates, DYS- IPs and IgG-IPs. 3-dystrobrevin (DTNA3) associates with dystrophin in the heart. Fold differences in -dystrobrevin abundance in cardiac vs. skeletal muscle DYS-IPs relative to dystrophin are shown (averages ± SD, N=3 biological replicates). B. Immunolabeling of wild type cardiac sections for 1 and -dystrobrevins. Scale bar: 50 m. C. Immunolabeling of mdx cardiac tissue section for -sarcoglycan. Scale bar: 50 m. D. Western blot analysis of - dystrobrevins in heart protein lysates from wild type (WT) and mdx mice. Fold differences in -dystrobrevin abundance in WT vs. mdx cardiac lysates relative to GAPDH are shown (averages ± SD, N=3 biological replicates). E. Western blot analysis of -dystrobrevins in DYS-IPs from human (H) and mouse (M) cardiac samples. Additional -dystrobrevin isoforms (arrow heads) are detected in human cardiac lysates and DYS-IP.

Figure 2.7: Novel cardiac-specific dystrophin-associated proteins. A. Western blot analysis of Cavin-1, Ahnak1, CRYAB, and Cypher in mouse cardiac and skeletal muscle lysates, DYS-IPs and IgG-IPs. B. Western Blot analysis of dystrophin in reverse immunoprecipitations from wild type cardiac muscle with antibodies to Cavin-1, Ahnak1, CRYAB, or Cypher. Control (Ctr) immunoprecipitations were species matched, not directed against muscle proteins. C. Cavin-1 and Ahnak1 co-purify with dystrophin in human cardiac samples. D. Western blot analysis of Cavin-1 and Ahnak1 in MANDAG2-IPs from wild type (WT) and mdx cardiac muscle. Expression levels of Cavin-1 and Ahnak1 are not decreased in mdx lysates compared to WT.

Figure 2.8: Mass spectra for DAPC members identified by a single peptide in cardiac DYS-IPs. Peaks matching the theoretical fragments ions are labeled as y ions ( green), b ions (red) and b* ions (blue). As shown in the spectra, precursor ions have correct charge status and the mass accuracy is <2.5ppm; the presence of b and - y ion sequential tag of five or more residues were also observed in the MS/MS spectra of these unique peptides. b* ions = nb-H20.

Figure 2.9: Mass spectra for DAPC members identified by a single peptide in skeletal muscle DYS-IPs. Peaks matching the theoretical fragments ions are labeled as y ions ( green), b ions (red). As shown in the spectra, precursor ions have correct charge status and the mass accuracy is <4.5ppm; the presence of b and -y ion sequential tag of five or more residues were also observed in the MS/MS spectra of these unique peptides.

Chapter 3: Identification of novel dystrophin-associated proteins links

dystrophin to membrane repair in the diaphragm.

3.1. Abstract

Dystrophin is the central organizer of a large protein complex in striated muscle which is essential for proper muscle function and integrity. Loss of dystrophin gives rise to a severe form of muscular dystrophy, Duchenne muscular dystrophy (DMD) which is the most common type of all muscular dystrophies. DMD is characterized by progressive muscle weakness and replacement of muscle with fibrotic and fat tissue. Ultimately DMD patients succumb to the disease in their second to third decade of life, where respiratory failure is the primarily cause of morbidity and mortality.

Similar to patients, the mdx mouse model for DMD, is characterized by muscle weakness and necrosis although symptomatically milder. The sole exception is the diaphragm which is the only muscle in the mouse that closely resembles the severe pathology seen in humans. Unfortunately, the reasons for this discrepancy are poorly understood. Using an immunoprecipitation approach, we identify here novel diaphragm specific protein interactions of dystrophin with cavin-1 and ahnak1, proteins recently suggested to function in membrane repair, and show that the localization of these proteins are specifically disrupted only in

77 the mdx diaphragm. Furthermore, we show that disruption of these proteins is further accompanied by a diaphragm specific disruption of dysferlin, an additional protein involved in membrane repair in skeletal muscle. Together, our study identified for the first time tissue specific differences in the protein associations of dystrophin between limb and diaphragm muscles. The association of these proteins with dystrophin and their disruption along with dysferlin suggests a new potential function of dystrophin in membrane repair in the diaphragm, and provides a likely explanation for the more severe phenotype observed in the mdx diaphragm.

3.2 Introduction

Duchenne muscular dystrophin (DMD) is the most common lethal neuromuscular disease caused by mutations in the DMD gene, the gene responsible for the dystrophin protein (Hoffman, Brown et al. 1987). Clinically, patients with DMD present with muscle weakness in their first decade of life.

Muscle weakness is progressive leading to loss of ambulation in the second decade of life primarily due to weakness of the proximal muscles. Pathologically,

DMD is characterized by chronic muscle degeneration and progressive replacement of muscle fibers with non-contracting fibrotic and fat tissue.

Ultimately, severe weakness and necrosis leads to loss in muscle function, and respiratory failure accounts for the leading cause of morbidity and mortality, typically in the third decade of life (Simonds 2002; Kaspar, Allen et al. 2009).

The genetic mouse model for DMD, the dystrophin deficient mdx mouse lacks dystrophin expression and is characterized by muscle weakness and degeneration of muscle fibers, although symptomatically milder compared to humans (Bulfield, Siller et al. 1984; Stedman, Sweeney et al. 1991). The exception to the mild phenotype observed in the mouse is the dystrophic diaphragm. The mdx diaphragm is characterized by progressive muscle degeneration and accumulation of fibrotic tissue (Stedman, Sweeney et al.

1991). Indeed, the diaphragm is the only muscle in the mdx mouse that closely resembles the pathology seen in DMD, and is therefore of particular interest for studying the mechanisms of disease (Stedman, Sweeney et al. 1991; Dupont-

Versteegden and McCarter 1992). Unfortunately, the reasons for the more severe phenotype observed in the mdx diaphragm remain unclear. However, studies directed towards understanding why the diaphragm is more severely affected may help explain the severity and disease mechanisms present in patients with DMD.

Dystrophin is a large cytoskeletal protein localized to the intracellular side of the plasma membrane in skeletal and cardiac muscle, and is an essential component of the muscle membrane. In striated muscle, dystrophin is the central organizer of a large protein complex known as the dystrophin-associated protein complex (DAPC) (Ervasti and Sonnemann 2008). Through the DAPC, dystrophin functions as a scaffolding protein by linking the actin cytoskeleton to the extracellular matrix, and thus protects the membrane from the shear forces generated by muscle contraction. Mutations disrupting dystrophin expression

79 results in a secondary loss of the DAPC perturbing the link from the cytoskeleton to the extracellular matrix. Absence of dystrophin therefore, results in membrane fragility and subsequent damage of the muscle membrane ultimately leading to muscle fiber necrosis. The functions of dystrophin, however are not limited solely to structural maintenance. Further studies have highlighted additional roles of dystrophin including signaling which is mediated through association with intracellular DAPC proteins, primarily the syntrophins and dystrobrevins (Albrecht and Froehner 2002). Therefore dystrophin is a multifunctional protein where the complete functions of dystrophin have likely not been fully elucidated.

Recently, we describe a novel proteomics approach for the identification of dystrophin-associated proteins (Johnson, Zhang et al. 2012). From that study, we identified novel dystrophin associated proteins that interact with dystrophin in the mouse and human heart but not mouse limb muscle. Despite their cardiac specific association, these proteins are highly expressed in skeletal muscle, and when disrupted have either been shown to directly cause or are associated with muscular dystrophy with overlapping symptoms to DMD (Selcen and Engel 2005;

Hayashi, Matsuda et al. 2009; Del Bigio, Chudley et al. 2011; Zacharias, Purfurst et al. 2011). Because of this we asked here if any of these proteins associate with dystrophin in the mouse diaphragm representing novel differences in the diaphragm DAPC compared to limb muscle. Identification of diaphragm specific interactions of dystrophin may explain why the diaphragm is more affected in the mdx mouse.

3.3. Results

3.3.1. The diaphragm has unique protein interactions not present in quadriceps muscle.

Previously we have described the association of novel proteins with dystrophin in the heart but not limb muscles (Johnson, Zhang et al. 2012).

Among them are ahnak1 and cavin-1 two proteins that have been shown to be important for membrane repair in skeletal muscle (Huang, Laval et al. 2007; Zhu,

Lin et al. 2011). Based on the more severe pathology present in the mdx diaphragm, we tested whether these proteins may differentially associate with dystrophin between diaphragm and limb muscles. Consistent with or previous study, immunoprecipitation of dystrophin from quadriceps muscle does not co- purify cavin-1 and ahnak1 (Figure 3.1) (Johnson, Zhang et al. 2012). The quadriceps muscle was chosen because it is a mixed fiber type muscle with similar proportions of slow and fast twitch fibers. However a similar result was also obtained from the gastrocnemious muscle (data not shown). By contrast, both ahnak1 and cavin-1 co-purify with dystrophin in the diaphragm indicating a diaphragm specific association (Figure 3.1). Importantly these proteins were not identified in control immunoprecipitations using an isotype matched non-specific antibody. Interestringly both cavin-1 and ahnak1 are expressed at comparable levels in the diaphragm and quadriceps muscles (Figure 3.1). Therefore, the association of cavin-1 and ahnak1 with dystrophin is not due to differences in total protein expression. Thus the specific association of cavin-1 and ahnak1 with

81 dystrophin in the diaphragm represents unique differences in the composition of the DAPC compared to limb muscle.

3.3.2. Co-localization of cavin-1 and ahnak1 with dystrophin in quadriceps and diaphragm.

Since differences in protein expression were not detected, we asked whether the localization of cavin-1 and ahnak1 differs between quadriceps and diaphragm muscles. Specifically we focused on localization of these proteins at the neuromuscular junction (NMJ) and the sarcolemmal membrane, the two cell structures where dystrophin is highly expressed. NMJs were identified by co- staining tissue sections with α-bungarotoxin (BTX) which specifically labels acetylcholine receptors. By immunofluorescence on tissue sections cavin-1 was highly enriched at the NMJ in wild type diaphragm and quadriceps muscles

(Figure 3.2). In the mdx mouse, loss of dystrophin disrupts the localization of cavin-1 at the NMJ in the diaphragm. Surprisingly, no disruption was observed in the mdx quadriceps highlighting a tissue specific disruption at NMJs. For ahnak1, faint although present staining was observed at the NMJ in both diaphragm and quadriceps sections (Figure 3.2). Similar to cavin-1, the localization of ahnak1 at the NMJ was disrupted in the mdx diaphragm but not quadriceps. Therefore, although both proteins co-localize with dystrophin at the NMJ the localization of cavin-1 and ahnak1 is disrupted only in the diaphragm.

In addition to the NMJ, cavin-1 localizes to the sarcolemmal membrane in wild type diaphragm and quadriceps muscle (Figure 3.3). In the mdx mouse, the

82 localization of cavin-1 was significantly disrupted at the sarcolemmal membrane in the diaphragm similar to what was observed at the NMJ. Additional interstitial punctate staining for cavin-1 was present in wild type tissue sections and was not disrupted in the mdx mouse (arrowheads; Figure 3.3). This is consistent with the expression of cavin-1 in non-muscle cells including endothelial cells(Davalos,

Fernandez-Hernando et al. 2010). Importantly, full length dystrophin does not co- localize with this interstitial staining, and as expected no loss in localization of cavin-1 was observed in the interstitium. In the mdx quadriceps cavin-1 immunofluorescence was clearly present and continuous throughout the entire sarcolemmal membrane although reduced. Similar to the diaphragm, punctate interstitial staining was present in the wild type quadriceps and preserved in the mdx.

Localization of ahnak1 was also present at the sarcolemmal membrane in both the wild type diaphragm and quadriceps tissue sections (Figure 3.4).

However, loss of dystrophin results in a significant loss in localization at the membrane of diaphragm muscle fibers. Continuous, although reduced membrane staining was detected in the mdx quadriceps. In the mdx diaphragm and quadriceps, strong staining for ahnak1 was observed in the interstitial space

(Figure 3.4; arrows). This is consistent with the expression of ahnak1 in the interstitial space in dystrophic skeletal muscle (Zacharias, Purfurst et al. 2011), and was not present in wild type muscles. Overall, the disruption of cavin-1 and ahnak1 at both the NMJ and sarcolemmal membrane in the diaphragm is

83 consistent with their selective association with dystrophin in diaphragm muscle lysates by immunoprecipitation (Figure 3.1).

3.3.3. Localization of cavin-1 and ahnak1 is not disrupted in skeletal muscle of patients with DMD.

The diaphragm is the only muscle in the mdx mouse that closely resembles the pathology seen in patients with DMD. We therefore asked if the association of cavin-1 and ahnak1 with dystrophin in the mouse diaphragm more closely resembled human limb muscle than the mouse. By immunofluorescence on human control tissue sections both cavin-1 and ahnak1 localize to the muscle fiber membrane similar to the mouse (Figure 3.5). However, in the absence of dystrophin, no disruption in localization was observed at the membrane for either cavin-1 or ahnak1 on DMD tissue sections. All sections were co-stained with

BTX, however no NMJs were observed, and therefore the specific disruption of these proteins at the NMJ could not be determined (data not shown). Therefore, the association of cavin-1 and ahnak1 appears to be specific to the mouse diaphragm. No human diaphragm biopsies were available.

3.3.4. Loss of dystrophin leads to disruption in the localization of dysferlin.

Recently ahnak1 and cavin-1 have been suggested to function in the membrane repair pathway in skeletal muscle (Huang, Laval et al. 2007; Cai,

Weisleder et al. 2009; Zhu, Lin et al. 2011). This has been suggested to be mediated through interaction with the dysferlin protein (Huang, Laval et al. 2007;

Cacciottolo, Belcastro et al. 2011). Dysferlin is a critical component of membrane repair pathway and mutations in the dysferlin gene gives rise to a type of muscular dystrophy referred to as dysferlinopathy with has overlapping phenotype with DMD (Han and Campbell 2007). Furthermore, double knockout of dysferlin and dystrophin leads to a more severe pathology than dystrophin alone(Han, Rader et al. 2011). Based on the disruption of both ahnak1 and cavin-

1, we asked if dysferlin was also disrupted in the mdx diaphragm. Surprisingly, by immunofluorescence, the localization of dysferlin is disrupted with loss of membrane staining in some fibers and decreased membrane staining accompanied by intense intracellular staining in other fibers (Figure 3.5). In wild type diaphragm muscle fibers dysferlin was highly expressed at the membrane with no intracellular staining . Finally, no disruption of dysferlin was observed in the mdx quadriceps consistent with previous reports (Ho, Post et al. 2004; Han,

Rader et al. 2011), and therefore disruption in dysferlin localization was unique to the diaphragm.

3.4. Discussion

While much is known about the role of dystrophin in striated muscle, the complete functions and binding partners of dystrophin have likely not fully been elucidated. In order to fully understand the roles of dystrophin in muscle, a better appreciation of the dystrophin interactome is essential.

The study presented here identified two novel proteins that selectively associate with dystrophin in the mouse diaphragm compared to quadriceps muscles. Interestingly we show that although these proteins are expressed in both quadriceps and diaphragm muscles with relatively comparable amounts, their association with dystrophin and subsequent disruption in the mdx mouse is specific to the diaphragm. Unfortunately, the reasons for this difference remain unknown. However, coupled with a previous study identifying differences in protein interactions of dystrophin between the heart and skeletal muscle

(Johnson, Zhang et al. 2012), it is becoming clear that the protein interactions of dystrophin vary between different tissues, even between different skeletal muscles. Because dystrophin functions are mediated through its interacting proteins, the tissue specific differences in the dystrophin interactome likely impacts the functions of dystrophin for each tissue type. Thus our understanding of the molecular mechanisms leading to muscle dystrophy is likely further complicated by tissue specific difference. Our findings presented here raise important questions as to the functions of these novel interactions in the diaphragm.

It is well established that dystrophin is essential for protecting membrane integrity, and that loss of dystrophin leads to membrane fragility and micro tears of the muscle membrane. The novel finding that dysferlin is disrupted in the mdx diaphragm represents the first time that dystrophin has been linked to membrane repair. Therefore, the disruption of dysferlin in the diaphragm likely represents a compounding phenotype where absence of dystrophin not only affects

86 membrane integrity, but further disrupts the membrane repair pathway needed for proper sealing of the membrane upon damage. This dual function of dystrophin represents an attractive model for the more severe phenotype observed in the mdx diaphragm and future studies will be aimed at defining the functions of dystrophin in membrane repair in the diaphragm.

In mdx mice, the diaphragm is the most affected muscle. It has been speculated that the reason for this dramatic difference compared to limb muscles is due to the either constant contraction needed for proper reparatory function

(Louboutin, Fichter-Gagnepain et al. 1993), reduced regenerative capacity

(Anderson, Garrett et al. 1998; Matecki, Guibinga et al. 2004), or increase inflammation (Demoule, Divangahi et al. 2005). However, our finding identifying diaphragm specific associations of ahnak1 and cavin-1 suggest that the possible lack in correlation between disease severities may be due to disruption of unique tissue specific protein interactions. Along these same lines, certain groups of muscles including the extraocular muscles in the mdx mice are relatively spared from disease pathology. While the complete mechanisms for this sparing in unknown, it has been shown using a double knockout mouse for dystrophin and its homolog utrophin that one potential mechanism for protection is due to higher levels of utrophin expression compared to other muscles (Porter, Rafael et al.

1998). While this provides evidence for unique protein interactions for sparing of skeletal muscle, our findings suggest tissue specific disruption of dystrophin- associated proteins can further worsen disease severity. Furthermore, our results represent the first time differences in dystrophin associations between different

87 skeletal muscles tissues have been identified, and provide a potential explanation for the differences in disease severity observed between the diaphragm and limb muscle of the mdx mouse. More studies are needed to elucidate the functions of these novel proteins in the diaphragm, and the consequence of their disruption in the mdx mouse.

3.5 Methods

3.5.1. Human ethics and tissue biopsies.

Human skeletal muscle material was obtained with informed written consent under our approved IRB protocol (IRB0502HSE046). Biopsies from two unrelated healthy patients and two patients with DMD were analyzed in this study. DMD patients were first diagnosed based on DNA sequencing.

3.5.1. Animals.

C57BL/6J wild type and dystrophin-deficient mdx5cv (referred to as mdx) mice between 12 and 20 weeks of age were used. Mice were provided with full access to food and water. Animal procedures were approved by the Institutional

Animal Care and Use Committees at Nationwide Children’s Hospital.

3.5.2 Antibodies.

Anti-dystrophin (MANDYS1) and isotype-matched control (MW8) antibodies were produced in-house from hybridoma cell lines (DSHB; University

88 of Iowa) and concentrated using the Amicon ultra-filtration cell (Millipore).

Antibodies for Western blot and immunohistochemistry include: anti-dystrophin

(clone MANDAG2, DSHB), anti-cavin-1 (ab48824, Abcam), anti-ahnak1 (clone

EM-09, Cedarlane), anti-αB-crystallin (ADI-SPA-223-D, Enzo Life Sciences), and anti-cypher (ab40840, Abcam).

3.5.3. Immunoprecipitations.

Immunoprecipitations were performed essentially as in chapter II. Briefly, quadriceps and diaphragm muscle were dissected, weighed and 100 mg of tissue were homogenized for protein extraction. This amount of tissue provided enough material for one experimental IP and one control IP from the same protein homogenate. Quadriceps muscles were chosen for analysis because of their mixed fiber type composition. Tissues were homogenized 1:10 w/v in ice cold homogenate buffer (1% digitonin, 0.05% NP-40) with protease and phosphotase inhibitors (Roche Diagnostics) using a polytron homogenizer

(Power Gen 700, Fisher Scientific). Proteins were extracted on ice for 1 hr and centrifuged to remove insoluble material. The resulting supernatant was pre- cleared with protein G agarose beads (Invitrogen), and protein concentrations were determined with the Dc Protein Assay (Bio-Rad). MANDYS1 or MW8 control antibodies were incubated with Dynal protein G magnetic beads in sodium phosphate buffer (pH 5.0) overnight at 4 °C, followed by cross-linked to immunoprecipitation beads. Antibody conjugated beads were incubated with 2-5 mg protein at 4 °C for 3 hr, washed in ice cold homogenate buffer without

89 digitonin. Following the last wash, proteins were eluted in Laemmli Reducing

Sample Buffer for Western blot analysis.

3.5.4. Immunoblots.

3.5.5. Immunohistochemistry.

Freshly dissected quadriceps and diaphragm from wild type and mdx mice were flash frozen in liquid nitrogen cooled isopentane, and 8 μm sections were cut. Sections were then fixed with 0.5% PFA for 15 min (2% for cypher) and permeabilized with 1% Triton-X (0.2% SDS in PBS for cypher). Sections were then blocked with 10% horse serum followed by an antibody block with rabbit anti-mouse fab fragment (315-007-003, Jackson Immuno) to block endogenous immunoglobulins. Primary antibodies were diluted in block and sections were incubated overnight at 4 °C. Following washing with PBS, appropriate secondary antibodies were diluted with fluorescently conjugated α-bungerotoxin. Images were captured using an Olympus BX61 microscope.

3.6. Tables

Diaphragm Quadriceps

wild type mdx wild type mdx Mem NMJ Mem NMJ Mem NMJ Mem NMJ

ahnak1 ++ + - - ++ + + +

cavin-1 ++ ++ - - ++ ++ + ++

αB-crystallin + ++ + - ++ ++ ++ -

cypher - - - - - ++ - -

Table 3.1: Localization of novel proteins in wild type and mdx skeletal muscle. Results from immunofluorescence on wild type and mdx diaphragm and quadriceps. Mem: sarcomeric membrane. NMJ; neuromuscular junction. ++ strong, + moderate, - absent staining.

3.7. Figures

Figure 3.1: Novel diaphragm dystrophin-associated proteins. Western blot analysis of dystrophin immunoprecipitations (DYS IP) from wild type diaphragm and quadriceps muscle. Membranes were cut and probed with antibodies to dystrophin (DYS), cavin-1, and ahnak1. No proteins were identified in control immunoprecipitations (Ctr IP).

Figure 3.2: Localization of novel proteins are disrupted at the neuromuscular junction in mdx mice. Immunofluorescence of wild type and mdx diaphragm and quadriceps for dystrophin, cavin-1, ahnak1, aB-crystallin (CRYAB), and cypher (POI – protein of interest) (red), and α-bungarotoxin (BTX) (green). Expression at the neuromuscular junction is disrupted for a subset of proteins. Individual channels and merged images are shown for localization of the neuromuscular junction.

Figure 3.3: Localization of cavin-1 is disrupted specifically in the mdx diaphragm. Immunofluorescence of wild type (WT) and mdx diaphragm and quadriceps for cavin-1. Cavin-1 expression is absent from the membrane of all muscle fibers in the diaphragm, while continuous but reduced staining is still present in the mdx quadriceps. Residual expression in the mdx diaphragm is still present in the interstitial space (arrowheads).

Figure 3.4: Localization of ahnak1 is disrupted specifically in the mdx diaphragm. Immunofluorescence of wild type (WT) and mdx diaphragm and quadriceps for ahnak1. Ahank1 expression is absent from the membrane of all muscle fibers in the diaphragm, while continuous but reduced staining is still present in the mdx quadriceps. Residual expression in the mdx diaphragm is still present in the interstitial space (arrow).

Figure 3.5: Localization of dysferlin is disrupted in the mdx diaphragm but not quadriceps. Immunofluorescence of wild type (WT) and mdx diaphragm and quadriceps for dysferlin. Dysferlin expression is absent from the membrane of a subset of muscle fibers in the diaphragm, while a separate subset of fibers are marked by intense intracellular staining. No disruption in the mdx quadriceps was observed.

Chapter 4: Characterization of dystroglycan complexes in wild type and dystrophic skeletal muscles.

4.1. Abstract

The dystroglycan complex is composed of the transmembrane protein β- dystroglycan and its interacting extracellular mucin-like protein α-dystroglycan. In skeletal muscle fibers, the dystroglycan complex plays an important structural role by linking the cytoskeletal protein dystrophin to laminin in the extracellular matrix. Mutations that affect any of the proteins involved in this structural axis lead to myofiber degeneration and are associated with muscular dystrophies and congenital myopathies.

Because loss of dystrophin in Duchenne muscular dystrophy (DMD) leads to an almost complete loss of dystroglycan complexes at the myofiber membrane, it is generally assumed that the vast majority of dystroglycan complexes within skeletal muscle fibers interact with dystrophin. The residual dystroglycan present in dystrophin-deficient muscle is thought to be preserved by utrophin, a structural homolog of dystrophin that is up-regulated in dystrophic muscles. However, we found that dystroglycan complexes are still present at the myofiber membrane in the absence of both dystrophin and utrophin. Our data show that only a minority of dystroglycan complexes associates with dystrophin in wild type muscle. Furthermore, we provide evidence for at least three separate

97 pools of dystroglycan complexes within myofibers that are differentially affected by loss of dystrophin and that are associated with different proteins. Our findings indicate a more complex role of dystroglycan in muscle than currently recognized and may help explain differences in disease pathology and severity among myopathies linked to mutations in DAPC members.

4.2. Introduction

The dystroglycan complex is comprised of a single-pass transmembrane protein, β-dystroglycan that anchors a highly glycosylated extracellular protein, α- dystroglycan, to the membrane (Ervasti and Campbell 1991; Ibraghimov-

Beskrovnaya, Ervasti et al. 1992). In skeletal muscle, the dystroglycan complex is an essential component of the larger dystrophin-associated protein complex

(DAPC) (Campbell and Kahl 1989). Within the DAPC, α-dystroglycan binds to extracellular matrix proteins including laminins while the short intracellular domain of β-dystroglycan interacts with dystrophin that in turn binds to F-actin

(Ervasti and Campbell 1993; Fabbrizio, Bonet-Kerrache et al. 1993; Senter, Luise et al. 1993; Lebart, Casanova et al. 1995; Rybakova, Amann et al. 1996).

Therefore in striated muscles the dystroglycan complex provides a link between the intracellular cytoskeleton and the extracellular matrix that is essential for protecting the myofiber membrane from the mechanical stress imposed by muscle contraction (Ibraghimov-Beskrovnaya, Ervasti et al. 1992; Ervasti and

Campbell 1993; Corrado, Mills et al. 1994; Jung, Yang et al. 1995). Indeed,

98 mutations that abrogate expression of dystrophin or impair binding of α- dystroglycan to the extracellular matrix lead to usually severe forms of muscular dystrophy associated with myofiber degeneration (Blake, Weir et al. 2002; Ervasti and Sonnemann 2008; Godfrey, Foley et al. 2011). These observations support to the notion that the DAPC, and in particular the dystroglycan complex within it, are performing an important structural function within muscle fibers. In addition, loss of dystrophin in the mdx mouse, a well characterized mouse model of

Duchenne muscular dystrophy, leads to drastic decrease in dystroglycan at the myofiber membrane (Ibraghimov-Beskrovnaya, Ervasti et al. 1992; Rafael,

Sunada et al. 1994; Rafael, Cox et al. 1996; Miller, Moore et al. 2012). Based on this finding, it has been suggested that the main function of dystroglycan is to anchor dystrophin at the membrane and link it to the extracellular matrix.

Therefore, the vast majority of dystroglycan complexes in myofibers are believed to interact with dystrophin.

However, dystrophin is not the sole intracellular binding partner of dystroglycan in muscle. At the neuromuscular junction (NMJ) a subset of dystroglycan complexes interact with utrophin, a homolog of dystrophin whose expression is normally restricted to the NMJ in adult muscles (Takemitsu, Ishiura et al. 1991; Matsumura, Ervasti et al. 1992). At this site dystroglycan plays an important role in the clustering and subsequent stabilization of acetylcholine receptors at the post-synaptic membrane (Jacobson, Montanaro et al. 1998;

Montanaro, Gee et al. 1998; Bartoli, Ramarao et al. 2001). Upon loss of dystrophin expression in mdx muscles, utrophin expression is up-regulated and

99 its expression is no longer restricted to the neuromuscular junction. In particular, utrophin is highly expressed in regenerating fibers throughout the membrane

(Tanaka, Ishiguro et al. 1991; Shim and Kim 2003), and in non-regenerating fibers it can extend beyond the boundaries of the neuromuscular junction

(Ohlendieck, Ervasti et al. 1991; Weir, Morgan et al. 2004). This up-regulation of utrophin expression in mdx muscles is believed to stabilize dystroglycan complexes allowing for partial functional compensation. Indeed, loss of both dystrophin and utrophin in double knockout (dko) mice leads to a much more severe muscular dystrophy than observed in mdx mice (Deconinck, Rafael et al.

1997; Deconinck, Rafael et al. 1998).

Although functional compensation of dystrophin by utrophin is an attractive concept, utrophin expression in dystrophic muscle reflects ongoing muscle regeneration rather than true functional compensation. Indeed, during muscle development, utrophin is expressed throughout the myofiber membrane and it is only later replaced by dystrophin as the myofibers mature (Pons, Robert et al.

1994; Lin, Gaschen et al. 1998; Radojevic, Lin et al. 2000). Subsequent to muscle injury, regenerating fibers are recognized in part by re-expression of embryonic proteins, including embryonic myosin heavy chain and utrophin (Lin,

Gaschen et al. 1998; Shim and Kim 2003; Stocksley, Chakkalakal et al. 2005), regardless of whether the injured muscle is dystrophin-deficient or wild type. As a result the vast majority of dystroglycan complexes that are “rescued” in mdx muscles by utrophin are those that are normally present in any regenerating muscle and would normally be associated with utrophin following any kind of

100 muscle injury. In non-regenerating fibers, utrophin is concentrated at the NMJ in both wild type and mdx mice. Its slight increase in expression at peri-junctional membranes is not sufficient to functionally compensate for loss of dystrophin throughout the myofiber membrane. Yet, residual dystroglycan expression can be observed at the membrane of myofibers in mdx muscles (Rafael, Cox et al.

1996; Whitehead, Pham et al. 2008; Yoon, Johnson et al. 2012). If utrophin is absent from extra-synaptic membranes in non-regenerating mdx myofibers, then what are these dystroglycan complexes binding to? A similarly important question is whether these residual dystroglycan complexes are normally present in wild type muscle or are a consequence of muscle adaptation to the pathological loss of dystrophin.

Establishing whether dystroglycan complexes not bound to either dystrophin nor utrophin do indeed exist and are present in wild type muscle is important because a growing number of myopathies are linked to mutations in genes that regulate the glycosylation and therefore the function of dystroglycan in muscle (Chandrasekharan and Martin 2010; Muntoni, Torelli et al. 2011). It is currently assumed that these mutations lead to muscle disease by impairing the structural functions performed by dystroglycan within the DAPC. However, the existence of dystroglycan complexes independent of the DAPC would imply that additional disease mechanisms may be involved in the highly variable clinical phenotype of dystroglycanopathies, with important implications for treatment avenues.

101

Here we provide evidence for the first time that a large number of dystroglycan complexes present at the myofiber membrane do not interact with dystrophin or utrophin in wild type muscles. In addition, we provide evidence that a subset of these new dystroglycan complexes that do not directly bind to dystrophin are destabilized in the absence of dystrophin, consistent with the drastic decrease in dystroglycan expression reported in mdx muscles. Finally, we have used our proteomics-based approach (Johnson, Zhang et al. 2012; Yoon,

Johnson et al. 2012) to identify proteins that interact with dystroglycan but are not associated with dystrophin.

4.3 Results

4.3.1. Dystroglycan is present at the membrane of myofibers in mdx and mdx/utrn-/- mice.

Early studies on the expression of dystroglycan in mdx muscle report its almost complete loss at the membrane of myofibers (Ibraghimov-Beskrovnaya,

Ervasti et al. 1992; Rafael, Sunada et al. 1994). However, more recent studies show low level expression of β-dystroglycan in mdx muscle fibers (Rafael, Cox et al. 1996; Whitehead, Pham et al. 2008; Yoon, Johnson et al. 2012). Therefore, we first sought to determine whether dystroglycan complexes are indeed retained at the membrane of both regenerating and non-regenerating myofibers in dystrophin-deficient muscles. Immunofluorescence for β-dystroglycan in sections of quadriceps wild type muscles revealed the expected continuous membrane

102 staining (Figure 4.1A). In mdx muscles, where dystrophin is absent, β- dystroglycan immunofluorescence was readily detected at the membrane of small regenerating fibers (Figure 4.1A, arrow head) as well as larger non- regenerating fibers (Figure 4.1A, asterisk). The intensity of staining was similar to wild type levels in regenerating fibers but decreased in non-regenerating fibers.

However, staining was continuous along all myofiber membranes. A similar result was observed in muscle sections from control versus DMD patient biopsies, where residual β-dystroglycan immunofluorescence was observed at the membrane of all myofibers (Figure 4.1B), although it was fainter than in control muscle.

Utrophin, a homolog of dystrophin, is up-regulated in mdx muscle and it has been proposed to functionally compensate for loss of dystrophin.

Immunofluorescence on wild type muscle sections with a monoclonal antibody to utrophin revealed the expected localization at neuromuscular junctions, with no labeling at the myofiber membrane (Figure 4.1A). In mdx muscles, utrophin was highly expressed at the membrane of small regenerating fibers where b- dystroglycan is also highly expressed. However, utrophin was not detected at the membrane of non-regenerating muscle fibers in mdx muscle. To confirm that the residual β-dystroglycan present in non-regenerating mdx myofibers did not depend upon utrophin for its localization at the membrane, we analyzed muscle sections from dystrophin/utrophin double knockout (mdx/utrn-/-) mice (Deconinck,

Rafael et al. 1997). Utrophin was not detected by immunofluorescence however

β-dystroglycan could still be detected at the membrane of both regenerating and

103 non-regenerating myofibers (Figure 4.1A). Expression levels were decreased compared to mdx muscle in regenerating fibers; however membrane staining remained continuous in all fibers.

These results indicate that a pool of β-dystroglycan is preserved at the muscle membrane in the absence of both dystrophin and utrophin. They also indicate that in mdx muscle utrophin is primarily responsible for high level expression of β-dystroglycan in regenerating but not in non-regenerating myofiber.

4.3.2. Core DAPC members are not major components of dystroglycan complexes in the absence of dystrophin and utrophin.

We next asked whether β-dystroglycan expressed in myofibers from mdx and especially mdx/utrn-/- muscles was still associated with core members of the

DAPC; specifically α-dystroglycan, the sarcoglycans, α-dystrobrevins and syntrophins. Prior immunohistochemical studies in DMD and mdx muscle have shown preserved, although generally reduced expression of some of these

DAPC proteins at the myofiber membrane (Tinsley, Potter et al. 1996; Harper,

Crawford et al. 2002; Compton, Cooper et al. 2005). However co-localization does not always imply that the proteins are interacting and form a complex. We therefore immunoprecipitated β-dystroglycan from wild type, mdx and mdx/utrn-/- muscles with the MANDAG2 monoclonal antibody using a mild detergent extraction protocol that preserves its interactions with DAPC proteins (Yoon,

Johnson et al. 2012). In order to isolate the entire pool of -dystroglycan, we

104 performed experiments where the amount of protein in the lysate before immunoprecipitation (Pre) was titrated against a set concentration of purified

MANDAG2 antibody conjugated to protein G beads. This allowed us to identify a protein concentration at which immunodepletion of -dystroglycan was consistently achieved for all genotypes studied. Western blot analysis on lysates following immunoprecipitation (Post) confirmed depletion of -dystroglycan in each sample (Figure 4.2). Controls for non-specific protein binding to immunoglobulins were performed using an irrelevant isotype-matched mouse monoclonal antibody on wild type protein lysates (Ctr IP). -dystroglycan was not detected in any control immunoprecipitation.

By immunoprecipitation, we isolated large amounts of -dystroglycan from wild type quadriceps as well as mdx and mdx/utrn-/- muscles (Figure 4.2). For all genotypes, α-dystroglycan was strongly associated with -dystroglycan indicating that the core dystroglycan complex is intact in both mdx and mdx/utrn-/- muscles.

In wild type muscle, dystrophin was highly abundant in -dystroglycan immunoprecipitations while only a small amount of utrophin was detected in agreement with its restricted association with dystroglycan at the neuromuscular junction (Nguyen, Ellis et al. 1991). By contrast, in mdx muscles, dystrophin was undetectable but utrophin was increased in -dystroglycan immunoprecipitations, consistent with their co-expression in regenerating fibers (Figure 4.1A). In agreement with a prior report (Li, Bareja et al. 2010), we found that utrophin- dystroglycan complexes in mdx muscle do not include neuronal nitric oxide synthase (Figure 4.2). Surprisingly we also found that utrophin was unable to

105 preserve the interactions mediated by dystrophin with three other DAPC proteins:

β-sarcoglycan, α1-dystrobrevin and α3-dystrobrevin (Figure 4.2). The inability of utrophin to associate with β-sarcoglycan and α1-dystrobrevin was further confirmed by immunofluorescence on quadriceps tissue sections (Table 4.1;

Figure 4.3). α1-dystrobrevin was undetectable at the membrane of both regenerating and non-regenerating fibers. Expression of β-sarcoglycan was severely reduced in all fibers but not completely abolished. Interestingly, the only site where both proteins were preserved at wild type levels in mdx muscles was at neuromuscular junctions. In addition, utrophin-dystroglycan complexes preferentially included β1-syntrophin compared to α1-syntrophin. These results were confirmed by immunofluorescence on mdx muscle sections (Table 4.1;

Figure 4.3), where α1-syntrophin expression was low in regenerating fibers and very low to undetectable in non-regenerating fibers. However, β1-syntrophin was highly expressed in regenerating fibers. Interestingly, loss of dystrophin expression led to the selective loss of β1-syntrophin at the neuromuscular junction (Table 4.2; Figure 4.3) suggesting that the synaptic localization of this syntrophin is mediated by dystrophin and cannot be compensated by utrophin.

Therefore, in mdx muscle utrophin mainly preserves the DAPC in regenerating fibers and at the neuromuscular junction; however the protein complexes it forms are different than those formed by dystrophin and lack several key members of the DAPC in addition to nNOS.

To determine whether the interactions of DAPC proteins with - dystroglycan in mdx muscle truly relied on utrophin, we performed a similar

106 analysis in mdx/utrn-/- mice. Importantly, none of the DAPC proteins was enriched in -dystroglycan immunoprecipitations (Figure 4.2) indicating a complete disruption of the DAPC complex in the absence of both dystrophin and utrophin.

However, some DAPC proteins were still detectable in β-dystroglycan immunoprecipitations, albeit at much more reduced levels than in mdx immunoprecipitations. These are β-sarcoglycan, α1- and α2-dystrobrevins, and

α1- and β1-syntrophins. By immunofluorescence, these interactions with β- dystroglycan are likely occurring at the membrane of non-regenerating fibers and/or at the neuromuscular junction where these proteins are still detectable

(Table 4.2; Figure 4.3). In addition, from the comparison of the immunofluorescence findings among all genotypes, two interesting observations can be drawn. First, additional proteins beside utrophin must exist in regenerating myofibers for localizing α- and β-dystroglycans, β-sarcoglycan, α1- and β1-syntrophins, and α2-dystrobrevin at the myofiber membrane. Second, the requirements for utrophin and dystrophin are different between the non-synaptic membrane and the neuromuscular junction. For example, loss of dystrophin leads to a near complete loss of β-sarcoglycan in non-synaptic membranes however its expression at the neuromuscular junction is entirely dependent upon utrophin expression. By contrast, synaptic localization of β1-syntrophin is entirely dependent on dystrophin expression however this syntrophin interacts with utrophin in regenerating fibers.

Together, these results indicate that the positive effects mediated by utrophin in mdx mice are due to its expression in regenerating fibers and at the

107 neuromuscular junction where it can assemble and stabilize DAPC proteins at the membrane. However, utrophin and dystrophin complexes differ in composition and are differentially regulated in synaptic and non-synaptic regions.

Furthermore, dystroglycan complexes that are preserved in the absence of dystrophin and utrophin still contain a subset of DAPC proteins. However, these proteins are of low abundance in β-dystroglycan immunoprecipitations from mdx/utrn-/- muscles, suggesting that either these complexes are heavily compromised or that they represent a previously unrecognized pool of dystroglycan complexes that include additional proteins still to be identified.

4.3.3. Only a small subset of dystroglycan complexes contain dystrophin in wild type muscles.

We reasoned that if dystroglycan complexes independent of dystrophin and utrophin do normally exist in wild type muscle, then we should be able to detect the existence of this free dystroglycan pool by comparing β-dystroglycan and dystrophin immunodepletions. Immunodepletion of the dystroglycan complex, either with the MANDAG2 antibody to β-dystroglycan or with wheat germ agglutinin (WGA) which binds to moieties on α-dystroglycan, resulted in near complete depletion of full length dystrophin (Figure 4.4A). This indicates that the vast majority of full length dystrophin in muscle is bound to the dystroglycan complex. By contrast, when dystrophin was immunodepleted with monoclonal antibodies directed against different epitopes along the protein, the vast majority of the dystroglycan complex did not co-purify but was detected in the post-

108 immunoprecipitation sample. This result indicates that only a minority of dystroglycan complexes contain dystrophin.

To obtain independent validation of this result, total mouse skeletal muscle lysates and crude rabbit membrane fractions were prepared, and the amounts of dystrophin, α-dystroglycan and its extracellular binding partner laminin were quantified by Western blot against a standard curve generated with purified recombinant proteins (Figure 4.4C). Quantifications confirmed that there is an excess of dystroglycan complexes compared to dystrophin in wild type skeletal muscle. Results indicated a ratio of about 1 to 40 between dystrophin and α- dystroglycan (Table 4.2). Interestingly, there was also an excess of dystroglycan compared to laminin with the calculated ratio of dystrophin to laminin to α- dystroglycan being 1:1:40 (Table 4.2). While these numbers can be affected by the relative efficiency of protein extraction and should not be taken as absolutes, our results support the existence of dystroglycan complexes that do not include full length dystrophin. They further suggest that laminin may not be the extracellular ligand of α-dystroglycan for the majority of these complexes.

4.3.4 The majority of dystroglycan in muscle is expressed by muscle fibers.

In muscle, dystroglycan is not only expressed in muscle cells but also in vasculature structures and nerves. At these locations, full length dystrophin is not expressed and dystroglycan interacts with utrophin or with smaller dystrophin isoforms, including Dp71 (Nguyen, Ellis et al. 1991; Nguyen, Helliwell et al. 1995;

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Tadayoni, Rendon et al. 2011). Therefore, it is conceivable that the excess of dystroglycan observed in dystrophin immunodepletions is derived from non- muscle cells. To test for this possibility, we took advantage of mice that lack dystroglycan expression specifically in hind limb myofibers and mononuclear myogenic cells (Jarad and Miner 2009; Singhal, Xu et al. 2012). We refer to these mice as P3Pro-Cre; Dag1lox/lox. Western blot analysis of gastrocnemious muscle lysates revealed undetectable levels of β-dystroglycan in P3Pro-Cre;

Dag1lox/lox mice compared to wild type (Figure 4.5A). However, β-dystroglycan could be detected in these same lysates after immunoprecipitation with the

MANDAG2 antibody (Figure 4.5B), indicating that β-dystroglycan was still expressed in non-muscle tissues but at very low levels compared to myofibers.

Importantly, in total lysates from mdx and mdx/utrn-/- mice where dystroglycan is still present at the myofiber membrane, β-dystroglycan was readily detected by

Western blot (Figure 4.5A).

To further confirm that non-muscle tissues do not significantly contribute to the pool of dystroglycan not bound to full length dystrophin, we tested whether a significant portion of dystroglycan was bound to Dp71, a short dystrophin form not expressed in myofibers but present in non-muscle cells and in muscle precursor cells (Tadayoni, Rendon et al. 2011). Probing of β-dystroglycan immunoprecipitations from wild type and dystrophic muscles using an antibody directed against the carboxyl-terminus of dystrophin showed only a faint band at

71 kDa in all lysates (Figure 4.5C).

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Together, these results strongly suggest that the vast majority of dystroglycan complexes that are not associated with full length dystrophin are present in myofibers.

4.3.5 Novel protein interactions of dystroglycan complexes in muscle

If a sizable number of dystroglycan complexes within myofibers do not interact with dystrophin, then these complexes may have different protein interactions. To identify these proteins, we took advantage of our ability to couple immunoprecipitation of dystrophin or β-dystroglycan with protein identification by shotgun proteomics (Johnson, Zhang et al. 2012; Yoon, Johnson et al. 2012).

Samples were analyzed on the LTQ Orbitrap and proteins identified in control immunoprecipitations were subtracted from the respective dystrophin or dystroglycan immunoprecipitation. Each experiment included two controls: an immunoprecipitation on the same lysate performed with an isotype-matched control antibody, or an immunoprecipitation with the antibody against dystrophin or β-dystroglycan on mdx or P3Pro-Cre; Dag1lox/lox muscle extracts, respectively.

No known DAPC proteins were identified in any control.

Following background subtraction, we specifically looked for proteins that co-purified with β-dystroglycan in at least 3 out of 4 biological replicates and had high confidence scores but were never detected in any of the three biological replicates for dystrophin immunoprecipitation. Four proteins satisfied these criteria and are listed in Table 3. Among these is β2-sytrophin which is known to interact with β-dystroglycan at the neuromuscular junction (Peters, Kramarcy et

111 al. 1994). This interaction is mediated by utrophin, not dystrophin, thus confirming the specificity and sensitivity of this approach. The remaining three proteins include synaptopodin-2 which we had previously identified as a dystroglycan binding partner in both wild type and mdx muscle (Yoon, Johnson et al. 2012), using an LTQ which is less sensitive than the LTQ Orbitrap. The remaining two proteins are Cavin-1 (a.k.a. PTRF) and Keratin 17. We had previously confirmed that Cavin-1 does not interact with dystrophin in quadriceps muscle (Johnson, Zhang et al. 2012). We therefore further pursued analysis of cavin-1 as potential dystroglycan-specific binding partner. Keratin 17 is not expressed in skeletal muscle and was therefore not further studied.

In addition to these 4 proteins, we detected several peptide matches to alpha and beta subunits of calcium channels in one β-dystroglycan immunoprecipitation (data not shown). Ion channels that contain multiple transmembrane domains and are difficult to extract are not easily detected by mass spectrometry, we therefore were not surprised that these proteins were not consistently detected in all immunoprecipitations. Because the activity of calcium channels is affected in mdx muscle and a link has been proposed with the DAPC

(Collet, Csernoch et al. 2003; Johnson, Scheuer et al. 2005), we decided to also test for an association of dystroglycan and dystrophin with calcium channels. We tested several commercially available antibodies to alpha subunits but they did not perform well on Western blots. By contrast, we had good results with antibodies directed against the β2- and β3-regulatory subunit, specifically the β2- regulatory subunit (Cavβ2) that regulates the activity and membrane expression

112 of several different voltage gated calcium channels (Buraei and Yang 2012).

Cavβ2 is expressed in skeletal muscle, including the neuromuscular junction where dystroglycan is concentrated (Buraei and Yang 2012). Cavβ2 also contains an SH3 domain and the cytoplasmic portion of β-dystroglycan can interact with SH3 containing proteins (Yang, Jung et al. 1995; Thompson, Kleino et al. 2008; Thompson, Moore et al. 2010). We therefore expanded our analysis to include the Cavβ2 of calcium channels.

Western blot analysis of protein lysates from wild type, mdx and mdx/utrn-/- quadriceps muscles were analyzed for expression levels of Cavβ2 and was found to be similar among all genotypes (Data not shown). We then performed immunoprecipitations to determine the association status with dystrophin and β- dystroglycan (Figure 4.6). Western blot analysis of dystrophin immunoprecipitations on wild type quadriceps lysates confirmed that cavin-1, and the Cavβ2 does not associate with dystrophin. By contrast, these proteins were present in β-dystroglycan immunoprecipitations, indicating that they are part of a dystroglycan complex that does not include dystrophin. None of these proteins were detected in control immunoprecipitations in wild type muscle using an isotype and species matched antibody. Interestingly, the association of cavin-1 with β-dystroglycan was disrupted in mdx muscles. This result suggests that at least a subset of dystroglycan complexes depend on dystrophin for their stability even though they do not directly bind to dystrophin. We next asked whether the

Cavβ2 association with β-dystroglycan is preserved in mdx muscle by utrophin.

Analysis of β-dystroglycan immunoprecipitations from mdx/utrn-/- quadriceps

113 showed that Cavβ2 was not disrupted in the absence of utrophin. These results confirm the existence of dystroglycan complexes in wild type muscle that do not contain dystrophin but associate with a subset of proteins distinct from the known

DAPC. They further indicate that a pool of dystroglycan complexes is independent of both dystrophin and utrophin for its presence at the membrane and may be associated with voltage-gated calcium channels.

4.4. Discussion

In this study, we demonstrate the existence of dystroglycan complexes that are not bound to dystrophin or utrophin at the membrane of wild type skeletal muscle fibers. We have further identified some of the proteins that associate with these alternate dystroglycan complexes. Our data support the existence of at least three separate pools of dystroglycan complexes in skeletal muscle (Figure

4.6). The first pool contains dystrophin as well as other known core DAPC proteins such as the sarcoglycans, dystrobrevins and syntrophins that co-purify with both dystrophin and dystroglycan (Johnson, Zhang et al. 2012). Based on the 1:1 ratio of laminin to dystrophin, this complex is likely linked to laminin in the extracellular matrix. A second dystroglycan pool does not include dystrophin but is dependent upon dystrophin expression for stability at the membrane and for interactions with cavin-1. This complex is likely destabilized in mdx muscle which may account for the large decrease in dystroglycan protein at the membrane of mdx myofibers. We currently do not know whether these dystroglycan complexes

114 contain any other core DAPC proteins nor how they are linked to dystrophin.

Finally, a third pool of dystroglycan complexes appears independent of both dystrophin and utrophin for its expression at the myofiber membrane and for its protein interactions. These dystroglycan complexes are preserved in dystrophic muscles and may function in the regulation of L-type calcium channels. An unexpected finding of this study is that the amount of dystroglycan complexes not bound to full length dystrophin or utrophin in wild type skeletal muscle is quite large. Although surprising, evidence of the existence of such a “free” pool of dystroglycan can be found in several prior studies. Indeed, sucrose-gradient fractionations of the DAPC after enrichment by WGA-affinity column show that a significant pool of dystroglycan is present in fractions that do not contain dystrophin or the sarcoglycans (Holt, Lim et al. 1998; Straub, Duclos et al. 1998;

Crosbie, Lebakken et al. 1999; Durbeej, Cohn et al. 2000). Although we have attempted to quantify the ratio of dystroglycan to dystrophin, this ratio should not be taken as an absolute value because it assumes that dystroglycan is extracted with similar efficiency by digitonin regardless of whether it is bound to dystrophin or not. This is likely not the case. Therefore, our protein quantifications together with our immunodepletion data should be taken as indicating that there is a significant amount of dystroglycan within muscle that does not interact with dystrophin and is therefore available to perform other functions.

The idea that dystroglycan may perform additional functions beyond structural integrity in skeletal muscle is not new. Several studies have identified intracellular binding partners for β-dystroglycan other than dystrophin in muscle

115 cells. These include caveolin-3 (Sotgia, Lee et al. 2000), grb2 (Yang, Jung et al.

1995), plectin 1f (Rezniczek, Konieczny et al. 2007), a subset of protein kinases

(Sotgia, Lee et al. 2001), rapsyn (Bartoli, Ramarao et al. 2001) and vinexin

(Thompson, Moore et al. 2010). Most of these interactions have been identified in vitro and some are expected to disrupt binding of β-dystrolgycan to dystrophin. It has therefore been questioned whether these protein interactions truly occur in vivo and have a significant functional role in mature myofibers where most of the dystroglycan is believed to bind to dystrophin to fulfill a structural function fundamental to myofiber integrity and survival. Our findings have shed a new light on this issue and demonstrate that association of these proteins with β- dystroglycan is not necessarily incompatible with the structural functions performed by the dystroglycan-dystrophin complex. Whether these interactions do occur in vivo with dystroglycan complexes separate from dystrophin remains to be confirmed since we did not detect them by our proteomic approach. In the case of Caveolin-3, we also did not detect this protein in our dystrophin or dystroglycan immunoprecipitations by Western blot analysis in a greement with a previous study (Crosbie, Yamada et al. 1998). Some of these interactions may be too labile to be detected by our approach such as transient interactions with kinases. Others may be too rare such as interactions occurring at the neuromuscular junction (i.e. rapsyn) or in a subset of muscle fibers (i.e. plectin

1f).

We have identified and confirmed new interactions of dystroglycan with cavin-1 and the β2-regulatory subunits of calcium channels. Cavin-1 is a protein

116 with a dual function in controlling caveolar dynamics and in initiation of membrane repair (Hill, Bastiani et al. 2008; Zhu, Lin et al. 2011). We show here that binding of cavin-1 to dystroglycan is disrupted in the absence of dystrophin.

Interestingly, dystrophin-deficient muscles show normal membrane repair

(Bansal, Miyake et al. 2003) but increased numbers and a general disorganization of caveolae in both mouse and human muscle fibers (Repetto,

Bado et al. 1999; Shibuya, Wakayama et al. 2002). It is therefore conceivable that the interaction of dystroglycan with cavin-1 is involved in regulation of caveolae. Finally, the association of a pool of dystroglycan independent of dystrophin and utrophin with the regulatory subunit of calcium channels suggests a function in the regulation of L-type calcium channels. Although this interaction is independent of dystrophin, studies in mdx skeletal muscle fibers indicate that some but not all properties of L-type calcium channels are perturbed (Collet,

Csernoch et al. 2003). Loss of dystrophin does not appear to affect expression levels of the channels in limb muscles (Pereon, Dettbarn et al. 1997). Instead, disturbances in calcium channel activity appear to be linked to the resulting disorganization of the sub-membranous cytoskeleton and deregulation of PKA signaling (Johnson, Scheuer et al. 2005). These observations indicate that alterations in L-type calcium channels are a secondary event to loss of dystrophin, a conclusion that is compatible with our biochemical findings.

An important question to be raised is the implication of our findings for our current understanding of disease mechanisms in muscular dystrophies and eventually for treatment development. We currently do not know the exact

117 composition of the “free” dystroglycan complexes we have identified here and the elucidation of their functions will require further studies. However, the observation that protein interactions within some of these “free” dystroglycan complexes are lost in the absence of dystrophin, suggests that they may contribute to muscle pathology in DMD. Characterization of these complexes could therefore reveal upstream disruptions in signaling pathways that are altered in muscular dystrophy. It will be equally important to determine whether these “free” dystroglycan complexes are affected in limb girdle muscular dystrophies and congenital myopathies arising from mutations in laminin, sarcoglycans, or proteins that glycosylate α-dystroglycan (Dalkilic and Kunkel 2003). Differences in the effects these mutations have on the “free” dystroglycan complexes could provide important information on their function and contribution to disease severity. Of particular relevance to treatment approaches, we have shown here that utrophin forms complexes that differ in composition from dystrophin complexes. We have further shown that the composition of both dystrophin and utrophin complexes changes depending on the location in the myofiber (synaptic versus extra-synaptic) or the developmental status of the fiber (regenerating versus non-regenerating). This suggests that there are mechanisms that are used by myofibers to regulate the temporal and spatial inclusion or exclusion of specific proteins from dystrophin and utrophin complexes. This is in agreement with our findings in heart versus limb muscles where we found that cavin-1, cypher, Ahnak1 and CRYAB are specifically excluded from the skeletal muscle

DAPC even though these proteins are highly expressed in both cardiac and

118 skeletal muscles (Johnson, Zhang et al. 2012). The question that arises is therefore what really happens when we over-express therapeutic constructs, such as utrophins or mini/micro-dystrophins (Tinsley, Potter et al. 1996; Harper,

Hauser et al. 2002)? Are we really reconstituting the correct dystrophin complex?

If not, what are the implications for muscle function and for the long term health of myofibers? Beyond the issue of stability of the therapeutic proteins

(Henderson, Belanto et al. 2011), altered composition of the DAPC and its ability to properly functionally integrate with the “free” dystroglycan complexes described here may be important new variables that influence the long term therapeutic performance of gene replacement approaches.

Overall, our study has revealed a greater complexity of regulation, composition and possibly function of the dystroglycan complex in adult muscle than currently appreciated. They also have revealed the usefulness of immunoprecipitation and proteomics in revealing brand new aspects of this complex. The challenges ahead will be to biochemically separate the different dystroglycan complexes in order to characterize their composition by proteomics and most importantly, to identify their functions in muscle maintenance and disease.

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4.5. Methods

4.5.1. Human ethics and skeletal muscle biopsies.

Human skeletal muscle biopsy material was obtained with informed written consent from guardians on behalf of the minor participants involved in the study under our approved IRB at Nationwide Children’s Hospital.

4.5.2. Animals.

Wild type mice were maintained as a C57Bl/6 strain. Mice deficient in dystrophin (mdx5cv, B6Ros.Cg-Dmdmdx-5Cv/J, stock 002379) were a kind gift of Dr.

Louis Kunkel, Harvard University and were maintained as a C57Bl/6J strain. Mice with hindlimb muscle deleted for dystroglycan (P3Pro-Cre; Dag1lox/lox ) were made by crossing P3Pro-Cre transgenic mice, which show a caudal to rostral gradient of Cre transgene expression in skeletal muscles, to Dag1loxP/loxP mice

(Jarad and Miner 2009; Singhal, Xu et al. 2012). Mice were bred and cared for in a clean barrier facility and all animal care and experiments were done under protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Nationwide Children’s Hospital.

4.5.3. Antibodies.

120 filtration cell (Millipore). Antibodies to DAPC members are: isoform specific anti-

α1-, or β1-syntrophin, and anti- α1- or α2-dystrobrevin antibodies (Peters,

Kramarcy et al. 1994; Peters, Adams et al. 1997; Peters, Sadoulet-Puccio et al.

1998) (a kind gift of Stan Froehner, University of Washington); clone IIH6C4 to α- dystroglycan (Upstate); anti-nNOS (#610308) and pan anti-α-dystrobrevin

(#610766, BD Bioscience); anti-β-sarcoglycan (clone 5B1, Leica Microsystems); anti-utrophin (DRP2, Leica Microsystems); anti-cavin-1 (ab40840), and anti-

Cavβ2 (ab93606).

4.5.4. Immunoprecipitations and WGA pull-downs.

Immunoprecipitations were performed from quadriceps muscles.

Immunoprecipitations for dystrophin and β-dystroglycan with the MANDYS1 and

MANDAG2 antibodies respectively were performed as previously described

(Peters, Adams et al. 1997). Proteins were extracted in a buffer containing 1% digitonin and 0.05% NP-40. Immunodepletions were performed similar to all immunoprecipitations except the amount of total protein incubated with antibody- conjugated beads was limited to 1 mg. Proteins were eluted in Laemmli

Reducing Sample Buffer for Western blot analysis or in 2% SDS or 100 mM DTT for LC-MS/MS analysis.

4.5.5. LC-MS/MS.

Proteomic analysis was performed on 3 dystrophin immunoprecipitations and 4 β-dystroglycan immunoprecipitations from wild type muscle. Each sample

121 is a separate biological replicate, not a technical replicate. To identify proteins that non-specifically co-purify with dystrophin or dystroglycan and subtract them from analysis, we also performed proteomic analysis on the following controls: 2 dystrophin immunoprecipitation on control mdx5cv muscles (background specific to the dystrophin antibody), 2 dystroglycan immunoprecipitations on control

P3Pro-Cre; Dag1lox/lox muscles (Background specific to the dystroglycan antibody), and 3 immunoprecipitations with the MW8 antibody to Huntingtin

(general IgG control for both dystrophin and dystroglycan immunoprecipitations).

Eluted proteins were chloroform/methanol precipitated, resuspended in 5X

Invitrosol protein solubilizer (Invitrogen), diluted with 25 mM ammonium bicarbonate to a final volume of 1X Invitrisol. The proteins were then reduced with 10 µL DTT (5 mg/mL solution in 100 mM ammonium bicarbonate) and carbamidomethylated with 10 µL Iodoacetimide solution (15 mg/mL in 100 mM ammonium bicarbonate). Trypsin (in 50 mM ammonium bicarbonate) was added to the protein solution with an enzyme to substrate ratio of 1:25 (w/w). Samples were incubated for 2 hr at 37 °C before quenching by acidification. Capillary- liquid chromatography-nanospray tandem mass spectrometry was performed on a Thermo Finnigan LTQ orbitrap mass spectrometer (Thermo Fisher Scientific,

San Jose CA) equipped with a microspray source (Michrom Bioresources Inc,

Auburn, CA) operated in positive ion mode. Samples were loaded onto a precolumn Cartridge (Dionex, Sunnyvale, CA) and desalted with 50 mM acetic acid for 10 minutes, then separated on the capillary column (0.2X150mm Magic

C18AQ 3µ 200A, Michrom Bioresources Inc, Auburn, CA) using an UltiMate™

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3000 HPLC system from LC-Packings A Dionex Co (Sunnyvale, CA). Mobile phases A and B were 0.1% formic acid in water and 0.1% formic acid in acetonitrile, respectively. Flow rate was 2 µl/min. Mobile phase B was increased from 2% to 50% in 250 min, then from 50% to 90% in 5 min, then kept at 90% for another 5 min The column was equilibrated at 2% of mobile phase B (or 98% A) for 30 min before the next sample injection. MS/MS data was acquired with a spray voltage of 2 KV and a capillary temperature of 175 °C. The scan sequence of the mass spectrometer was based on the TopTen™ method. The full scan mass resolving power was set at 30,000 to achieve high mass accuracy MS determination. The CID fragmentation energy was set to 35%. Dynamic exclusion is enabled with a repeat count of 3 within 30 s, a mass list size limit of

200, exclusion duration of 350 s and a low mass width of 0.50 and high mass width of 1.50 Da.

4.5.6. Peptide sequence analysis.

Resulting .mgf files were searched using Mascot Daemon (version 2.3.2, Matrix

Science, Boston, MA) against the Swissprot mouse database (version 57.0,

428,650 sequences; 154,416,236 residues). Trypsin was used as the enzyme

123 and three missed cleavages were permitted. Considered variable modifications were oxidation (Met) and carbamidomethylation (Cys). The mass accuracy of the precursor ions was set to 10ppm and the fragment mass tolerance to 0.5 Da.

Accidental picking of one 13C peak was included into the search. The significance identity threshold was set at p<0.05 for valid protein identification.

False discovery rates (FDR) for peptide matches were estimated using the target-decoy search strategy (Elias and Gygi 2007; Elias and Gygi 2010). All reported results are for peptides with less than 5% FDR. Proteins with a Mascot mowse score of 25 or higher containing a minimum of one unique peptide with a

-b or -y ion sequence tag of five residues or better were accepted. However, identifications of proteins with one unique peptide were considered to be true positives only if: 1) the precursor ion has correct charge status and the mass accuracy is <10ppm. 2) b and -y ion sequential tag of five or more residues were present following manual validation of MS/MS spectra.

4.5.7. Immunohistochemistry Quadriceps were flash frozen in isopentane cooled in liquid nitrogen and

4-8 μm serial sections were cut. Immunofluorescence labeling of tissue sections was performed as previously described [53]. Images were captured using an

Olympus BX61 microscope or a Leica TCS-NT confocal microscope.

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4.5.8. Immunoblots.

Membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) and enhanced.

4.5.9. Molar Concentrations

Various amounts of purified proteins from rabbit skeletal crude surface membranes were separated by gel electrophoresis along with known amounts of purified protein standards and probed by Western blot analysis. The amount of protein loaded was plotted against integrated optical intensity and linear regression was applied to generate a standard curve for protein standards.

Optical density of the protein sample from surface membranes was then compared to the standard curve for each protein to calculate the molar concentration of each protein.

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4.6. Tables

wild type mdx mdx/utrn-/- Non- Non- Non- DAPC protein regen. NMJ regen. NMJ regen. NMJ regen. regen. regen. -sarcoglycan +++ N/A +++ + + +++ + + - +++ 1-syntrophin +++ N/A +++ +/- ++ +++ - ++ * 1-syntrophin +++ N/A +++ - +++ - - ++ - 1- + N/A +++ - - +++ - - +++ dystrobrevin 2- +++ N/A +++ ++ ++ +++ - + + dystrobrevin

Table 4.1: Localization of DAPC proteins in wild type, mdx and mdx/utrn-/- mouse muscles. Summary of immunohistochemical analysis on tissue sections for the indicated DAPC proteins. Representative micrographs are shown in Figure 4.1 and Figure 4.3. Non-regen: non-regenerating myofiber; Regen.: Regenerating myofiber; NMJ: Neuromuscular junction. +++: High expression at the membrane, similar to wild type levels; ++: slightly reduced expression; +: very low expression; +/-: very low expression in some fibers and no detectable expression in others; - : no expression detected. * α1-syntrophin expression at NMJs from mdx/utrn-/- mice varied and was either at levels similar to wild type or undetectable.

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BL6 MDX DKO Non- Non- Non- regen. NMJ regen NMJ regen. NMJ regen. regen. regen. - +++ N/A +++ + + +++ + + - sarcoglycan 1- +++ +++ N/A +++ +/- ++ +++ - ++ syntrophin * 1- +++ N/A +++ - +++ - - ++ - syntrophin 1- + N/A +++ - - +++ - - +++ dystrobrevin 2- +++ N/A +++ ++ ++ +++ - + + dystrobrevin

Table 4.2: Summary of protein concentrations for Laminin, α-dystrolgycan and dystrophin in mouse skeletal muscle extracts (SKM) and rabbit crude surface membrane preparations (CSM). The concentrations are shown as mean ± standard error with the unit of nmol per g of total mouse skeletal muscle protein (SKM) or rabbit crude surface membrane protein (CSM).

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GENE SWISSPROT ID DESCRIPTION Expt SCORE PEP. COV. FUNCTION ID Polymerase I 1 50 2 3.8 and transcript 2 207 9 16.6 Membrane repair, PTRF_MOUSE Ptrf release factor caveolar formation 3 172 7 7.9 (Cavin-1) 4 45 2 2.3

1 109 6 3.1 Beta-2- 2 290 14 12.5 Adaptor protein, SNTB2_MOUSE Sntb2 syntrophin 3 78 7 3.7 signaling 4 161 11 7.3 1 227 20 13.6 128 Keratin, type I Cytoskeletal K1C17_MOUSE Krt17 2 151 11 6.2

cytoskeletal 17 protein 3 301 10 13.6 Synaptopodin-2 Z-disc protein, SYNP2_MOUSE Synpo2 1 90 2 1.6 (myopodin) actin bundling

Table 4.3: Proteins identified by proteomics in β-dystroglycan but not dystrophin immunoprecipitations from wild type quadriceps muscles.Expt: Experiments where the protein was detected in at least 3 out of 4 individual biological replicates; Score: Mascot protein confidence score; Pep: number of unique peptides identified; Cov: percent protein sequence coverage.

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4.7. Figures

Figure 4.1: β-dystroglycan is present at the membrane of myofibers in human DMD muscle biopsies and in mdx and mdx/utrn-/- mouse muscles. A. Immunolabeling of wild type, mdx and mdx/utr-/- (DKO) skeletal muscle sections for β-dystroglycan and utrophin. Arrowheads and asterisks denote non- regenerating and regenerating fibers respectively. β-Dystroglycan staining was continuous at the membrane of all muscle fibers for all tissue types. B. Immunolabeling of tissue sections from control and Duchenne muscular dystrophy (DMD) biopsy samples for β-dystroglycan. Scale bars: 50 µm.

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Figure 4.2: Analysis of DAPC members that associate with β-dystroglycan in wild type, mdx and mdx/utrn-/- mouse muscles. Western blot analysis of β- dystroglycan immunoprecipitations (IP) from wild type, mdx and mdx/utr-/- (DKO) skeletal muscle lysates. Lysates before (Pre) and after (Post) immunoprecipitation confirmed immunodepletion of β-dystroglycan from skeletal muscle lyastes. No proteins were detected in non-specific antibody controls (Ctr IP). Blots were cut at appropriate molecular weight sizes and probed with antibodies to indicated proteins. nNOS – neuronal nitric oxide synthase.

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Figure 4.3: The localization of DAPC proteins show unique patterns of disruption in mdx and mdx/utr-/- skeletal muscle. Immunolabeling of wild type, mdx, and mdx/utr-/- (DKO) skeletal muscle tissue sections. Arrowheads and asterisks denote non-regenerating and regenerating fibers respectively. Tissue sections were serial cut and Arrowheads and asterisks correspond to the same skeletal muscle fibers in figure 4.1A.

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Figure 4.4: A significant pool of dystroglycan complexes are not bound to dystrophin in wild type muscle. A. Western blot analysis of β-dystroglycan immunoprecipitation (βDG IP; top) or wheat germ agglutinin pull down (WGA; bottom) of α-dystroglycan on wild type skeletal muscle lysates. Blots were probed for dystrophin (DYS), β-dystroglycan (βDG), and α-dystroglycan (αDG). Lysates before (Pre) and after (Post) were used to confirm immunodepletion of all proteins. B. Western blot analysis of dystrophin immunoprecipitations (DYS IP) using two separate antibodies against different regions of dystrophin. Immunodepletion of dystrophin from lysates after immunoprecipitation does not immunodeplete β-dystroglycan or α-dystroglycan. Controls immunoprecipitations (Ctr IP) for both a. and b. used non-specific antibodies on wild type lysates. C. Protein quantification of dystrophin (DYS), α-dystroglycan (αDG), and laminin (lam). Standard curves were generated using recombinantly expressed protein, and the amount of proteins were calculated by comparing plotting the intensity of Western blot bands of endogenous expressed proteins obtained from crude skeletal muscle membrane preparations (CSM).

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Figure 4.5: Non muscle cells within adult muscle do not significantly contribute to overall β-dystroglycan expression. A. Western blot analysis of total skeletal muscle lysates from wild type (WT), dystroglycan deficient (Dag1-/-), mdx and mdx/utr-/- (DKO) for β-dystroglycan (βDG). GapDH was used as a loading control. B. Western blot analysis of β-dystroglycan immunoprecipitations from wild type or dystroglycan deficient skeletal muscle lysates blotted for β- dystroglycan. C. Western blot analysis of β-dystroglycan immunoprecipitations (βDG IP) from wild type, mdx and DKO skeletal muscle lysates. Only small amounts of DP71 was purified by IP. Importantly no proteins were detected in non-specific antibody control immunoprecipitations (Ctr IP).

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Figure 4.6: New dystroglycan-interacting proteins are differentially affected in mdx and mdx/utrn-/- muscles. Western blot analysis of dystrophin and β- dystroglycan immunoprecipitations (DYS IP and βDG IP respectively) on skeletal muscle lysates from wild type (WT), mdx, and mdx/utr-/- (DKO). Blots were cut at appropriate molecular weight sizes and probed with antibodies to β-dystroglycan (βDG), dystrophin (DYS), cavin-1, and beta 2 subunit (Cavβ2). No proteins were detected in control non-specific antibody immunoprecipitations on wild type lysates (Ctr IP).

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Figure 4.7: Proposed model for dystroglycan complexes in wild type and dystrophin-deficient muscle. A. Schematic representation of β-dystroglycan complexes localized at the membrane of wild type skeletal muscle fibers. B. Schematic representation of β-dystroglycan complexes at the membrane of mdx skeletal muscle fibers. Complexes that are perturbed in mdx skeletal muscles are represented by dashed lines. The mechanisms of association

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5.1. General Discussion

Even from the initial identification of the DMD gene and its protein product dystrophin, it was apparent that the functions of dystrophin and its associated proteins in striated muscle were extremely complex. Indeed, despite several years of intense research our understanding of the exact roles and mechanisms of disease for dystrophin and the DAPC are still poorly understood. A major road block facing the field was the inability to purify intact dystrophin-associated protein complexes, and identify interacting proteins in a sensitive and reproducible manner. This was likely due to several compounding factors including dystrophin’s large size, multimeric composition of the DAPC, and the complex nature of the protein associations including multiple direct and indirect proteins interactions. Presented here is a novel method for the isolation of dystrophin and associated proteins by immunoprecipitation and their identification by high throughput proteomics. Using this approach we were able to not only highlight key muscle type specific differences in the composition of the core DAPC, but further identified novel dystrophin interacting proteins in both the heart and diaphragm. These proteins represent new promising tissue specific functions of dystrophin and are prime for exploration.

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Besides dystrophin, our studies focusing on dystroglycan have led to the discovery of multiple dystroglycan complexes at the sarcolemmal membrane that are distinct from the dystrophin-containing DAPC. This includes the identification of new dystroglycan complexes and binding partners that potentially link dystroglycan to additional cellular functions in striated muscle. While much work is needed to fully understand the mechanism of interaction for the proteins described here and their function in muscle, these proteins highlight potential new roles for dystrophin and dystroglycan, and shed light on the mechanisms of disease leading to muscular dystrophy.

5.1. New pathways for dystrophin function

Our proteomic analysis of dystrophin-associated proteins identified several novel proteins. The identification of these proteins opens up new avenues of research investigating pathways that likely require dystrophin expression.

Specifically, in the heart we identified four proteins: ahnak1, cavin-1, αB- crystallin, and cypher. Furthermore, we have shown that for at least two of these proteins, ahnak1 and cavin-1 their interaction is evolutionarily conserved in the human and mouse heart. The association of these proteins with dystrophin is particularly interesting has they have all been suggested to have cardiac functions and when disrupted give rise to cardiac disease. In addition to the heart, the association of ahnak1 and cavin-1 is further conserved in the mouse diaphragm where dystrophin may have overlapping functions in these tissues.

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Ahank1 was originally identified as a tumor-related phosphoprotein, but recent studies have implicated it in cardiac function (Haase, Alvarez et al. 2005;

Haase 2007) . Ahnak1 is localized to the intracellular side of the cardiomyocyte membrane, intercalated discs, and the T-tubules (Hohaus, Person et al. 2002), in addition to the sarcolemmal membrane in skeletal muscle (Huang, Laval et al.

2007). In cardiomyocytes ahnak1 has been suggested to function in actin bundling were it is believed to be an important link between the L-type calcium channel Cav1.2 at T-tubules and the actin cytoskeleton (Hohaus, Person et al.

2002; Haase, Pagel et al. 2004). Possibly the most interesting function of ahnak1 relative to dystrophin in the heart is its role as a regulator of calcium influx via L- type calcium channels (Hohaus, Person et al. 2002; Haase, Alvarez et al. 2005).

Multiple direct binding sites for the β2 subunit (Cavβ2) have been characterized in the C-terminus of ahnak1 (Hohaus, Person et al. 2002; Haase, Alvarez et al.

2005), where upon binding the Cavβ2, ahnak1 regulates calcium channel acitivy by suppressing the open time of L-type calcium + channels. However, upon phosphorylation of ahnak1 its binding affinity for Cavβ2 is decreased, releasing the inhibition and resulting in prolonged activation of calcium channels (Hohaus,

Person et al. 2002; Alvarez, Hamplova et al. 2004). Therefore, dystrophin may be needed for the proper localization and/or function of ahnak1 in the heart, where loss of dystrophin disrupts ahnak1 and releases the inhibition on Cavβ2. This would then lead to prolonged activation of the L-type calcium channels in the heart and result in increased intracellular calcium. In support of this, studies investigating the properties of the L-type calcium channel Cav1.2 in mdx

138 myocytes have shown delayed inactivation of the calcium current and increased calcium influx (Sadeghi, Doyle et al. 2002; Woolf, Lu et al. 2006). Calcium deregulation including increased intracellular calcium has long been proposed as a primary contributor to cardiac failure in DMD, and the association of ahnak1 provides a new mechanism to explain the increased intracellular calcium levels in

DMD. Further studies are needed to truly understand the exact function of the dystrophin-ahank1 interaction and its role in calcium channel homeostasis.

Only recently has ahnak1 been investigated in skeletal muscle. By using immunoprecipitation coupled with proteomics, ahnak1 was identified as a direct binding partner of dysferlin (Huang, Laval et al. 2007). Furthermore, the membrane localization of ahnak1 in skeletal muscles is disrupted in dysferlinopathies (Huang, Laval et al. 2007). Using a similar approach as to the heart we identified ahnak1 as a dystrophin associated protein in diaphragm.

Furthermore, in the absence of dystrophin, ahnak1 localization at the sarcolemmal membrane is lost only in the diaphragm. Surprisingly, the localization of dysferlin is also disrupted specifically in the mdx diaphragm. Taken together, these studies suggest that dystrophin is also involved in membrane repair in the diaphragm and potentially the heart. Together, these studies identify potential new functions of dystrophin in the heart and diaphragm, that include not only structural maintenance of the membrane but also possibly calcium regulation and membrane repair.

Another novel dystrophin-associated protein in the heart and diaphragm is cavin-1. Cavin-1 is as a key component in the formation and stabilization of

139 caveolae (Briand, Dugail et al. 2011). Caveolae are small invaginations of the plasma membrane and are specialized lipid microdomains which have been suggested to function in lipid storage, cell signaling and endocytosis (Briand,

Dugail et al. 2011). In striated muscle little is known about the function of cavin-1 although recent studies have highlighted its role in membrane repair (Zhu, Lin et al. 2011). Cavin-1 was shown to be an important factor in the membrane repair process where it directly binds MG53, a protein that concentrates at sites of membrane damage and facilitates sealing of the plasma membrane in skeletal and cardiac muscle (Wang, Xie et al. 2010; Zhu, Lin et al. 2011). Therefore, cavin-1 functions to recognize sites of membrane damage, and localizes proteins required for proper membrane sealing (Zhu, Lin et al. 2011). RNAi knockout of cavin-1 in skeletal muscle prevents the translocation of MG53 to the site of damage preventing rapid sealing of membrane tears (Zhu, Lin et al. 2011).

Therefore, disruption of cavin-1 in the heart and diaphragm of mdx mice may represent yet another mechanism of disease for impaired membrane stability and repair. Indeed, loss of dystrophin in the diaphragm results in a complete loss in localization of cavin-1 at the sarcolemmal membrane. Unfortunately, little else is known about the function of cavin-1 in striated muscle. However, cavin-1 mutations have been identified in patients with lipodystrophy and ventricular arrhythmias (Rajab, Straub et al. 2010; Shastry, Delgado et al. 2010) making cavin-1 a prime candidate for futures studies in mdx mice.

Possibly the best studied protein in respects to muscle function that was identified as a novel dystrophin-associated protein in the heart was cypher.

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Cypher is a cytoskeletal protein, which binds to α-actinin at the Z-line of both skeletal and cardiac muscle (Zheng, Cheng et al. 2009). In striated muscle three isoforms of cypher are expressed, one short and two long isoforms which are developmentally regulated (Huang, Zhou et al. 2003). Disruption of all cypher isoforms in striated muscles is postnatally lethal and mice display severe congenital myopathy and dilated cardiomyopathy (Zheng, Cheng et al. 2009).

Specific knockout of long isoforms still gives rise to myopathy and late-onset dilated cardiomyopathy with delayed mortality, and is accompanied by disorganization of the z-lines. Interestingly, knockout of the long isoforms of cypher in the heart results in upregulation of several DAPC proteins including dystrophin (Cheng, Zheng et al. 2011). Although the mechanism of interaction with dystrophin in the heart is not known, cypher has been shown to bind myotilin and in turn filamin-c which binds the sarcoglycans (van der Ven, Wiesner et al.

2000; Guyon, Kudryashova et al. 2003; Zheng, Cheng et al. 2009). Therefore, through the intermediate filament network, dystrophin may indirectly associate with cypher at the z-disk. It is well established that mdx mice have force defects

(Lynch, Hinkle et al. 2001; Janssen, Hiranandani et al. 2005; Beastrom, Lu et al.

2011), and the association of cypher could functional link dystrophin to maintenance and stabilization of the contractile apparatus, particularly the z- disks.

The final protein that was identified as a dystrophin associated protein in the heart was αB-crystallin (CRYAB). CRYAB is a member of the small heat shock protein family and was originally identified in the lens, although abundantly

141 expressed in both cardiac and skeletal muscle (Dubin, Ally et al. 1990). Heat shock proteins bind unfolded proteins preventing aggregation and degradation, and CRYAB has been shown to bind microtubules and intermediate filaments specifically desmin and cytosolic actin (Launay, Goudeau et al. 2006). Upon ischemic injury CRYAB translocates to the z-disk in the heart helping to stabilize the sarcomeres (Golenhofen, Htun et al. 1999). By binding structural proteins,

CRYAB helps to maintain the integrity of the cytoskeleton against stress

(Goldfarb and Dalakas 2009). Knockout of CRYAB in the mouse heart results in diastolic dysfunction (Pinz, Robbins et al. 2008), and in humans mutations give rise to skeletal muscle weakness and heart failure, a type of muscular dystrophy termed α-crystallinopathy (Sanbe 2011). Unfortunately, the function of CRYAB with respects to dystrophin is not known, although it could be hypothesized that together they may help to stabilize the cytoskeleton and the link between the contractile apparatus and costameres similar to cypher.

Together out studies identify potential novel function of dystrophin in cardiac and diaphragm muscles, specifically regulation of intracellular calcium and membrane repair. Furthermore, our studies identify additional dystrophin associated proteins in the heart that strengthen the role of dystrophin as a structural protein in cardiac muscle. Interestingly, the disruption of these novel functions, particularly those similar between the heart and diaphragm could potentially lead to aberrant calcium influx and deregulation, a major hallmark and contributor to disease pathology in DMD. Currently, the exact mechanisms of calcium deregulation are poorly understood, however it is generally believed that

142 the major source of calcium influx is through micro-tears in the sarcolemmal membrane. In support of this, use of a membrane-sealing poloxamers are able to quickly seal membrane micro-tears thus reducing calcium influx and helping to protect the membrane and improve force output (Yasuda, Townsend et al. 2005;

Ng, Metzger et al. 2008). However recently, additional mechanisms of calcium influx have been elucidated. Specifically, dystrophin has been linked to the regulation of transient receptor potential cation (TRPC) channels which are deregulated in mdx skeletal muscles leading to increased intracellular calcium

(Sabourin, Lamiche et al. 2009). Our results here identify potentially new mechanisms of calcium influx which have not been previously shown. First, disruption in membrane repair can lead to improper or slowed sealing of micro- tears and thus a larger influx of extracellular calcium. Second, loss of dystrophin leads to disruption of ahnak1 at the sarcolemmal membrane, and therefore would be unable to bind and sequester the Cavβ2. This in turn would lead to prolonged activation of the L-type calcium channels and increased intracellular calcium.

Together, disruption of these functions potentially provides additional sources of increased intracellular calcium and help to elucidate the mechanism of disease leading to disrupted calcium homoeostasis for the dystrophinopathies.

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5.2 Novel functions of β-dystroglycan in skeletal muscle.

Previously, the functions and localization β-dystroglycan in skeletal muscle were primarily believed to be mediated through the inclusion in the DAPC.

However, our studies here identified novel β-dystroglycan complexes that exist at the membrane in wild type muscle and are not components of the skeletal muscle DAPC. Specifically, this includes the association of β-dystroglycan with cavin-1 and Cavβ2. Interestingly, while both of these proteins associate with dystrophin in cardiac and diaphragm muscle, we show that their association in limb muscle is specific to β-dystroglycan. Never the less, the association of these proteins identifies potentially novel β-dystroglycan functions in skeletal muscle, and possibly links β-dystroglycan to both membrane repair and calcium homeostasis. Interestingly, although cavin-1 does not associate with dystrophin in limb muscle, absence of dystrophin leads to a selective loss of the β- dystroglycan-cavin-1 complex. This finding further highlights the complex nature of β-dystroglycan, and adds an additional layer of complexity to not only the function of β-dystroglycan but also dystrophin in skeletal muscle. Similar to the heart and diaphragm, disruption of the dystroglycan-cavin-1 complex may lead to an impaired ability to properly seal the membrane and further calcium deregulation. As for the β-dystroglycan-Cavβ2 complex, the association of Cavβ2 is independent of both dystrophin and utrophin, and represents yet another potential link to the regulation of calcium homeostasis, although through a distinct mechanism being regulation of L-type calcium channels. While loss dystrophin

144 disrupts the DAPC and the association of cavin1, the absence of β-dystroglycan further disrupts additional β-dystroglycan complexes including ones containing

Cavβ2 and is likely the reason for the more severe muscle pathology observed compared to mdx. Indeed, skeletal muscle specific ablation of β-dystroglycan in mice results in a more severe pathology than absence of dystrophin, and is in support of our findings.

Taken together the results here provide compelling evidence for novel functions of both dystrophin and β-dystroglycan in cardiac and skeletal muscle.

These studies provide a critical first step toward our goal to fully elucidate the roles of dystrophin and β-dystroglycan, and identify novel pathways towards our understanding of the molecular mechanisms leading to muscular dystrophy.

5.3 Plasticity of the dystrophin associated protein complex.

One of the major findings from the studies presented in chapter II-IV is the plasticity of the DAPC and β-dystroglycan between different muscle tissues.

Specifically, we have identified several novel differences in the composition of these protein complexes including; the cardiac versus skeletal muscle DAPC, the diaphragm versus limb muscle DAPC, and the skeletal muscle DAPC versus β- dystroglycan complexes. These finding are graphically summarized in figure 5.1.

With respect to the DAPC, it is often portrayed as a static complex and is generally depicted as being highly similar for all striated muscles. In agreement with this, we show in that in cardiac and skeletal muscle the composition of the

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DAPC does not significantly differ for the majority of proteins. However, with the identification of novel cardiac specific proteins, the general assumption that the

DAPC is relatively similar for all muscle no longer holds true. Indeed, the studies presented here highlight the unique differences in the composition of the DAPC between different muscle types, and suggests to tissue specific functions of dystrophin. One recent example of this includes gene therapy strategies utilizing truncated mini- and micro-dystrophin constructs. Initial studies were thought to functionally rescue the heart with relative similar efficacy as in skeletal muscle.

However, subsequent studies testing the ability of these constructs to rescue the heart revealed only partial restoration of cardiac function where not all parameters were corrected (Townsend, Blankinship et al. 2007; Bostick, Yue et al. 2008). Because of the ability of the mini- and micro-dystrophin constructs to restore the basic structural function of dystrophin, it appears that the some other unknown function of dystrophin, likely mediated by unique proteins interactions must exist in cardiac muscle. Indeed, the studies presented in chapter II identify novel cardiac-specific dystrophin associated proteins that are not present in limb muscles.

As described in chapter III, the plasticity of the complex is not restricted to cardiac versus skeletal muscle, but also differs between diaphragm and limb muscles. This is of particular interest as the diaphragm is the most affected muscle in the mdx mouse and is the only muscle that closely resembles the pathology present in patients with DMD. Unique differences in the composition of the DAPC in the diaphragm, highlight yet additional tissue specific

146 differences of the DAPC. As previously mentioned these protein interactions are more similar to the heart than limb muscle, and potentially link dystrophin to membrane repair and calcium homeostasis. Therefore, disruption of these proteins in the mdx diaphragm will like compound disease mechanisms and is likely a causative factor for the more severe pathology observed in the diaphragm. Further studies are needed to address whether the human DAPC in diaphragm and limb muscles more closely resemble the mouse diaphragm.

Finally, our studies identifying novel β-dystroglycan complexes in skeletal muscle further add to the plasticity of members of the DAPC. Interestingly, although cavin-1 does not interact with dystrophin in limb muscle, it does associate with β-dystroglycan. Furthermore, this association is somehow dependent upon dystrophin. Currently the reasons for this difference are not understood. Never the less, the association of cavin-1 and Cavβ2 identify yet another tissue specific difference in the organization of function of these proteins in striated muscle. Future studies are needed to elucidate if these novel β- dystroglycan complexes are present in diaphragm and cardiac muscle.

5.4 High throughput mutational analysis

The majority of disease causing mutations in the dystrophin gene abolish protein expression and clinically give rise to DMD where all functions of dystrophin are lost. However for BMD, mutations do not completely disrupt expression but seemingly disrupt some specific function of dystrophin.

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Unfortunately, the exact mechanisms by which the majority of these mutations result in loss of function are not known. In striated muscle the functions of dystrophin are primarily mediated through protein interactions, and it is likely that the full scope of dystrophin interactions have yet to be identified. Therefore it could be inferred that for a subset of BMD mutations for which a truncated protein is still expressed, muscle pathology may be due to disruption of as yet unidentified protein interactions. Unfortunately, current studies on patient biopsy samples are limited to the analysis of the core DAPC.

The approach described in chapter II has successfully been applied to the analysis of human cardiac biopsy samples by immunoprecipitation and shotgun proteomics. Utilizing this approach, it would be possible to perform proteomics on

BMD patients for mutations that meet two criteria: 1) do not disrupt the recognition of MANDYS1 antibody and 2) maintain expression of a truncated dystrophin protein. For mutations meeting both criteria, profiles of the dystrophin interactome could be generated, and compared to a library of proteins from control tissue. This could then be used to assess how a particular mutation affects the protein interactions of dystrophin and potentially provide insights into the molecular mechanisms leading to disease. A specific advantage to using a biopsy sample is that it provides direct information on human disease mechanisms as opposed to the generation of mouse models mimicking human mutations, that also take longer to generate. Mutations that would be excellent candidates for proteomic profiling including those that disrupt regions of dystrophin not know to have a particular function but seemingly disrupt

148 unidentified functions of dystrophin. One specific example of this includes the

T279A missense mutation corresponding to hinge 1 (see intro) that does not disrupt expression of dystrophin but clinically gives rise to severe early onset dilated cardiomyopathy (Berko and Swift 1987; Towbin, Hejtmancik et al. 1993).

The results of these studies could further be coupled with a phenotype analysis to potentially decipher the functional consequence of losing specific protein interactions.

Another possible application of our approach could be directed towards understanding how mutations affecting the rod domain lead to myopathy. A mutational “hot spot” for dystrophin includes exons 45-55 which disrupt the spectrin-like repeats flanking hinge 3 (Kaspar, Allen et al. 2009). Currently, little is known about the function of the rod domain, and it has been proposed that the majority of the rod domain is dispensable (Harper, Hauser et al. 2002). This originates from a mutation that clinically resulted in mild BMD although the majority of the rod domain was not expressed (England, Nicholson et al. 1990).

In further support, mini- and micro-dystrophin constructs lacking the majority of the rod domain have proven capable of rescuing skeletal and cardiac muscle

(Harper, Hauser et al. 2002; Yue, Li et al. 2003). However, comparison of mini- and micro-dystrophin constructs in transgenic mice yield some interesting findings. Some of these constructs when expressed either transgenically or by viral delivery show significant improvement in histological studies including reduced centrally located nuclei, fibrosis, and Evans blue dye uptake (Harper,

Hauser et al. 2002). However, with the exception of the larger mini-dystrophin

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(ΔH2-R19), these constructs only partially restore muscle strength (Harper,

Hauser et al. 2002). To further complicate these findings, the ability to recue both diaphragm and limb muscle was inconsistent for a subset of constructs tested

(Harper, Hauser et al. 2002). Together, this would suggest that the function of the rod domain is not completely dispensable and may differ between tissues. Using the approach described in chapter II, mini- and micro-dystrophin could be immunoprecipitated, and libraries of dystrophin associated proteins could be generated for each construct. The results of these studies would be twofold: 1) it would allow for identification of novel proteins localized to the rod domain, and 2) by comparison to wild type and alternative constructs, limitations in protein binding for each construct could be assessed. Indeed a similar approach has been performed specifically focused on the interaction of nNOS. Using a subtractive approach of spectrin-like repeats, nNOS was shown to require repeats 16 and 17 for association with the DAPC (Lai, Thomas et al. 2009).

However, this approach was limited only to the study of nNOS. By using the approach described in chapter II, it would be possible to rapidly analyze the entire interactome of dystrophin, while simultaneously evaluating each construct for disrupted protein interactions and possible functional limitations. The information gained from these studies could further be applied to exon skipping strategies directed at skipping large regions of the rod domain currently with unknown implications for protein function.

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5.5 Considerations of the approach

The approach described here yields a wealth of information towards our understanding of the dystrophin interactome. However, there are some important factors that need to be considered when performing proteomic studies. Possibly the most significant limitation is the lack of mechanistic information. Proteomic analysis produces large datasets of interacting proteins but does not provide any information towards how these interactions occur. Furthermore, no direct conclusions can be drawn for direct versus indirect binding, which becomes challenging when studying dystrophin interactions due to the complexity of the

DAPC. Never the less, hints can be inferred from the LC-MS/MS results with caution. Protein identification is biased towards enriched proteins that generate more peptides thus creating higher MS scores. Therefore in can be inferred that proteins with consistently high scores are likely tightly associated proteins as opposed to transient associations. Examples of these include the α1-syntrophin and α-dystrobrevin which were always among the top scoring proteins for all proteomic runs. However, caveats to this observation do exist and must be keep in mind including proteins that do not digest well producing low amounts of peptides for LC-MS/MS analysis. Examples of these include transmembrane proteins and highly glycosylated proteins such as α- and β-dystroglycan which directly binds dystrophin but often scored lowest compared to the majority of other known DAPC proteins. Additionally, the transmembrane sodium channel

Nav1.5 which binds dystrophin through the syntrophins was not identified by LC-

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MS/MS. Western blot analysis of cardiac dystrophin IPs confirmed that Nav1.5 is present in our samples. This is likely due to compound factors including indirect interaction and transmembrane localization.

Unfortunately, in order to determine the exact mechanisms of association, we are limited to current strategies that rely on more traditional biochemical approaches. For dystrophin associated proteins, certain techniques can be used to better analyze how these interactions are occurring. The DAPC contains several protein binding motifs that have been well studied and characterized. Utilizing both known binding domains for proteins of interest along with protein databases and programs that predict putative domains, it is possible to better predict how these interactions are occurring, and can be used as a guide to characterizing the mechanisms of association. Furthermore, use of knockout mouse models for individual components of the DAPC, which do not disrupt dystrophin expression and localization can be used with a subtractive approach to better gain insight into the mechanisms of protein association.

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5.5 Figures

Figure 5.1: Summary of the tissue specific differences in the DAPC and dystroglycan complexes in wild type muscles. Proposed schematic representation illustrating the plasticity of the DAPC and dystroglycan complexes that were identified between cardiac and skeletal (diaphragm and limb) muscles. The mechanisms of association for these novel proteins are currently not known. The existence of additional dystroglycan complexes in cardiac and diaphragm muscles has not been studied.

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