Functional alterations and rhythmic disturbances by pan-histone deacetylase inhibition in the .

Item Type Dissertation

Authors Patel, Dakshesh

Rights Attribution-NonCommercial-NoDerivatives 4.0 International

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Link to Item http://hdl.handle.net/20.500.12648/1811 FUNCTIONAL ALTERATIONS AND RHYTHMIC DISTURBANCES BY PAN-HISTONE DEACETYLASE INHIBITION IN THE HEART

Dakshesh Patel

A Dissertation in Pharmacology

Approved

Date

Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Graduate Studies of the State University of New York, Upstate Medical University.

Acknowledgements PhD is a journey and I was directed, guided and surrounded by multiple people along the path of this journey. Each of them had defining role in my professional as well as personal development. First and foremost, I want to thank my family especially my parents and my sister (Dhruti Patel) for believing in me and supporting my decision to pursue graduate school. They are my pillars of survival in this world. I am grateful to God for bestowing HIS blessings via wonderful parents and supportive sister. Next, my salutation goes to my mentor Dr Richard Veenstra. He is the ultimate source of motivation and inspiration for me in science. I have always respected his work ethic and hardworking persona. He is a good teacher and I am grateful to him for giving me a project which spans from single electrophysiology to whole animal ECG recordings. Most importantly, I learnt patch-clamping which is just not a technique but it’s an experience which he gladly shared with me. I want to thank my committee members Dr George Holz, Dr Arkady Pertsov, Dr Eduwardo Solessio, Dr Steve Taffet for serving on my thesis defense committee. I want to especially thank Dr Taffet for believing in me and supporting me at every step of my graduate career with his valuable suggestions and inputs. I can never forget his assistance and encouragement during my graduate school. I am deeply grateful to Dr Peter Brink for driving from New York City and serving as an external examiner for my thesis dissertation. Most importantly, it’s the lab members who hone your skills and train you with nitty- gritties to achieve your goals. I am indebted to Li Gao for her relentless support and training for valuable techniques in the laboratory. I want to thank Dr Qin Xu (for initially helping me acclimatize to laboratory rubrics), Xian Zhang (for valuable scientific discussions), Christian Cassara (for his amicable political and scientific discussions) and Siyu Wei. Also, I can’t forget the valuable suggestions and motivation from Wanda Coombs, Aaron Glass and Liz Snyder. Good friends are indispensable for sound mental balance especially when you pursuing your graduate career away from the family. I want to personally thank Dr Anup Srivastava, Dr Geeta Negi, Dipmoy Nath, Disharee Das, Subhrajit Bannerjee, Avik Dutta, Anushree Gulvady and Stuti Sharma for lending an ear to my frustrations and providing emotional support since the I came to this country. Sacrifice of the neonatal and adult mice were the foundations of my project. I can never thank them enough for their contribution in my research project. Last but not the least, I want to thank Pharmacology Department and SUNY Upstate Medical University for providing me with resources and collegial environment at an excellent place for my career.

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LIST OF ABBREVIATIONS

AF KO Knockout AV node atrioventricular node LQTS Long QT syndrome AP MAPK Mitogen activated kinase APD Action potential duration MEF2 Myocyte enhancer factor-2 CaM Calmodulin MLC2v Myosin light chain 2v CTCL Cutaneous T-cell lymphoma NMVM Neonatal mouse ventricular Cx myocytes DAD Delayed After depolarization Ncad N-cadherin EAD Early after depolarization PKA Protein kinase A ECG Electrocardiogram PKC Protein kinase C FK228 Romidepsin, Istodax PTCL Peripheral T-cell lymphoma GJ PVC Premature Ventricular contractions gj Gap junctional conductance RTPCR Real time polymerase chain

Gj Normalized gap junctional reaction conductance RyR HAT Histone acetylases SA node sinoatrial node HDAC Histone deacetylase SR Sarcoplasmic reticulum I L-type calcium current TQT thorough QT Ca,L TSA trichostatin A Ij Junctional current USFDA United States Food and Drug INa Voltage gated sodium current - Administration Nav1.5 V Trans-junctional voltage INa,L Voltage gated sodium late current - j

Vm Membrane voltage Nav1.5 ICH International Council for VT Ventricular Tachychardia Harmonization VF Ventricular Fibrillation

Kd Dissociation constants -MHC -myosin heavy chain

Kfast and Kslow On-rates for fast and fast and slow The decay time constants slow inactivation for fast and slow inactivation

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Table of Contents List of Figures ...... 3 List of Tables ...... 4 Abstract ...... 5 CHAPTER 1: INTRODUCTION AND BACKGROUND ...... 7 Overview of HDACs and HDAC inhibitors ...... 8 Biological functions of HDACs ...... 8 HDAC inhibitors ...... 11 Role of HDACs and HDAC inhibition in heart ...... 14 Cardiotoxicity of HDAC inhibitors ...... 19 and Electrocardiogram ...... 22 Mechanisms of Cardiac ...... 29 Reentry ...... 30 Triggered activity ...... 33

Late Sodium current (INa,L) and arhythmogenesis ...... 37 Cardiac Safety Testing of Oncologic Drugs ...... 39 References ...... 42 CHAPTER 2: FUNCTIONAL ALTERATION OF PANOBINOSTAT IN VENTRICLES...... 52 Introduction ...... 53 Materials and Methods ...... 58 In vivo ECG monitoring ...... 58 Cardiomyocyte cultures ...... 60 Solutions ...... 61 Dual whole cell patch clamp recording ...... 62 Whole cell patch clamping...... 63 HDAC activity assays ...... 64 Real-time PCR ...... 64 Western blotting ...... 66 Statistics ...... 67 Results ...... 68 Electrocardiographic changes in conscious mice treated with pan-HDAC inhibitor 68 Panobinostat dose dependent inhibition of HDACs in neonatal mouse ventricular cardiomyocytes (NMVMs) ...... 69 2

Transient and steady-state cardiac ionic K+ currents during HDAC inhibition...... 69

HDAC inhibition reduces the magnitude of cardiac peak sodium current (INa) ...... 70 HDAC inhibitor does not alter the magnitude of L-type cardiac calcium current .... 71 HDAC inhibition induced action potential shortening ...... 72 Reduction in gap junction coupling by HDAC inhibition ...... 72 Regulation of , calcium handling, and gap junction by HDAC inhibition ...... 73

Reduction in Cx43 and Nav1.5 protein expression induced by HDAC inhibition.... 74 Discussion ...... 75 References ...... 107 CHAPTER 3: FUNCTIONAL ALTERATIONS OF HDAC INHIBITION IN MOUSE ATRIUM...... 114 Introduction ...... 115 Materials and Methods ...... 118 In vivo ECG monitoring ...... 118 Primary atrial culture ...... 118 Whole cell patch clamping...... 118 Real-time PCR ...... 119 Statistics ...... 119 Results ...... 121 Electrocardiographic changes in atria of conscious mice treated with pan-HDAC inhibitor ...... 121 Transient and steady-state cardiac atrial ionic K+ currents during HDAC inhibition...... 121 Regulation of ion channel, calcium handling, and gap junction genes by HDAC inhibition in atrial culture ...... 122 Discussion ...... 124 References ...... 136 CHAPTER 4: SUMMARY AND FUTURE STUDIES ...... 142 Summary ...... 142 Future Studies ...... 149 References ...... 156 CHAPTER 5: APPENDIX ...... 160

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List of Figures

Figure 1: Correlation of Electrocardiogram with Action potential...... 27

Figure 2: Examples of EAD and DADs ...... 37

Figure 3: Panobinostat dosing regimen...... 59

Figure 4: Electrocardiographic changes in mouse heart with panobinostat (pan-HDAC) inhibition...... 85

Figure 5: Dose dependent reduction in HDAC activity by panobinostat in neonatal mouse ventricular myocytes...... 89

Figure 6 : Transient and Steady State K+ currents before and after acute treatment of panobinostat 1µM in neonatal mouse ventricular myocytes...... 91

Figure 7 : Transient and Steady State K+ currents recorded after 24 hour treatment with 100 nM panobinostat in neonatal mouse ventricular myocytes...... 93

Figure 8: Dose dependent reduction in sodium current (INa) with various doses of panobinostat in neonatal mouse ventricular myocytes for 24 hours...... 95

Figure 9: I-V relationship of L-type calcium currents in neonatal mouse ventricular myocytes with various doses of panobinostat...... 97

Figure 10 : Shortening of action potential duration by treatment of neonatal mouse ventricular myocytes with 100 nM panobinostat for 24 hours...... 98

Figure 11: Dose dependent decrease in Gj conductance (nS) between neonatal mouse ventricular myocytes treated with panobinostat...... 99

Figure 12: Non-significant change in capacitance (pF) of neonatal mouse ventricular myocytes with increasing doses of panobinostat...... 100

Figure 13: Changes in mRNA expression of various LQT genes caused by 100 nM panobinostat treatment for 24 hours...... 103

Figure 14: Dose dependent reduction in Cx43, Nav1.5 levels by panobinostat in NMVMs & mouse treated with panobinostat 30 mg/kg intraperitoneally for two weeks...... 106

Figure 15: Atrial fibrillation in mice on with pan-HDAC inhibitor, panobinostat...... 130

Figure 16: Transient outward and steady state K+ currents of primary atrial culture treated with panobinostat for 24 hours...... 132

Figure 17: Changes in mRNA expression in primary neonatal atrial culture with panobinostat 100 nM treatment for 24 hours...... 134 4

List of Tables

Table 1 : Classification of HDAC inhibitors based on chemical moiety ...... 12

Table 2 : Congenital LQT syndrome types, mutated gene and ionic currents affected. ... 35

Table 3: External Pipette Solutions for recording various ionic currents ...... 61

Table 4 : Internal Pipette Solutions for recording various ionic currents...... 62

Table 5: Primer sequences of murine genes used for real time PCR ...... 65

Table 6: Table summarizing absolute and normalized ECG parameters of mice treated with panobinostat analyzed using Ponemah 5.2 ...... 86

Table 7: Table summarizing absolute and normalized ECG parameters of mice treated with panobinostat analyzed manually (Blind fold) ...... 87

Table 8 : Summary of arrhythmic events at 14th and 7th day...... 88

Table 9 : Summary of alterations in calcium, sodium currents and Gj coupling with different doses of panobinostat...... 101

Table 10: Primer sequences of murine genes used for real time PCR ...... 120 5

Abstract

Histone acetyl transferases (HATs) and histone deacetylases (HDACs) maintain a dynamic balance of acetylation and deacetylation of histone and non-histone .

HDAC inhibitors are small molecules anti- therapeutics that exhibited dose limiting cardiac toxicities during clinical and preclinical trials. Multiple instances of abnormal T-waves, ST segment depression, QT prolongation, grade 3 sinus bradycardia and non- circumstantial deaths have been observed in patients. The underlying electrophysiological and molecular mechanism of these cardiac side-effects are poorly understood. In our in vivo ECG monitoring using Data Science International® telemetry transmitters, mice injected with panobinostat showed ventricular tachychardic and atrial fibrillation episodes with significant prolongation of ST, QT and QTc intervals. In whole cell patch clamp studies, we observed no significant change in transient and steady state

K+ currents in myocyte ventricular cultures suggesting no role of hERG currents in ventricular arrhythmias. The majority (>90%) of congenital and drug induced QT prolongation is caused by alterations of hERG (IKr, Kv11.1) current. Interestingly, we observed significant reductions of INa and gap junctional conductance along with reductions in protein expression of Nav1.5 & Cx43 in vivo and in vitro. We conclude that pan-HDAC inhibition reduced cardiac INa density and gap junctional coupling with

+ unaltered late INa and K currents explaining the cardiac abnormalities exhibited by pan-

HDAC inhibitors. Decreased gap junctional coupling can enhance triggered activity by limiting electrotonic inhibition, combined with reduced INa density which can lead to 6

slow myocardial conduction. Both of them taken together, increases the vulnerability to reentrant arrhythmias.

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CHAPTER 1: INTRODUCTION AND BACKGROUND

DNA and histones are building blocks of nucleosomes. Over fifty years ago, Allfrey et al identified acetylation and deacetylation as possible post-translational mechanisms for regulating transcription in nucleosomes. DNA, being a negatively charged macromolecule, is tightly bound with the positively charged of histones.

Neutralization or retention of this positive charge by addition or removal of an acetyl group (-COCH3) leads to “active” or “inactive” forms of DNA ultimately affecting . Acetylation is regulated by two sets of enzymes; 1) Histone Deacetylases

(HDACs), that remove the ‘acetyl’ group and 2) Histone Acetyl Transferases (HATs) that add ‘acetyl’ groups. Addition of acetyl groups to the ɛ-amino terminals of at histone tails neutralizes the positive charge residues inducing a conformational change in chromatin structure leading to unwrapping of histones from the DNA. A dynamic balance of acetylation or deacetylation of histone and is maintained by HATs and HDACs

(Haberland et al., 2009). This dynamic equilibrium of protein acetylation or deacetylation induces or inhibits expression of key genes which regulate , cell differentiation and tissue development (Reichert et al., 2012). Currently there are 18 members of the

HDAC family and they are categorized into 4 different classes, which differ in structure, subcellular localization, enzymatic function, and expression pattern. Class I HDACs include HDACs 1,2,3,8, which are ubiquitous but preferentially localize inside the nucleus and are related to yeast RPD3 deacetylase. Class II HDACs include HDACs

4,5,6,7,9,10 and are homologous to Hda1 (histone deacetylase) in yeast. Class II HDACs 8

are further divided into two subclasses, IIa (HDACs 4, 5,7,9) and IIb (HDACs 6 and 10)

(Xu et al., 2007). Class IIa HDACs are primarily present in but can shuttle between nucleus and cytoplasm in response to intracellular cues by binding to 14-3-3 protein (Xu et al., 2007). Both Class I and Class II enzymes use Zinc-dependent inhibition at their active site (McKinsey, 2012). Class III HDACs encompass SIRT1 to

SIRT7 and need NAD+ for enzymatic activity (Blander & Guarente, 2004). HDAC11 is the only known HDAC of the Class IV HDAC family which uses Zinc-dependent inhibition at the active site. Although, it is found to be localized in , heart, muscle, kidney and testis, very little is known about the localization based function of HDAC11

(Gao et al., 2002a). HDACs lack an intrinsic DNA binding capacity but they are recruited to chromatin and are known to interact with a multitude of transcriptional coactivators and promoters. Classically, they are considered as transcriptional repressors, since they condense chromatin by removal of acetyl group from histones.

Overview of HDACs and HDAC inhibitors Biological functions of HDACs

Class I HDACs do not function independently but they work in concert with a catalytic core of multiple protein complexes like NCoR, SMRT, MEF, MeCP2 which are coactivators or corepressors (Glaser, 2007). Class I HDACs are known transcriptional co- repressors and various siRNA and knock out studies have implicated their role in , , apoptosis, and survival (Glaser et al., 2003; Wang et al., 2007).

Increased expression of Class I HDACs have been found in ovarian as compared to normal ovarian tissues with no change in expression of Class II HDACs (Su et al., 9

2008). Expression and function of both HDAC1 and HDAC2 is similar in several tissues and their roles are redundant in numerous physiological processes. They play an essential role in development of central and peripheral nervous system (Haberland et al., 2009).

Moreover, they have a redundant role in cardiac growth, adipogenesis, hematopoiesis, and epidermis development (Thaler & Mercurio, 2014). The role of HDAC1 and HDAC2 in cancer development and progression has been extensively studied. Santoro et al. described an interesting role for HDAC1 and HDAC2 as onco-suppressors during tumorigenesis and tumor maintenance in leukemic and pre-leukemic mice (Santoro et al.,

2013). In another study, HDAC3 knock out mice were shown to cause hepatocellular carcinoma signifying their role in liver development and tumor inhibition (Bhaskara et al., 2010).

Unlike most of the Class I HDACs, which are localized in the nucleus (except HDAC3, which has been found in sarcomeres), Class II HDACs get transported to the cytoplasm depending on intracellular signals. They have elongated N-terminals which are conserved binding sites for myocyte enhancing factor 2 (MEF2) and chaperone protein 14-3-3.

Phosphorylation of these HDACs by protein kinase D (PKD) and calcium/calmodulin dependent kinase (CAMK) causes binding of MEF2 and 14-3-3 to these N-terminal extensions eventually shuttling them from nucleus to cytoplasm.

HDAC5 and HDAC7 have been found in brain, muscle and heart (Haberland et al., 2009) and HDAC7 is highly expressed in endothelial cells while HDAC4 was principally found in growth plate of skeleton and brain (Shurong Chang et al., 2006; Haberland et al.,

2009). Class IIa HDACs are involved in several key developmental and differentiation 10

processes (Thaler & Mercurio, 2014). For example HDAC4 plays a central role in skeletal formation, synaptic plasticity and memory formation (Sando et al., 2012).

HDAC4 knock out mice died in their first week due to ectopic ossification of endochondral cartilage (Haberland et al., 2009). HDAC7 has been shown to selectively bind to hypoxia inducible factor α (HIFα) and regulate transcription (Yang & Gre, 2005).

Further, its localization in mitochondria has been linked with its role in intrinsic apoptotic pathway. Genetic ablation of HDAC5 and HDAC9 mice show abnormalities in growth and maturation of cardiomyocytes (Haberland et al., 2009). Class II HDACs have also been shown to promote cancer progression. For instance, HDAC5 has been described to be critical for differentiation (Clocchiatti et al., 2011), HDAC9 for medulloblastoma cell growth, and HDAC7 promotes cell growth via its regulation of c-Myc (Zhu et al., 2011).

Classically, histones are considered as primary targets of HDACs, however, recently more than 1700 non-histone proteins have been found to be post-translationally modified by HDACs (Bush & McKinsey, 2010; Choudhary et al., 2009). Class IIb HDACs are distinct in this regard because HDAC6 primarily is localized in cytosol, has been shown to deacetylate various cytoplasmic proteins like α-tubulin both in vitro and in vivo (Thaler

& Mercurio, 2014). HDAC6 plays a critical role in regulating cytoskeletal stabilization, , and cell-cell interaction by deacetylation of α-tubulin and cortactin, an actin remodeling protein. Moreover, another noteworthy target of HDAC6 is heat shock protein 90 (HSP90), altering its chaperone function. Interestingly, HDAC6 is the only

HDAC to have two independent catalytic deacetylase domains and although HDAC10 belongs to the same class IIb HDAC family, it has only one catalytic domain. HDAC10 is 11

the most recent of all discovered HDACs and little is known about its physiological functions. Recent studies suggest its involvement in metastasis by regulating matrix metalloproteinase 2 and 9 expression (Song et al., 2013). Silencing of

HDAC11, a known class IV HDAC, has been shown to induce in several solid tumor cell lines along with its increased abundance in numerous cancerous cell lines suggesting HDAC11 may be one of the potential targets for anti-malignancy (Gao et al.,

2002b).

HDAC inhibitors

A number of small molecules have been identified which can inhibit HDACs so far.

Class I, II and IV HDACs have zinc at the catalytic domain. This fact has formed the basis for the mechanism of action for most of the HDAC inhibitors developed till now.

Most of them are competitive inhibitors i.e the structure of the inhibitor resembles that of the substrate. They follow a “cap-linker-chelator” pharmacophore model, which is comprised of a ‘cap’ group that usually binds at the rim of the catalytic site, a ‘linker’ group consisting of a hydrophobic chain which resides along the tunnel of the enzyme, followed by a ‘chelator’ or Zinc binding group at the catalytic site.

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Table 1 : Classification of HDAC inhibitors based on chemical moiety (Katoch et al., 2013) (* Approved by FDA) Structural Class Examples

Short chain fatty acids Sodium butyrates, Valproic Acid

Benzamides CI - 994, Entinostat (MS-275), Mocetinostat

Hydroxamic Acids Trichostatin A, Vorinostat* (Suberoylanilide hydroxamic

acid), M-carboxycinnamic acid bishydroxamide, Azelaic

bis-hydroxamic acid, Dacinostat (LAQ824), Abexinostat,

Belinostat (PXD101) *, SB939, Resminostat, Givinostat,

Pentobinostat, CUDC-101, Panobinostat* (LBH589) ,

Oxamflatin,

Cyclic tetrapeptides Romidepsin* (FK228), Apicidine

Epoxyketones Trapoxin, Depudecin

Due to the association of HDACs in differentiation, cell proliferation, metabolism, and various critical physiological cellular processes, inhibition of these HDACs has shown promise in treatment of multiple types of cancers. At least 2-20% of the total gene expression have been shown to be altered by HDAC inhibition (Katoch et al., 2013).

Various HDAC inhibitors have been shown to induce terminal differentiation of transformed cells, cell cycle arrest, apoptotic cell death-involving both intrinsic and extrinsic pathway as well as autophagic and reactive oxygen species facilitated cell death

(Xu et al., 2007). The HDAC inhibition efficacy varies depending on cell type, 13

concentration, exposure time and the nature of HDAC inhibitor. Based on the anti-cancer effects of HDAC inhibition, HDACs have been attractive targets for drug discovery for the last few decades. Currently they are a part of more than 600 clinical trials, 25% of them are in active phase, in various malignant cancer and tumor indications

(clinicaltrials.gov). Over 400 PCT (Patent Cooperation Treaty) patent applications have been filed based on HDAC inhibitors. United States Food and Drug Administration

(FDA) approved vorinostat (trade name ZolinzaTM), a hydroxamate compound for treatment of cutaneous T-cell lymphoma (CTCL) in October 2006. In November 2009, romidepsin (FK228 or depsipeptide or trade name, IstodaxTM, a cyclic peptide, was also approved for CTCL, its indication was later expanded to peripheral T-cell lymphoma

(PTCL) in November 2011. Another hydroxamate, belinostat (trade name BELEODAQ®) was licensed to be prescribed for refractory and relapsed PTCL in July 2014. Recently, in

February 2016, panobinostat (LBH589 or trade name Farydak®), a hydroxamate too, was registered in combination with bortezomib and dexamethasone for multiple myeloma.

Currently, all these drugs alone or in combination therapy are being investigated in innumerable clinical trials for solid tumors, pancreatic cancer, , non-small cell and thyroid cancers. One such example is entinostat (MS-275), a benzamide, which in combination with exemestane (an aromatase inhibitor) has been given the ‘breakthrough status’ by the FDA because it has been extremely effective for patients with estrogen receptor positive breast cancer in Phase II clinical trials. All the approved HDAC inhibitors are pan-HDAC inhibitors except entinostat, which is a class I specific HDAC inhibitor. 14

Besides, exploiting HDAC inhibitor’s potential in oncological and cancers, they are also being investigated for neurodegenerative diseases, immune disorders and . Their mechanism of action can either be an alteration of signal transduction pathways, gene expression or modulation of post-translational acetylation of non-histone proteins, on a case to case basis (Katoch et al., 2013).

Over the last decade, numerous reports have indicated beneficial role of HDAC inhibitors in cardiovascular diseases such as atherosclerosis, myocardial infarction, , fibrosis, and heart failure. So, in the next few sections I will summarize the role of

HDACs in the heart and some examples of HDAC inhibitors that have shown to be beneficial in heart pathophysiology.

Role of HDACs and HDAC inhibition in heart

In the last few years, several studies have described the role of Class I and Class II

HDACs in cardiac cell proliferation, development and differentiation. While HDAC1 specific knockout mice died before embryonic day 10.5, there are contradictory reports regarding viability of HDAC2 knock out mice (Montgomery et al., 2007). One study found that global deletion of HDAC2 mice caused death in 24 hours after birth due to severe cardiac development issues while, on the other hand, Trivedi et al showed HDAC2 knock out mice were viable (Trivedi et al., 2007). These contradictory results were attributed to differences in exon deletions in individual studies. Alternatively, cardiac specific overexpression of HDAC2 causes a mild hypertrophic (Eom et al.,

2011). As mentioned earlier, the roles of HDAC1 and HDAC2 has been shown to be redundant in various tissues, as genetic deletion of HDAC1 or HDAC2 did not yield any 15

cardiac , however deletion of both HDAC1 and HDAC2 simultaneously caused neonatal lethality along with cardiac arrhythmias, dilated cardiomyopathy and upregulation of skeletal muscle contractile specific and genes

(Montgomery et al., 2007). Although, cardiac specific HDAC3 knock out mice showed abnormal lipid storage in the heart, these mice showed thickening of ventricular wall and intraventricular septum suggesting cardiac hypertrophy at the end of 3-4 months

(Montgomery et al., 2008). The initial drive to identify the role of Class II HDACs in cardiac hypertrophy came from the fact that they bind to MEF2 (a known hypertrophy marker), which assists their transport from nucleus to cytoplasm. In contrary to Class I

HDACs, Class IIa HDACs negatively regulate cardiac hypertrophy except the nuclear transport of HDAC4 and HDAC9 which induces hypertrophy. The physiological roles of

Class IIa HDACs are known to be redundant because deletions of individual class II

HDACs do not show any major phenotypes. For example mice lacking HDAC5 or

HDAC9 were viable and normal but a double knock out mice with dual ablation of

HDAC5 and HDAC9 showed thinning of ventricular walls and ventricular septal defects.

MEF2 is an essential factor which drives the pathological cardiac hypertrophic stress response. Hypertrophic factors stimulate the expression of MEF2 which binds to the extended N-terminals of Class II HDACs (HDAC5 and HDAC9) leading to their shuttling from nucleus to cytoplasm. Knock out mice devoid of HDAC5 and HDAC9 showed thinning of heart ventricular walls, which may be due to non-responsiveness of the intracellular compensatory mechanisms which involve transport of Class II HDACs from nucleus to cytoplasm (Chang et al., 2004). Although two classes of HDACs work in 16

opposite manner for inducing cardiac hypertrophy, the anti-cardiac hypertrophic activity of pan HDAC inhibitors like trichostatin A (TSA) is attributed to their Class I HDAC inhibition. This theory was further strengthened by recent studies showing effects of

Class I specific HDAC inhibitors like entinostat (Cavasin et al., 2012), apicidin (Gallo et al., 2008), and SK7041 (Kee et al., 2008) in cardiac hypertrophy. Class II HDACs show anti-hypertrophic activity in an enzyme independent manner. Stress activated protein kinases like calmodulin kinase (CAMK), protein kinase D phosphorylate class II

HDACs, MEF2 or chaperone protein 14-3-3 bind to them eventually shuttling them from nucleus to cytoplasm. Hence, their role in cardiac hypertrophy is attributed to their transcriptional regulation rather than enzymatic activity.

Cardiac fibrosis is also usually associated with cardiac hypertrophy. HDACs have been associated with differentiation of fibroblasts to myo-fibroblasts (which are activated fibroblasts, responsible for deposition of collagen) in cardiac fibrosis. More especially,

HDAC2 which has been found to be upregulated during scar formation (Fitzgerald

O’Connor et al., 2012).

HDACs have also been shown to be involved in regulating gene expression induced after ischemia-reperfusion (I/R) injury (Zhao et al., 2007). HDAC inhibitors downregulate

PGC-1α, which is a regulator of fatty acid oxidation and mitochondrial biogenesis in I/R injury (Ramjiawan et al., 2013). HDAC4 inhibition has been shown selectively to be involved in improving vascular permeability and reduce infarct size after injury in vivo and in vitro (Granger et al., 2008). Moreover, intraperitoneal injection of TSA 24 hours before I/R injury has been shown to reduce infarct size and improve contractile function, 17

mainly by blocking expression of hypoxia inducing factor α (Hif-α) and vascular endothelial growth factor (VEGF) (Ramjiawan et al., 2013). There are some controversial reports of HDAC involvement in atherosclerosis. TSA treatment in low density lipoprotein knock out mice caused deposition of fatty streaks in the aortic sinuses while

Okamoto et al reported TSA inhibited the proliferation of vascular smooth muscle cells, one of the most important factors for atherogenesis. TSA was shown to inhibit vascular smooth muscle cell proliferation by increasing the expression of potent negative regulator of cell cycle, p21WAF1/Cip1. Both of these reports underscore the essential role of HDACs in atherosclerosis (Ishibashi et al., 1993; Okamoto et al., 2006).

Studies focusing on the relevance of pharmacological HDAC inhibition in arrhythmias are limited. Ismat et al showed that overexpression of HopX, a gene which codes for a homeodomain protein which does not bind to DNA but is essential for cardiac growth and development, induced prolongation of PR intervals while PR intervals tend to be shorter in the HopX-/- mice (Ismat et al., 2005). TSA treatment was shown to be beneficial for atrioventricular conduction abnormalities in the HopX-/- mouse mainly by reducing the expression of connexin 40. It is uncertain if the anti-arrhythmic roles of TSA in these mice is due to alteration of connexin 40 expression or modifications in HopX gene especially when it was found that HDAC2 is associated with HopX (Liu et al.,

2008). Interestingly, a study reported increased expression of calcium channel subunits in the HDAC1 and HDAC2 knock out mice (Montgomery et al., 2007). Additionally, increased L-type calcium current is considered as one of the predominant factors leading to triggered arrhythmias (Weiss et al., 2010a). Moreover, global gene expression studies 18

of mice overexpressing HDAC2 reported dysregulation of ion channel genes. The study described decreased expression of type 2 α1 subunit (Scn2a1) and calcium channel beta 2 subunit (Cacnb2) while increased expression of sodium channel type 3 beta subunit (Scn3b) and one of the Ikr subunit (Kcne1) (Eom & Kook, 2014).

Congenital in Scn3b have shown to cause while, malfunctioning of Kcne1 have been strongly associated with long QT syndrome, with higher propensity to arrhythmias (Monteforte et al., 2012). In 2010, Colussi et al reported

5mg/kg/day administration of vorinostat for 90 days reduced arrhythmias in muscular dystrophic mouse (Colussi et al., 2010). Later, the same group in 2011 showed that post translational acetylation of connexin 43 by vorinostat leads to dissociation of gap junction followed by lateralization at intercalated discs (Colussi et al., 2011). Clearly, HDACs dysregulate expression and localization of various ion channel genes and gap junctions

(GJs) which can lead to cardiac arrhythmias.

Very little is known about the role of HDACs in hypertension, one or two elegant studies suggest a definite functional role of HDAC3 and the therapeutic value of HDAC inhibitors in hypertension (Lee et al., 2007). Another report specified that HDAC4 induces high blood pressure through increased vascular inflammation and TSA can rescue this phenomenon which ameliorates high blood pressure (Usui et al., 2012).

In summary, multiple HDACs are involved in various aspects of cardiovascular physiology and patho-physiology and a lot of interest has been generated over the last few years to understand the effect of HDAC inhibitors in cardiovascular diseases. HDAC inhibitors in multiple instances have shown promise in ameliorating pathological 19

conditions like cardiac hypertrophy, I/R injury, arrhythmias, fibrosis and hypertension.

The efficacy of HDAC inhibitors may be due to regulation or dysregulation of multiple gene transcription or by modification of signal transduction pathways or due to altered post-translational acetylation of non-histone proteins. Thus, their involvement in cardiovascular physiology can’t be underestimated. Along with the investigation of

HDAC inhibition for oncological indications, a new era for their exploration in cardiovascular diseases is on the rise.

However, due to participation of individual HDACs in a myriad number of signaling pathways and processes during pathophysiology, non-specific side effects of pan-HDAC inhibition are unavoidable. One of the most critical roadblocks to the development of

HDAC inhibitors as potential anti-cancer drugs or as drugs for any other indication is their dose limiting cardio-toxicities. I will summarize some of the cardiac side effects of pan-HDAC inhibitors in the following section.

Cardiotoxicity of HDAC inhibitors

HDAC inhibitors approved in the market have similar mechanisms of action, which explains the similarity in their toxicity profiles. The most common side-effects of pan-

HDAC inhibitors are fatigue, gastrointestinal disturbances like diarrhea, and dose-related hematological side effects like thrombocytopenia. Most importantly, HDAC inhibitors approved by FDA for respective indications have been shown to cause adverse cardiac side-effects in patients as well. In 2006, Shah et al discussed a Phase 2 clinical trial, where romidepsin was used for treatment in neuroendocrine tumors. The clinical trial was prematurely aborted due to cardiac side effects induced by romidepsin. One out of fifteen 20

patients died due to , possibly attributed to romidepsin, although patient characteristics, drug interactions, or electrolyte imbalance cannot be ruled out

(Shah et al., 2006). In August 2005, the romidepsin clinical trial initiated by the National

Cancer Institute involving 450 patients reported the sudden death of 5 patients (Byrd et al., 2005). In another study, Lynch Jr. et al in 2012 published a case report showing QT interval prolongation and in a patient with one week of 900 mg/m2 vorinostat treatment (Lynch et al., 2012). Extensive clinical and preclinical studies on mice, rats and beagle dogs treated with various doses of romidepsin, showed cardiac- related side effects. Beagle dogs, after i.v infusion of romidepsin over 2 weeks or more, exhibited elevated cardiac enzymes, prolonged QTc changes, and histo-pathological changes. A phase 1 clinical trial of romidepsin required extensive cardiac monitoring in patients. Reports suggest that romidepsin caused a 75% increase in the incidence of ECG changes and a more than 10% occurrence of cardiac events including QT prolongation, ventricular tachycardia and sudden cardiac death (Morganroth et al., 2010). Apart from romidepsin, severe and fatal cardiac arrhythmias and ECG abnormal changes have occurred in patients receiving panobinostat as well. Cardiac toxicities like abnormal T wave, ST segment depression, QT prolongation and grade 3 sinus bradycardia have been observed in panobinostat treated patients (Sharma et al., 2013). Arrhythmias occurred in

12% of patients receiving panobinostat compared to 1% in control. Electrocardiographic abnormalities such as ST-segment depression and T-wave abnormalities also occurred more frequently in patients receiving panobinostat (22% versus 4% and 40% versus

18%). Instances of prolonged cardiac ventricular repolarization have also been reported 21

(Novartis, 2015). Both romidepsin and panobinostat are prescribed in the market with a black box warning advising physicians to avoid these drugs in patients with history of previous cardiac abnormalities and to regularly monitor cardiac parameters during their treatment. Risk evaluation and mitigation strategy (REMS) is an initiative by FDA where drug companies are required to design a strategy to manage potential expected side effect of drugs. Panobinostat (trade name Farydak) is recommended by FDA to be prescribed along with a REMS to make sure its benefits outweigh the risks.

Moreover, atrial fibrillation (AF), which is the most common type of arrhythmia occurring with HDAC inhibitors (Subramanian et al., 2010), has been reported during the clinical trials of belinostat (Steele et al., 2008), dacinostat (de Bono et al., 2008), romidepsin (Shah et al., 2006) and panobinostat (Ellis et al., 2008).

Unfortunately, despite frequent occurrences of these cardiac side effects, the electrophysiological and molecular mechanisms that accrue these manifestations are unknown. This forms the foundation of our study which is to identify molecular and electrophysiological mechanisms explaining these cardio-toxicities of HDAC inhibitors.

Romidepsin and panobinostat are highly potent HDAC inhibitors that work at nano-molar concentrations. Their non-specific cardiac side effects limit their dose in the clinical setting. Our objective is not to challenge drugs which have been approved by FDA, but to identify potential mechanisms for these toxicities to help identify and design HDAC inhibitors with a broader therapeutic index. In order to get a better understanding of various mechanisms of arrhythmia, in the next few sections I will discuss the cardiac action potential and its correlation with different stages of the cardiac cycle. 22

Cardiac action potential and Electrocardiogram

Synchronous conduction throughout various regions of heart with contractile function ensures a continuous blood flow across the body. The selective differences in intracellular and extracellular ionic composition (Na+ and K+) and permeability leads to a potential difference of 80 mV across the of heart cell with a higher negative charge on the inner side of the membrane than the outside. This creates a resting membrane potential of -80 mV across the membrane. Normal cardiac excitability is characterized by a delicate balance of depolarizing current (which shift the cell membrane potential towards positive voltage) and repolarizing current (which shifts the cell membrane potential towards negative voltage). Myocardial activity in the heart is generated at the sinoatrial node (SA node) and passes to the atrioventricular node (AV node), after a brief delay the conduction reaches the bundle of His at the base of the ventricles and then spreads into the working myocardium via the bundle branches and the

Purkinje fibers. The shape of a cardiac action potential is divided in various phases (refer

Figure 1):

Phase 0 – This phase is characterized by rapid upstroke of action potential (AP) in the atrial, ventricular and His- Purkinje cells except nodal cells. Fast Na+ is the main current responsible for this rapid AP upstroke. INa is carried by voltage gated sodium channel

(NaV1.5) encoded by the gene Scn5a. This current is considered fast due to its rapid activation and inactivation kinetics. Depolarization by INa brings the membrane potential to 20 – 30 mV in ventricular cells, at this point INa approaches zero (it never reaches zero 23

+ because of membrane permeability to K ions), and by this time most of the Nav1.5 are inactivated (Nerbonne & Kass, 2005).

+ Phase 1 – Phase 1 repolarization consists of transient outward K current (Ito). Ito rapidly activates at the end of Phase 0 and inactivates with the start of Phase 2 repolarization.

This quick activation and inactivation of Ito produces a notch at the peak of AP. APs recorded from the epicardial layer (outer) of ventricles have a prominent notch in their

APs as compared to endocardial layer (inner). This is due to relative differences in expression of Ito across various regions of heart. Ito is composed of two currents Ito1 and

+ Ito2. Ito1 is further divided into fast (Ito,f) and (Ito,s). Ito1 is a K current which lacks calcium dependence but is sensitive to 4-aminopyridine (known as Ito blocker) while Ito2 is a calcium dependent chloride current less sensitive to 4-aminopyridine with higher sensitivity to salt (TEA). Subunits of Ito,f (Kv4.2/4.3) are coded by genes KCND2 and KCND3 while KCNA4, KCNA7 & KCNC4 are translated to subunits for Ito,s (Kv1.4/1.7/3.4) (Grunnet, 2010).

Phase 2 – It is also called the plateau phase. There is a short interval in this phase when the membrane potential stays relatively constant which gives the name, “plateau phase”.

L-type calcium current is the most prominent inward current for Phase 2. During the end of Phase 2, a late activating K+ current is also activated which is called a ‘delayed

+ rectifier’. In addition to ICa, L and K , INa may also be present during the plateau phase.

Usually, most of INa gets inactivated at the end of Phase 0 depolarization. Only a small fraction (1%) of this current does not get completely inactivated which is involved in the plateau phase is called, ‘sodium window current’. Classically, the calcium current can be 24

of two types, L-type (‘long lasting’ or ‘large’) and T-type (‘transient’ or ‘tiny’). They are encoded by genes CACNA1c and CACNA1g respectively. Entry of intracellular calcium during this phase stimulates calcium release from intracellular stores (sarcoplasmic reticulum) and is eventually responsible for mechanical contraction of cardiomyocytes.

This phase of cardiac AP links the electrical component to the mechanical function of heart cells (Bers & Perez-Reyes, 1999).

Phase 3- This is the final phase of action potential repolarization and is comprised of activation and inactivation of multiple subtypes of majorly K+ currents. There is great diversity in expression, subtypes and electrophysiological characteristics of K+ currents across different species as well as their regional distribution across heart (Nerbonne,

2000). Delayed rectifier K+ currents at the end of Phase 2 plateau phase are responsible for Phase 3 repolarization. Prominently, delayed rectifiers have been subdivided into rapid activating (IKr) and slow activating (IKs). IKs is composed of four subunits Kv7.1

(tetramer), encoded by gene KCNQ1 (also known as KvLQT1) and a non-pore forming β- subunit called, MinK, which is the product of gene KCNE1. Similarly, IKr is composed of

α-pore forming subunit of the channel Kv11.1, also known as ERG1 encoded by Kcnh2

(Grunnet, 2010). Two types of non-pore forming β-subunits are known to interact with

Kv11.1 and which can be MinK (encoded by KCNE1) or Mirp1 (encoded by KCNE2)

(Nerbonne & Kass, 2005). ERG1 is commonly referred to as human ether a-go-go related gene, hERG. IKr activates and inactivates rapidly and is a non-specific target of multiple classes of drugs like , etc. (classic hERG blockers). Since, IKr and IKs form the major currents during cardiac repolarization, unintentional off-target blockade 25

of hERG has been shown to cause life threatening cardiac arrhythmias. Hence they are a potential target for drug screening during pre-clinical studies. Apart from IKr and IKs,

+ another ultra-rapid delayed activator K (IKur) current also contributes to Phase 3 repolarization. It plays a major role in repolarization of atrial action potential rather than ventricular AP. IKur is carried by Kv1.5/3.1 and is encoded by KCNA1 and KCNA5 gene.

As the membrane potential approaches the resting potential, one more inwardly rectifying

+ K currents is opened due to unblocking of polyamines, which is called IK1. Kir2.X proteins form the subunits of IK1. There are four members of this family Kir2.1

(IRK1/KCNJ2), Kir2.2 (IRK2/KCNJ12), Kir2.3 (IRK3/ KCNJ4) and Kir2.4

(IRK4/KCNJ14) (Grunnet, 2010). Apart from IK1, IKATP and IK,ACh are other subtypes of inward rectifiers, where rectification is dependent on ATP and acetylcholine (secondary messenger pathway) respectively. The fundamental difference between inward rectifiers and transient outward or delayed rectifiers is that inward rectifiers are not voltage sensitive and the rectification of the current is known to be modulated by magnesium ions and polyamines from the intracellular side.

Phase 4 - This is the resting potential (RP) phase. In most cell types, the magnitude of IK1 current determines the RP. The significance of IK1 can be underscored by the fact that ventricles have a RP of -80 mV due to high IK1 while nodes devoid of IK1 have a maximum diastolic potential of -50 mV. These inwardly rectifying IK1 channels during the Phase 4 of AP sets the resting membrane potential which marks a diastole in a contracting heart. The total time taken by a cell to depolarize, repolarize and to come back to rest is called an action potential duration (APD). 26

These action potentials can be measured by placing an electrode on a single cardiomyocyte or by placing an electrode on the surface of the body. However, the surface electrocardiogram (ECG) measures the sum of all electrical activity in the heart.

In a standard lead II configuration, an electrode is placed on the right arm of the patient and the left leg to measure the electrical activity across the vertical axis of the heart. By convention, if the wave of depolarization goes towards the positive electrode, a positive signal is measured and vice versa. Each event on an ECG is denoted in alphabetical order starting with ‘P’. Atrial activation starts at the SA node which is represented as a positive deflection in ECG known as a “P-wave”. Atrial depolarization is followed by ventricular depolarization which is represented as QRS complex in ECG. ‘Q’ is small dip in deflection, followed by an ‘R’ at the peak. An ‘S’ is the signal that approaches the isoelectric baseline. Ventricular repolarization follows the ‘S’ wave that shows up on

ECG trace as a ‘T wave’. The ECG reflects the state of heart at a particular moment and any alterations in timings or characteristics of these waves serve as indicators of existing cardiac rhythm disorders and heart conditions (refer Figure 1) (Fermini & Fossa, 2003). 27

Figure 1: Correlation of Electrocardiogram with Action potential. A) Representative ECG trace showing QRS complex, PR interval, ST segment and QT interval B) Representative action potential showing all the phases 0 depolarization, Phase 1 (rapid repolarization), Phase 2 (plateau phase) and Phase 3 repolarization (adapted from Fermini & Fossa, 2003).

Heart Rate

It is the rate at which the heart beats. It can be determined using the RR intervals, which is the time between two consecutive ventricular contractions/beats.

PR intervals

A PR interval is defined as the time taken between the atrial activation and the onset of ventricular activation, which is the beginning of the P wave to the start of the QRS 28

complex. The PR interval gets prolonged due to defects in atrioventricular conduction.

On the other hand, shortening of PR intervals indicates faster impulse propagation from

SA node to AV node. This occurs in Wolff-Parkinson-White Syndrome.

QRS complex

It is measured as the beginning of the Q till the end of the S wave when the signal reaches the isoelectric baseline. Prolongation of QRS complex occurs during right or left bundle branch block (Becker, 2006).

ST segment

It represents the flat isoelectric baseline corresponding to ‘plateau phase’ of Phase 2, before the beginning of T-wave. ST segment elevation has been a fundamental diagnostic marker for myocardial infarction. ST segment gets wide spread in pericarditis (Di Diego

& Antzelevitch, 2014).

QT interval

The QT interval is the time from the start of the QRS complex to the end of the T wave and marks the time required by the ventricles to completely repolarize before getting ready to contract again. Abnormal prolongation of QT intervals has been shown to cause severe ventricular arrhythmias, especially torsades de pointes. These abnormalities may or may not revert back to sinus rhythm and can lead to sudden cardiac death.

Prolongation of QT intervals can occur due to congenital mutations or can be drug induced. Detailed mechanisms regarding the basis of QT prolongation will be discussed in the next section. 29

Since QT intervals tend to be shorter during faster heart rate and longer during bradycardia, they can be corrected depending on the RR intervals. The most common type of correction is Bazett’s correction and it is calculated by:

QTc = QT/ (RR) 0.5

Although Bazett is the most commonly used correction method it is non-reliable at very fast or slow heart rates. Hence, several other correction methods have been used. Some of them are as follows (Luo et al., 2004):

Friderica: - QTc = QT/ (RR) 0.333

Framingham: - QTc = QT + 0.154(1 - RR)

Hodges: - QTc = QT + 105 (1/RR – 1)

The relationship between RR intervals and QT is very complex, especially in mice due to its high HR. In our study, we have used the Bazett formula, because it is the most reliable and prevalent correction used for mice in the field (Roussel et al., 2016).

In the next section I will discuss principle mechanisms causing cardiac arrhythmias and we will test a few of them to identify potential mechanisms for cardiac toxicities of

HDAC inhibitors.

Mechanisms of Cardiac Arrhythmias The human heart beats normally at a rate of 72 beats each minute with a cardiac output of

70 ml each beat, pumping approximately 5 liters of blood every minute. Considering the average blood volume of 7.5 liters, total blood is recirculated throughout the body in approximately 1-2 min. This enormous task requires synchronous conduction in the heart along with a smooth transition from electrical to mechanical contractile function. Both 30

excitation of impulse along with recovery are important for this critical task. Disruption in the regular electrical activity can lead to arrhythmias. ‘Arrhythmia’ as the name suggests means without a rhythm. Dysrhythmic heart will have a reduced cardiac output

(product of heart rate and stroke volume) causing reduced blood flow to vital organs and eventually causes death unless recovered back to sinus rhythm. Several mechanisms have been proposed for arrhythmia and traditionally most of them have been divided into two main types a) Abnormal Conduction (Rentry); b) Abnormal automaticity (Triggered). I will discuss these mechanisms in brief below:

Reentry

Reentry occurs when an excitation impulse fails to quench and it excites a part of a tissue which has recovered from refractoriness. Unextinguished excitation can be due an anatomical obstacle in the path of an impulse (circus type), like an infarct, hypertrophy or a scar formation or 2) non-anatomical obstacle called as a functional obstacle (phase 2 reentry), which could be a core of refractory tissue (Allessie et al., 1973). In order for reentry to occur three conditions need to be met: i) a unidirectional block to prevent propagation wave from self-decaying; ii) slow conduction velocity; and iii) conduction time should be greater than the refractory period (Tse & Yeo, 2015). If these three conditions are met then a single trigger or stimulus can lead to multiple wavelets of propagation disrupting the heart rhythm. The wavelength of an excitation wave (λ) is defined as the product of conduction velocity (θ) and refractory time (tr) (Kléber & Rudy,

2004). Reduction in conduction velocity can be due to decreased membrane excitability which in turn can be a result of decreased availability of sodium channels (or diminished 31

INa). Several pathological conditions like acute ischemia (Downar et al., 1977), electrical remodeling (Pu & Boyden, 1997), or treatment with anti-arrhythmic drugs can reduce INa in turn reducing excitability. Congenital genetic mutations can also cause a loss of function of sodium channel function like in Brugada Syndrome (Antzelevitch &

Dumaine, 2011). The excitable cell serves as ‘source’ of electrical charge for the neighboring cell, ‘sink’, which gets excited when enough charge is transferred from the source to depolarize the cell to threshold. In the heart, cardiomyocytes are end to end connected by GJs especially at the intercalated discs. GJs consists of hexameric proteins, called connexons that dock from end to end to form a conduit between two neighboring myocytes. Each connexon is composed of six connexin protein as subunits. In the cardiac tissue, GJs play a critical role for the propagation of impulse from source to sink which can be determined by the extent of electrical coupling between cardiomyocytes. Regional differences in the properties of individual subtypes, their distribution and localization can disturb the conduction pathway significantly. Each ventricular myocyte is connected by

10 neighboring myocytes via intercalated discs (Jongsma & Wilders, 2000). Further, heart is a 3-dimentional tissue, and cardiac cells are arranged in a ‘brick stone’ type of configuration with regional differences in cell type, cell size and expression of ion channel proteins across heart. Innumerable studies estimating the direction of conduction velocity have determined that impulse propagation along the long cell axis (longitudinal) is 4 times higher than in transverse direction indicating an anisotropy in propagation

(Kléber & Rudy, 2004). The anisotropy ratio varies from 10 in the atria to 2 in the ventricles emphasizing the significance of varied factors in controlling propagation. 32

Connexin43 (Cx43) is the major connexin present in the heart among Cx30.2, Cx40 and

Cx45. Cx40 is predominantly found in the atria and in the His-Purkinje system while

Cx45 is expressed at the AV node and the conduction pathway (Fontes et al., 2012).

These differ in their single channel conductance as well as voltage gating properties which are further modulated by post-translational modifications like phosphorylation (Lampe et al., 2000), pH (Morley et al., 1996) or intracellular calcium levels (Xu et al., 2012). Apart from their individual subtype properties, they tend to co- localize together forming heteromeric or heterotypic channels, further increasing the complexity in predicting their cumulative or individual effect on impulse propagation.

Moreover, the pattern of localization of Cx43 in ventricles is altered in diseased states.

Normally, GJs are known to be localized end to end at the intercalated discs, however in certain pathological conditions like hypertrophic, dilated, ischemic cardiomyopathy and muscular dystrophy, Cx43 expression has been shown to be heterogeneous and relocated to the transverse side of the myocyte called, ‘lateralization’. This creates a source to sink mismatch in the flow of propagation and multiple wavelets generated can create a phase difference around regions of heart to create arrhythmic phenotype (Colussi et al., 2011).

Recently, a number of studies suggest that Nav1.5 and GJs co-localize to form a

‘connexosome’ at intercalated discs along with several other junctional proteins (Agullo-

Pascual, Cerrone, et al., 2014). C-terminal deletion of Cx43 has been shown to reduce INa and conversely heterozygous deletion of SCN5A showed heterogeneous expression of

Cx43, indicating the fact that altered expression and function of one affects the other

(Agullo-Pascual, Lin, et al., 2014; van Veen et al., 2005). Hence, both reduced cell-cell 33

coupling and decreased excitability are important determinants of conduction velocity.

Moreover, in most instances, there is slow conduction in one heart region with intact conduction around it causing heterogeneity in smooth conduction across the heart. This disturbance when met with other conditions acts as a substrate for reentry, eventually leading to reentrant arrhythmias.

Triggered activity

Triggered activity can be defined as unwanted, premature or delayed activation of cardiac tissue before complete repolarization or after a preceding action potential. This can occur before full repolarization which is called, Early after Depolarization (EAD) or after full repolarization, referred to Delayed after Depolarization (DAD).

EADs are considered as the primary mechanism for promoting arrhythmias in long QT syndrome (LQTS) (both congenital and acquired), polymorphic ventricular tachycardia, and ventricular fibrillation. In most instances, EADs are associated with prolonged action potential duration (APD), which can increase accumulation of intracellular calcium sufficient enough to raise the membrane potential causing a triggered beat as Phase-2

EADs. In addition, increased intracellular calcium enhances the sodium-calcium

+ 2+ exchanger current (exchanges 3 Na ions for one Ca , INCX) which resists repolarization.

With enough calcium accumulation, more calcium may get released through the sarcoplasmic reticulum (calcium induced calcium release). INCX increases further resisting repolarization in turn facilitating phase-3 EADs (Weiss et al., 2010b). Prolonged

APD can be due to an increase in the inward currents (INa and ICa) or reduction in the outward repolarizing currents. Prolongation of APD which correlates to QT interval on 34

ECG is found to be caused by congenital loss/gain of function mutations in major ionic currents of an AP, primarily repolarizing currents. 75% of LQTS causing autosomal loss of function mutations have been found in KCNQ1 (30-35% cases, IKs) and KCNH2 (25-

30%, IKr). These are major repolarizing currents in a human AP (Lehnart et al., 2007).

Loss of function mutations cause a reduction in repolarizing currents which in turn prolongs the AP. Conversely, LQT3 and LQT8 are caused by congenital mutations in depolarizing sodium and calcium channel subunits respectively. Interestingly, mutations in proteins that affect the anchorage of ion channels, pumps and exchangers on membrane can also cause LQTS. For example, LQT4 occurs due to defects in Ankyrin B.

Throughout the literature, exercise, emotional stress and sleep have been identified as triggers for arrhythmic episodes in subjects that possess LQTS genetic mutations. The degree of impact of these triggers varies depending on subtype of LQTS. For instance, exercise is the most intense trigger for LQT1 subjects, affecting 62% as compared to emotional stress (26%) and sleep (3%). In contrast, arrythmogenic episodes that occur in

LQT3 patients occur more frequently during sleep (39%) than during exercise (13%).

Fatal cardiac events due to emotional stress in LQT2 patients can occur in up to 43% of cases (P J Schwartz et al., 2001). A brief summary of type of LQTS, chromosomal , gene, protein and currents affected is provided in Table 2.

35

Table 2 : Congenital LQT syndrome types, mutated gene and ionic currents affected. (Lehnart et al., 2007) Protein/Ion Type of Chromosomal Mutated Gene Current LQTS Locus Affected KVLQT1 or KCNQ1 LQT1 11p15.5 K 7.1 α(I )↓ (heterozygotes) v Ks LQT2 7q35-36 hERG, KCNH2 Kv11.1 α (IKr)↓ LQT3 3p21-24 SCN5A Nav1.5 α(INa)↑ Ankyrin-B LQT4 4q25-27 ANK2, ANKB (INa,K ↓, INCX↓ ) KCNE1 (heterozyg LQT5 21q22.1-22.2 minK β(I )↓ otes) Ks LQT6 21q22.1-22.2 MiRP1, KNCE2 MiRP1 β (IKr)↓ LQT7* 17q23.1-q24.2 KCNJ2 Kir2.1 α (IK1)↓ ψ CaV1.2α1c LQT8 12q13.3 CACNA1C 2+ (ICa ,L)↑ LQT9 3p25.3 CAV3 Caveolin-3(INa)↑ LQT10 11q23.3 SCN4B Nav1.5 β4(INa)↑ LQT11 7q21-q22 AKAP9 Yotiao(IKs)↓ LQT12 SNTAI α-syntrophin (INa) Kir3.1and Kir3.4 LQT13 KCNJ5 (IK1) LQT14 RYR2 Ryanodine *also called, Anderson Syndrome Ψ also called,

Moreover, numerous drugs unintentionally prolong QT interval by blocking the primary repolarizing current in human (IKr) carried by the hERG channel. The list of drugs is not limited to anti-arrhythmic drugs, but includes anti-histaminics like astemizole, antibiotics like erythromycin, opiates like methadone, and antifungals as well as anticancer drugs

(Peter J. Schwartz & Woosley, 2016). QT prolongation is usually associated with torsades de pointes, which is a form of polymorphic ventricular tachycardia with abnormal QRS complex and characteristic twisting of isoelectric baseline. Usually, it 36

terminates in few seconds but can also transform into ventricular fibrillation, precipitating to sudden death (Morita et al., 2008). Although QT prolongation is not the only primary marker for pro-arrhythmic risk, it is used as a surrogate marker for screening drugs during preclinical trials and has been a common factor for withdrawal of multiple drugs from the market.

Delayed after Depolarizations (DADs) develop after complete repolarization mainly due to accumulation of intracellular calcium overload which is stimulated by exposure to digitalis, or catecholamines. All the processes that increase intracellular Na+, increase

Ca2+ entry, decrease Ca2+ efflux, stimulate the release of calcium from sarcoplasmic reticulum or reduce IK1 during diastole can be causative factor for DADs. Both EADs and

DADs have similar ionic mechanisms but one major difference between them is the timing of the triggered depolarization is during phase 2 or phase 3 in case of EADs and phase 4 in case of DADs. Moreover, the significant difference among them is that DADs occur at faster heart rates while EADs occur preferentially during bradycardia

(Antzelevitch & Burashnikov, 2011; Tse, 2016; Weiss et al., 2010b). (Refer: Figure 2)

37

Figure 2: Examples of EAD and DADs A) Representative traces showing Phase 2 & 3 early after depolarization (EADs) & B) Example trace of DAD & DAD induced extrasystole. Adapted from Antzelevitch & Burashnikov, 2011.

Late Sodium current (INa,L) and arhythmogenesis Late sodium currents were first identified in 1979, but their relative importance in causing arrhythmia was appreciated only after the identification of ‘gain of function’ of

SCN5a in LQT3 patients (Wang et al., 1995). These congenital mutations slowed inactivation of Nav1.5 potentiating INa,L in these subjects. Phase 0 depolarization caused activation of INa,peak which inactivated quickly, however inactivation is not complete and it gives rise to a tiny residual sodium current called as INa,L. Although the amplitude of

INa,L is 0.5 % of the INa,peak it lasts for hundreds of milliseconds, this causes an increase in

+ + Na accumulation (up to 30%) as compared to Na entry during INa,peak (Makielski &

Farley, 2006). Various pathological conditions like hypoxia, myocardial infarction, heart failure, numerous toxins and pharmaceutical drugs have been shown to alter the inactivation of Nav1.5 and to increase INa,L (Toischer et al., 2013). Significant increase in 38

INa,L during phase-2 has been associated with reduced repolarization reserve eventually causing prolongation of APD. INa,L blockers like and have been shown to reduce APD in Purkinje fibers and ventricular myocytes. This proves the idea that increasing INa,L can prolong APD (Shryock et al., 2013). Moreover, increased INa,L during the plateau phase of AP can tremendously increase the risk of EADs and DADs.

EADs are usually associated with prolonged AP, which have an enhanced calcium window current during the plateau phase. Accumulated calcium causes further increase in intracellular calcium due to calcium induced calcium release and also activates INCX in forward mode (starts exchanging 3 Na+ ions for 1 Ca2+ ion out). This positive inward current (INCX) along with increased INa,L can serve as a trigger for depolarization during the AP facilitating EADs (Shryock et al., 2013).

DADs, however, are triggered after complete repolarization. Increased INa,L may not serve directly in eliciting DADs, but it sets the stage by overloading cells with unwanted

2+ sodium, which activates INCX in reverse mode, leading to transport of Ca back to the cell. This augmented calcium during diastole may trigger DADs (Shryock et al., 2013).

Moreover, amplified INa,L can also cause beat to beat variability and transient regional heterogeneity across the myocardium, in addition to regional differences in AP properties that already exist across regions of the heart (Banyasz et al., 2015). In summary, INa,L can cause prolongation of APD and can directly or indirectly contribute to the generation of

EADs and DADs which have a potential role for arrythmogenesis. 39

Cardiac Safety Testing of Oncologic Drugs Cardiac safety is one of the most primary aspects of the drug development process.

Various drugs classes like anti-histaminics (astemizole, terfanidine), antibiotics

(sparfloxacin) anti-motility drug (cisapride) and many others have been withdrawn from the market due to their pro-arrhythmic potential like QT prolongation and incidences of torsades des pointes (Nachimuthu et al., 2012). Most of the drug induced QT prolongation is due to blockade of hERG. This promiscuity in binding of hERG is due to its wide central cavity compared to other K+ channels. This is how drugs get trapped inside the pore after closure of activation gate. Another distinct feature of the hERG channels is the presence of two aromatic rings in the central pore which support hydrophobic π-π interactions with functional groups present on the drug (Sanguinetti &

Mitcheson, 2005). Over the last few years it has become evident that growing number drugs for malignant indications have shown cardiac related drug toxicities including QT prolongation. These include HDAC inhibitors, multi-targeted tyrosine kinase inhibitors,

Src/Abl kinase inhibitors, multidrug resistance modulators and several others. In 2005,

The International Council of Harmonization (ICH) released guidelines S7B (non-clinical) and E14 (clinical) for evaluating cardiac safety addressing the pro-arrhythmic and QT prolongation of non-antiarrhythmic drugs (Morganroth et al., 2010). These guidelines were narrowly focused on hERG current block (IKr, primary repolarization current in humans) and QT prolongation by the test drug. ICH S7B guideline prescribed two core tests for proarrythmic risk assessment of potential drug, a) in vitro hERG assay, which can be studied via hERG expressed in human embryonic kidney cells or Chinese Hamster 40

Ovary cells and b) in vivo QT prolongation study in a suitable rodent and non-rodent species each.

By contrast, ICH E14 dictated a mandatory clinical trial (called as, ‘thorough QT trial’,

TQT) in healthy human volunteers evaluating the effect of novel molecules on QT intervals (Curigliano et al., 2008). E14 guidelines acknowledged that it can be difficult to administer drugs to healthy volunteers on safety and tolerability issues like for pediatric population or anti-cancer drugs. So most of the TQT studies for anti-cancer drugs are undertaken amongst the patient population. This posed a challenge in evaluating the QT interval liability of the test anti-cancer agent, independent of . Also, the outcome can be confounded by subject variability including comorbidities, electrolyte disturbances, and co-medications.

Another challenge posed was the FDA’s prime focus on acute effects of the drugs. A majority of the drugs block hERG in minutes or few hours while most of the anti-cancer drugs act by altering transcription or trafficking of hERG which takes 24-48 hours. This fact was not considered during the enactment of ICH S7B. For instance, drugs like arsenic trioxide used to treat promyelocytic leukaemia, pentamidine (anti-protozoal) and cardiac glycosides alter the trafficking of hERG without blocking hERG (Dennis et al.,

2007).

Moreover, relying on hERG blockade and QT prolongation liability of drug was unjustified for determining the pro-arrhythmic potential because drugs like , phenobarbital and tolterodine cause QT prolongation but were not found to be pro- arrhythmic. In addition, is a potent hERG blocker but it does not cause QT 41

prolongation possibly due to its L-type calcium channel blockade. is another example which prolongs QT but does not cause torsades de pointes. In a nutshell, non- hERG mechanisms can span from disturbed trafficking proteins to altered post- translational mechanisms to changed depolarizing/repolarizing currents due to congenital mutations or drug induced mechanisms. Any of these causative factors can predispose a patient to abnormal QT intervals (Sager et al., 2014). So, in order to overcome these limitations of previous guidelines USFDA in 2014 release new guidelines under Critical

Path Initiative called Comprehensive in vitro Pro-arrhythmia assay (CiPA). The three core guidelines dictated by them are: a) Determine the functional alterations in multiple cardiac ionic currents and not just hERG by the investigational drug; b) determine potential changes by the drug in ventricular action potential in silico; and c) in vitro assessment of drugs using human induced pluripotent stem cell cardiomyocytes (Sager et al., 2014). These inclusions will broaden the focus for preclinical screening of drugs with better cardiac safety, where huge impetus previously was only on evaluating a surrogate marker for pro-arrhythmia. The target date for implementation is December 2017.

My dissertation thesis in the following two chapters will focus on identifying electrophysiological and molecular mechanism of cardiac toxicity of currently approved

(for multiple myeloma) pan-HDAC inhibitor, panobinostat. The key question we want to address here is whether cardio-toxic effects of panobinostat during preclinical and clinical trials are due to hERG. If not then, we want to investigate other major ionic currents that may be involved.

42

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Zhao, T. C., Cheng, G., Zhang, L. X., Tseng, Y. T., & Padbury, J. F. (2007). Inhibition of histone deacetylases triggers pharmacologic preconditioning effects against myocardial ischemic injury. Cardiovasc. Res., 76(3), 473–81. http://doi.org/10.1016/j.cardiores.2007.08.010

Zhu, C., Chen, Q., Xie, Z., Ai, J., Tong, L., Ding, J., & Geng, M. (2011). The role of histone deacetylase 7 (HDAC7) in cancer cell proliferation: regulation on c-Myc. J. Mol. Med. (Berl)., 89(3), 279–89. http://doi.org/10.1007/s00109-010-0701-7

52

CHAPTER 2: FUNCTIONAL ALTERATION OF PANOBINOSTAT IN VENTRICLES.

# Data in this chapter include work done by Dakshesh Patel and Xian Zhang

Xian Zhang only performed experiments for changes in gap junctional coupling with 500nm and 1µM panobinostat in neonatal mouse ventricular myocytes

The contents of this chapter will be anticipated for a publication as, “Functional

Alterations of Ionic Currents and Rhythm Disturbances in the Heart by pan-Histone deacetylase inhibition”

53

Introduction Reversible acetylation and deacetylation of histone tails bound to DNA maintain a dynamic balance of active and inactive transcription, resulting in robust regulation in expression of key genes involved in cellular metabolism (Choudhary et al., 2014), differentiation, tumorigenesis (Santoro et al., 2013), angiogenesis (Yoon & Eom, 2016) and cell division (Haberland et al., 2009). Histone acetyl transferases (HATs) transfer

‘acetyl’ groups while histone deacetylases (HDACs) remove ‘acetyl’ groups, to lysines of histones and eventually maintain this dynamic homeostatic balance in transcription of crucial genes. HDACs can be divided into 4 distinct classes based on their sequence, localization, and function; Classes I, II, III and IV. Class II HDACs are further divided into IIa with an extended C-terminal domain and IIb which possess two deacetylase domains. Classes I, II, IV HDACs have Zn2+ in their catalytic cores while Class III

HDACs depend on nicotinamide adenine dinucleotide for their deacetylase catalytic function. Apart from regulation of transcriptional activity through reversible histone acetylation, HDACs also control post-translational acetylation of non-histone proteins.

More than 1700 non-histone protein targets including transcription factors like p53, c-

Myc and Nf-κB; signal transducers such as Stat3 and Smad7; proteins like paxillin, cortactin; α-tubulin; and molecular chaperones like heat shock protein 90

(HSP90), have been mentioned in the literature (Singh et al., 2010). Acetylation of proteins can alter their cellular localization, stability, protein-protein interactions and function.

Owing to HDACs’ crucial regulation in multiple gene transcription and pathways, HDAC inhibition has been widely studied and explored in the last decade. 54

Class I HDACs have been shown to be overexpressed in gastrointestinal cancer (Choi et al., 2001), prostate (Halkidou et al., 2004) and breast cancer (Zhang et al., 2005).

Alternatively, Class II HDACs are aberrantly upregulated in cervical, uterine and gastric cancers (Yoon & Eom, 2016). HDAC inhibition has been shown to upregulate p21

(potent cell growth arrester) and reduce expression of cell proliferation gene cyclin D1

(Alao et al., 2006). HDAC inhibitors induce the expression of pro-apoptotic genes like

Bax, Bak and Apaf1, thereby stimulating apoptosis in cancer cells (Marchion & Münster,

2007). Currently, HDAC inhibitors, alone or in combination with other drugs, are the subject of 610 ongoing clinical trials (135 are active) for various oncologic indications like multiple myeloma, cutaneous and peripheral T-cell lymphoma (CTCL, PTCL) and acute lymphoblastic leukemia (clinicaltrials.gov). To date, 4 HDAC inhibitors; vorinostat

(Zolinza®), romidepsin (Istodax®), belinostat (Belinodaq®) and panobinostat

(Farydak®); have been approved by United States Food and Drug Administration

(USFDA). Vorinostat, romidepsin, and belinostat were approved for CTCL and PTCL while panobinostat can be prescribed for treatment of multiple myeloma in combination with bortezomib (proteasome inhibitor) and dexamethasone. Interestingly, all of these

HDAC inhibitors cause pan-HDAC inhibition and have been shown to cause cardiac side effects in patients during preclinical and clinical studies. Unfortunately, no study exists which can illuminate the mechanisms of HDAC inhibitor induced cardiac toxicity. This dearth of information has created a roadblock in the path of HDAC inhibitors’ success story (Gryder et al., 2012). Therefore, the central focus of our study is to identify molecular and electrophysiological mechanisms for cardiotoxicity of HDAC inhibitors. 55

Although, there are multiple pan-HDAC inhibitors available commercially for this study, most of the experiments are based on the recently approved HDAC inhibitor, panobinostat or LBH589.

Panobinostat is a potent, cinnamic hydroxamic acid derived pan-HDAC inhibitor with an

IC50 lower than HDAC inhibitors in the same class (IC50 ≤ 13.5 nM for all HDACs except HDAC4, HDAC8 and HDAC7, IC50 is 203-500 nM) (Shao et al., 2008).

Panobinostat is 10 fold more potent than vorinostat and is currently the most potent

HDAC inhibitor in drug development. It is approved by USFDA for patients of multiple myeloma at a dose of 20 mg orally on 1st, 3rd and 5th day of the first two weeks of a 3 week cycle, for 8 weeks with or without food. It is prescribed with a black boxed warning for severe diarrhea and severe and fatal cardiac ischemic as well as arrhythmic events. In case of toxicities, USFDA recommends dosage to be reduced in units of 5 mg and be discontinued if the dosage is reduced to 10 mg three times a week. Approval is in combination with bortezomib and dexamethasone for multiple myeloma patients who have received two lines of standard therapy (Novartis, 2015). Panobinostat is currently undergoing clinical trials in combination for multiple other malignancies such as acute and chronic lymphocytic leukemia, chronic myeloid leukemia, neuro-endocrine tumors, prostate and renal cancer. Interestingly, all the clinical trials with panobinostat alone as the investigational drug have ceased.

Panobinostat has absolute oral bioavailability of 21% with a Tmax of 2 hours and a linear correlation of both Cmax and AUC with increasing doses of panobinostat. It is moderately bound to plasma protein (≈ 90%) and has an elimination half-life of ≈ 37 hours based on 56

patients with advanced cancer (Garnock-Jones, 2015). Concomitant administration of drugs which are strong CYP3A inhibitors or inducers should be avoided and appropriate dose adjustment is recommended for patients.

The most common adverse effect of panobinostat in patients is diarrhea (42% of patients compared to placebo), peripheral neuropathy, fatigue, thrombocytopenia and pneumonia

(San-Miguel et al., 2014). Apart from these adverse reactions, the most unexpected were the dose-dependent cardiac toxicities. Few patients receiving panobinostat had cardiac adverse reactions including, severe ischemia (1% of patients to control), severe and fatal cardiac arrhythmias (12% patients), ST- wave depression (22% compared to 4% in control), and significant T-wave abnormalities (40% compared to 18% control) (Novartis,

2015). In a Phase 1 dose escalation clinical trial of panobinostat, QT interval duration was prolonged by >30 milliseconds in ≈15% of patients with panobinostat 11.5 to 20

2 mg/m (based on surface area) achieving a Cmax of 565 to 784 ng/ml (≈ 1.6-2.2 μM)

(Sharma et al., 2013). It is strongly recommended to avoid panobinostat in patients with a previous history of cardiac diseases or co-administering it with QT prolonging drugs.

Adverse unexpected cardiac arrhythmias due to drug induced LQTS is the most common reason for withdrawal of drugs from the market. The majority (90%) of the drug induced

+ LQTS is caused by blockade of hERG (IKr), the primary rapid delayed rectifier K current responsible for repolarization in humans. Thus, we propose to identify if hERG blockade is the potential mechanism for panobinostat exhibited cardiotoxicity. If not, then we will evaluate other major ionic currents that may be altered by panobinostat leading to its cardiac side effects. Since the QT interval is the total time taken for depolarization and 57

complete repolarization of ventricles, in this study we investigated alterations in major depolarizing sodium current (INa) and calcium current (ICa,L). Moreover, we also addressed the molecular mechanisms that are responsible for cardiac electrophysiological changes induced by panobinostat.

58

Materials and Methods In vivo ECG monitoring

All procedures and experiments on mice were done according to the protocols approved by the Institutional Animal Care and Use Committee (IACUC). One day before the surgery, transmitters (ETA-F10) were taken out of sterile packaging and both the leads white (negative) and red (positive) were cut approximately to 55 mm and 42 mm respectively. The length of these leads depends on the size of the mouse. The insulation on the terminal ends of these leads were removed using a razor, exposing a tiny portion of bare electrode outside. A tiny portion at the extreme end of the leads was left covered with the insulation and was tied with an Ethicon® nylon silk suture. The transmitter was then incubated in saline at 37°C overnight. The same day C57BL6/J mice weighing no less than 17 g were selected and hair was removed from the abdomen and below left front limb using a hair removal cream (Nair®). On the day of surgery, the mouse was anesthetized with isoflurane and a nose cone was attached to the mouth for continuous anesthesia during the surgery. The mouse was kept on a 37°C heat pad throughout the implantation procedure. Breathing and heart beat was monitored visually throughout the surgery. Using sterile surgical instruments, the red lead was sutured to the abdominal wall on the right side using non-absorbable Ethicon® nylon suture (Ethicon, 4-0, K871) making sure that the bare lead touched the abdominal wall. Similarly, the white lead was passed subcutaneously towards the pectoral cavity and was sutured to the pectoral muscle. The transmitter resided in the abdominal cavity. The wounds were closed using an absorbable suture (Ethicon, 5-0, 1845). The mouse was allowed to recover for two weeks and appropriate post-operative care was provided. In total, 9 C57BL6/J mice were 59

implanted with transmitters each for three doses of panobinostat 10, 20 and 30 mg/kg intraperitoneally (n=3 each) with the following dosing regimen (Figure 3). At the end of

12 day, animals were sacrificed and ventricles and atria were collected for western blotting and RT-PCR analysis.

Figure 3: Panobinostat dosing regimen. Panobinostat 10, 20 and 30 mg/kg intraperitoneally was injected for the first five consecutive days of a week (1 week ON), with no injections for the next 2 days (2 days OFF) followed by five days in the second week (2nd week ON). ECG data collection was initiated 2 days before drug administration (basal recordings) and was continued throughout for two weeks. The daily recording time was 5-6 hours.

Mice ECG recordings were initiated 2 days before panobinostat treatment. Data was collected using a Data Exchange Matrix and acquisition software, Dataquest® ART. Data was analyzed using Ponemah 5.2® for calculating heart rate, PR, RT, ST, QT intervals and QRS complex duration. Detailed procedure for the surgery, animal care and maintenance was followed according to (McCauley & Wehrens, 2010). 60

Cardiomyocyte cultures

Newborn C57BL/6 mice [Charles River Laboratories] were anesthetized using isoflurane and the were excised in accordance with procedures approved by the Institutional

Animal Care and Use Committee (IACUC). The excised hearts were separated into atria and ventricles and kept in DMS8 (in mM: 116 NaCl, 5.4 KCl, 1.0 NaH2PO4, and 5.5 dextrose). The hearts were cut longitudinally and horizontally to maximize exposure of collagenase solution with the interiors of the ventricles. They were then placed in a 25-ml

Erlenmeyer flask containing 5 ml collagenase dissociation medium, which contained 2 mg/ml collagenase (type 2; Worthington), 5.5 µg/ml DNase I (Worthington), and 1 mg/ml BSA (fraction V; Sigma Chemical, St. Louis, MO) dissolved in DMS8. The atria were placed into the 5 ml collagenase dissociation medium. After 25 seconds of oxygen perfusion, the tissues were placed in a 37 ° C for 10 min. The supernatant from each 10-min dissociation cycle was filtered through a 70-µm cell strainer (Falcon,

Franklin Lakes, NJ) into 5 ml of M199 cell culture media supplemented with 10% FBS

(GIBCO BRL, Gaithersburg, MD) and 100 U/ml penicillin-streptomycin (Invitrogen,

Grand Island, NY). After the fourth dissociation cycle at 37°C, the remaining tissue was gently pipetted, and the cell suspension was centrifuged at 500g for 5 min. The pellet was suspended in 12 ml culture media and pre-plated in a 100-mm culture dish for 30 min to get rid of the fibroblasts by differential cell adhesion at 37°C. The supernatant was collected, centrifuged and re-suspended in 1 ml/heart culture media. The primary cells were plated into 96-well plate for HDAC activity assay, 35 mm culture dishes for patch 61

clamp electrophysiology, or fibronectin-coated 18 mm diameter glass coverslips for imaging and various other experimental purposes.

Solutions

Please refer Table 3 for detailed composition of the external pipette solution for individual ionic currents, gap junctional coupling and action potential duration experiments.

Table 3: External Pipette Solutions for recording various ionic currents INa – Sodium current, ICa,L – L-type calcium current , Gj coupling – Gap junction coupling

IK (transient Action Reagents outward and INa ICa,L Potential Gj coupling Steady State) Duration NaCl (mM) 140 50 140 140 CsCl (mM) 110 120 4 TEACl (mM) 20 2 NaH2PO4 (mM) 1 1 KCl (mM) 1.3 1.3 5.4 1.3 CaCl2 (mM) 1.8 1.8 1.8 2 1.8 MgSO4 (mM) 0.8 0.8 MgCl2 (mM) 1 1 1 CdCl2 (mM) 0.1 HEPES (mM) 10 10 10 10 10 Dextrose (mM) 5.5 10 5.5 10 5.5 Sodium Pyruvate 2 (mM) Tetrodotoxin 30 (μM) (μM) 1 1

pH 7.4 7.4 7.4 7.4 7.4

Titrated with NaOH CsOH CsOH NaOH NaOH

62

Please refer Table 4 for detailed composition of the internal pipette solution for individual ionic currents, gap junctional coupling and action potential duration experiments.

Table 4 : Internal Pipette Solutions for recording various ionic currents. INa – Sodium current, ICa,L – L-type calcium current , Gj coupling – Gap junction coupling

IK (transient Action Reagents outward and INa ICa,L Potential Gj coupling Steady State) Duration NaCl (mM) 10 10 CsCl (mM) 135 120 TEACl (mM) 20 KCl (mM) 140 140 140

CaCl2 (mM) 3 2 1.8 3

MgSO4 (mM)

MgCl2 (mM) 1 1 1 1 1 EGTA (mM) 5 5 BAPTA (mM) 5 5

HEPES (mM) 25 10 20 10 25 ATP (Magnesium 3 4 Salt, mM) pH 7.4 7.4 7.4 7.2 7.4 Titrated with KOH CsOH CsOH KOH KOH

Dual whole cell patch clamp recording

Gap junction currents (Ij) were recorded in the dual whole cell configuration according to previously published methods (Veenstra, 2001). Upon establishing a dual whole cell 63

patch, Ij was recorded using a 30 second, 20 mV trans-junctional voltage protocol. All dual whole cell current recordings were low-pass filtered at 500 Hz and digitized at 2

KHz using pClamp 8.2 and graphical analysis performed using Origin 7.5 software as previously described (Xu et al., 2013).

Whole cell patch clamping

Single whole cell patch electrode voltage clamp experiments were performed on neonatal mouse ventricular myocytes (NMVMs) using conventional procedures with an Axon

Instruments Axopatch 1D or 200B patch clamp amplifier, Digidata 1320A or 1440 A/D converter, and pClamp8.2 or 10.1 software (Molecular Devices). Transient (peak) and steady state outward potassium (IK,to and IK,ss) currents were recorded during 1 sec voltage steps from a holding potential (Vh) of -100 mV to +60 mV in 10 mV increments. (Refer:

Table 3 and Table 4 for solutions, Figure 7 for protocol). Voltage-gated sodium currents

(INa) were elicited from a holding potential (Vh) of -120 mV during voltage steps from -

90 mV to +50 mV in 5 mV increments for 150 ms using reduced NaCl solutions (Refer:

Table 3 and Table 4 for solutions, Figure 8 for protocol)

Alembic VE-2 amplifier was used for up to 99% series-resistance compensation. In some experiments, 30 μM tetrodotoxin (TTX) was added to the bath to verify that the inward current was TTX-sensitive.

2+ L-type Ca currents (ICa,L) were elicited with a Vh of -50 mV by voltage steps ranging from -40 mV to +60 mV for 500 ms in 10 mV increments in the presence of 30 μM TTX.

The rest interval between pulses was 2010 ms. 1 μM nifedipine was added to the bath to verify that the inward current was dihydropyridine-sensitive (refer Table 3 & Table 4 for 64

solutions, Figure 9 for voltage protocol). Ventricular action potential duration were recorded from neonatal mouse ventricular myocyte cultures in the whole cell current clamp configuration. Action potentials were stimulated by application of 100 µsec, 100 pA depolarizing current pulses at a frequency of 1 Hz. The internal and external solutions were used as mentioned in Table 3 & Table 4 respectively.

HDAC activity assays

Aliquots of 2 x 105 ventricular myocytes or HeLa cells per well (96-well plate) were grown in 200 μl of 200 μM bromodeoxyuridine (BrDU)/M199 or N2a culture media, exchanged daily. Cell densities were counted with a hemocytometer and seeded into the wells. Wells were incubated with 2,000 pmoles of the acetylated Fluor-de-Lys® substrate for 8 hours during HDAC inhibition. Cells were incubated with varying doses of HDAC inhibitor, panobinostat, for 24 hours. Protocols for HDAC activity assay were developed according to manufacturer’s directions and background subtracted relative fluorescence unit (RFU) counts were acquired with a BIO-TEK Synergy 2 plate reader (360 nm excitation, 460 nm emission). Panobinostat was commercially obtained from Selleck

Chemicals, LLC.

Real-time PCR

Cellular RNA was extracted with a Qiagen RNeasy® mini kit, quantified by UV absorption (260 nm & 230 nm), and 500 ng of the RNA was reverse-transcribed with

Superscript®III™ (Qiagen). The RT-PCR was carried out on 384 well plates using

LightCycler® 480 Real-Time PCR System (Roche). 1 μl of the 1:5 diluted cDNA reaction, 1 μl of 50 nM custom forward and reverse RT-PCR primer mixes, 5 μl of 65

enzyme and SYBR Green dye mix and 3 μl of water was added in a total volume of 10 μl

(LightCycler® 480 SYBR Green I Master Superscript®) in duplicates. All results were normalized to GAPDH and control samples. cT values were determined by the apparatus and the quality of the PCR product was confirmed by analyzing the melt-curve (Tm). RT-

PCR primers for murine Cacna1c, Cacna1g, Kcnd2, Kcnd3, Kcnh2, Kcnj2, Kcnq1,

Kcne1, Kcne2, Scn5a, Scn4b, Ank2, Slc8a, Cdh2, and Gja1 were designed to span exon- intron regions of the gene of interest. All forward and reverse primers were purchased from realtimeprimers.com or Life Technologies. The murine ion channel forward and reverse primer sequences are listed in Table 5 below:

Table 5: Primer sequences of murine genes used for real time PCR Gene Forward Primer Sequence Reverse Primer Sequence Gapdh 5'- TGCCACTCAGAAGACTGTGG-3' 5'-AGGAATGGGAGTTGCTGTTG-3'

Gja1 5'-GAGAGCCCGAACTCTCCTTT-3' 5'-TGGAGTAGGCTTGGACCTTG-3'

Kcne1 5'-AGACACACTGAGGCTCCAAG-3' 5'-CAGTCTCTGTCCTGGCATCT-3'

Kcnh2 5'-CAGGCTGACATCTGCCTACA-3' 5'-GAGGCTCTCCAAAGATGTCG-3'

Kcnd3 5'-CATCCCTGCATCTTTCTGGT-3' 5'-CCCTGCGTTTATCTGCTCTC-3'

Kcnd2 5'-CAGAACTGGGCTTCTTGCTC-3' 5'-ACCCAAAAATCTTCCCTGCT-3'

Kcnq1 5'-CAGCAGTGTAGGGCTTCTTCC-3' 5'-GGGTTGGAAGTGTTTCGTGT-3'

Kcnj2 5'-TTGCTTCGGCTCATTCTCTT-3' 5'-AGAGATGGATGCTTCCGAGA-3'

Kcne2 5'-AATTTGACCCAGACACTGGA-3' 5'-ACCACGATGAACGAGAACAT-3'

Cacna1c 5'-CAGCTCATGCCAACATGAAT-3' 5'-TCTTGGGTTTCCCATACTGC-3'

Cacna1g 5'-CCTCACCACCTTCAACATCC-3' 5'-TGAGCGTCCTGTGTGAACTC-3'

Scn5a 5'-CTTCACCAACAGCTGGAACA-3' 5'-GACATCATGAGGGCGAAGAG-3'

Scn4b 5'-TCCTCTTGAGTGACCTGGAG-3' 5'-CCAGGATGATGAGAGTCACC-3'

Ank2 5'-CAGAAACCACAAACCCACTC-3' 5'-AGCACGGATGCTCAAAATAG-3' 66

Cdh2 5'-TATGTGATGACGGTCACTGC-3' 5'-GAAAGGCCAT AAGTGGGATT-3'

Slc8a 5'-TGAATCTTGCACGCTCATTA-3' 5'-CCAGGCACATCCAAAGTATC-3'

Western blotting

Ventricular myocytes were cultured at high density in 35 mm culture dishes for four days in 3 ml of BrDU/M199 media, harvested, and lysed with 1% Triton X-100 extraction buffer (50 mM Tris pH 8.0, 150 mM NaCl, 0.02% Sodium azide, 1.0 mM PMSF, 1 µg/ml

Aprotinin, 1% Triton X-100, 1 mM Na3VO4, 50 mM NaF) with protease inhibitors

(Roche). One dish from each primary culture served as a control sample and a second dish was treated with panobinostat for 24 hours prior to harvesting. Sonicated samples

(three 30 sec pulses) were incubated on ice for 30 min, centrifuged at 14,000 rpm (10 min

@ 4 ºC), transferred to new tubes, and protein concentrations were measured using the coomassie blue protein assay (Bio-Rad). Total protein/sample was heated (55 ºC) and loaded onto an SDS-PAGE gel and electrophoresed for 90 min at 110 V in 4X Nupage sampling buffer and 10X Nupage reducing buffer (Bio-Rad). The protein gels were transferred onto polyvinylidine difluoride (PVDF) membranes for 90 min at 4 ºC (110

V), blocked with 5% nonfat milk for 1 hour at room temperature, and incubated overnight at 4 ºC with primary antibodies against Cx43, N-cadherin, Nav1.5 or α-tubulin in PBS-T with 5% nonfat milk. Acetylated α-tubulin and acetylated H3 were used as cytoplasmic and nuclear marker for acetylation in myocytes. The membranes were washed 5 min  4 with PBS-T, incubated with HRP labeled secondary antibody (1:5000) at room temperature in PBS-T with 5% nonfat milk for 30 min, washed again 5 min  4 with 67

PBS-T, and developed using the ECL™ Western Blot Detection Reagents (Bio-Rad). The image was detected using the Biorad imager. The density of the bands was quantified using ImageJ.

Primary antibodies used in this study include rabbit anti-Cx43 (Millipore), mouse anti-

Cx43 (Millipore AB1727), mouse anti-α-tubulin (Sigma# T5168), rabbit anti-acetylated

H3 (Millipore#06-599), rabbit anti-acetylated-α-tubulin (Enzo), rabbit anti-NaV1.5

(Alomone- ACC-001) and N-cadherin (Biomol#610921).

Statistics

Averaged values are presented as the Mean ± SEM. Statistical analyses were performed with the Normality and one-way ANOVA tests using the Bonferroni method in Origin

8.6. Data with p < 0.05 was considered significant unless specified. REST 2009® was used to analyze RT-PCR data, using two internal controls Gapdh and Rpl13a as internal controls.

68

Results Electrocardiographic changes in conscious mice treated with pan-HDAC inhibitor

To determine the in vivo ECG changes in a conscious mouse by pan-HDAC inhibition, panobinostat was injected intraperitoneally based on the regime shown in Figure 3. We recorded a statistically significant 31% and 25% prolongation of ST and QT intervals

(22% for corrected QT, based on Bazette’s formula) by panobinostat 30 mg/kg (n=3)

(Figure 4b, G & H). Multiple instances of extrasystole (premature ventricular contractions) were also recorded in ventricles (Figure 4b, D), summarized in Table 8.

Ventricular tachycardia episodes were induced by panobinostat 20 and 30 mg/kg which reverted back to sinus rhythm as shown in Figure 4a (B&C) as compared to control

(Figure 4A) which had normal sinus rhythm. Figure 4b (E) shows a representative ECG from the same mouse before and after at 12th day injected with 30mg/kg panobinostat.

Clearly, we can see prolonged PR, ST and QT intervals along with severe bradycardia.

One of the three 30 mg/kg mice developed 3rd degree AV Block and died less than 3 minutes later (Figure 4b, F). Table 6 summarizes the ECG parameters of all treatment groups on their last day normalized to their respective values before treatment.

Panobinostat 30 mg/kg treatment group shows higher variability because of excessive prolongation of QT and ST intervals which is outside the defined constrained values of automated software used to analyze data (Ponemah 5.2). Hence, we calculated ECG parameters manually in a blind fold fashion, indicating much higher statistical significant prolongation of QT and ST intervals by 30 mg/kg i.p (Table 7). 69

Panobinostat dose dependent inhibition of HDACs in neonatal mouse ventricular cardiomyocytes (NMVMs)

By performing a flourometric HDAC activity assay, we found that panobinostat has a high capacity, high affinity site (56 nM) and a low capacity, low affinity site (8.9 µM, respectively) in NMVMs suggesting that these different Ki’s might correlate with differential affinities for ClassI/IIb and ClassIIa HDACs (Figure 5). These differential affinities can be correlated with the off-target effects of pan and class-selective HDAC inhibitors.

Transient and steady-state cardiac ionic K+ currents during HDAC inhibition.

The majority of ‘congenital’ or ‘drug’ induced LQTS are caused primarily by reduced repolarization. Since, hERG (IKr) and IKs are the major currents involved in repolarization of cardiac AP, we studied acute and long-term changes in steady state and transient outward K+ currents in NMVMs treated with panobinostat. We recorded insignificant changes of steady state and transient outward K+ currents after acute (Figure 6) and long term (Figure 7) (24 hours) treatment with 100 nM panobinostat. This dose was selected on the basis of HDAC activity assay performed in the previous experiment. Although, myocytes look ‘stringy’ and elongated after 24 hour drug treatment, there was no change in input capacitance (Cin) with increasing doses of panobinostat (Figure 12). These currents were recorded in presence of 30 M TTX, 100 M CdCl2, and 1 M nifedipine to isolate K+ currents specifically and were clamped as shown in Figure 6C & Figure 7C.

The unaltered steady-state and transient outward K+ currents by 100 nM panobinostat 70

indicated that non-hERG or other mechanisms apart from K+ currents may be involved in drug induced cardiac side effects.

HDAC inhibition reduces the magnitude of cardiac peak sodium current (INa)

Sodium current (INa) is one of the primary currents responsible for excitability of cardiac myocytes and alterations in macroscopic INa have been shown to cause arrhythmias

(Rook et al., 2012). Therefore, we clamped INa current in NMVMs treated with 24 hours using the internal, external solutions and protocols mentioned in Table 3, Table 4, and

Figure 8D respectively. In contrast to ICa,L and IK currents which did not change with panobinostat treatment, there was a 59.3% reduction in INa with 100 nM panobinostat

(-146.5 ± 26 pA/pF) & 55% with panobinostat 500 nM (-135.9 ± 20.4 pA/pF)) as compared to control (-247.2 ± 42.8 pA/pF) (Figure 8F). We did not see any change in input capacitance (Cin) with panobinostat treatment (Control: 40.7 ± 5.3 pF, n=25; 1 μM panobinostat: 32.9 ± 4.2 pF, n=9) (Figure 12, Table 9). We also observed +2 mV shift in steady state activation (Boltzmann charge-voltage fit for activation, V1/2 for, control:

-58.87 ± 1.35 mV (n=8); 100 nM panobinostat: -55.66 ± 0.47 mV (n=8); 500 nM panobinostat: -57.05 ± 0.44 mV (n=10)) of INa with panobinostat treatment while insignificant effect on steady state activation (Boltzmann charge-voltage fit for activation, V1/2 for control: -16 ± 1.8 mV (n=8) ; 100 nM panobinostat: -20.7 ± 3.1 mV

(n=8); 500 nM panobinostat: -14.6 ± 1.9 mV (n=10)) was observed (Figure 8H). This observation is consistent with other pan-HDAC inhibitors tested in our laboratory (data not shown). The time-dependence of INa inactivation was also examined by curve-fitting the decaying phase of the INa traces with a first-order exponential function. The Vm- 71

dependent inactivation time constants (h) were insignificantly slower in the -60 to 10 mV range for 100 nM panobinostat (Figure 8E) while the inactivation rate was significantly faster at 5 & 10 mV for 500 nM panobinostat. Increased late sodium current

(INa,L) during the plateau phase of AP has been shown to tremendously increase the risk of EADs and DADs. Thus, we determined the steady state INa at 100 milliseconds in both control and treatment groups. The whole cell current at 100 milliseconds from -120 mV to 40 mV voltage steps was determined and were fitted linearly using Origin 8.6 to reveal that there was no significant changes in slopes and intercepts (pA) of fitted values of late

INa (Control: 0.08, 7.76 pA; 100 nM panobinostat: 0.08, 9.0 pA; 500 nM panobinostat:

0.06, 7.18 pA) (Figure 8G).

HDAC inhibitor does not alter the magnitude of L-type cardiac calcium current

L-type Ca2+ channels are responsible for phase 2 entry of Ca2+ in ventricular myocytes.

Excitation-contraction coupling, calcium induced calcium release and several other physiological roles of intracellular Ca2+ are widely known. LQTS8, although rare is caused by of Cacna1c gene encoding for L-type Ca2+ channel in myocytes

2+ (Morita et al., 2008). Therefore, L-type Ca current (ICa,L) were recorded using the solutions mentioned in Table 3 and Table 4; patch clamp protocol is shown in Figure 9E.

In contrast to other pan-HDAC inhibitors tested in our laboratory like vorinostat, trichostatin A, and romidepsin that showed more than 2 fold stimulation in magnitude of

ICa,L, surprisingly, there was no dose dependent stimulation of ICa,L by panobinostat in

NMVMs. A family of traces is displayed in Figure9 A, B, C, D and magnitude of currents are summarized in Table 9. 72

HDAC inhibition induced action potential shortening

After clamping individual ionic currents, we wanted to determine the effect of action potential duration induced by HDAC inhibition. Thus, myocytes were current clamped and action potentials were elicited by 100 µsec, 100 pA depolarizing stimulus at a frequency of 0.5 Hz or 1 Hz. Significant shortening (≈50%) of APD90 was recorded in ventricular myocytes treated with 100 nM panobinostat for 24 hours (42.6 ± 6.9 milliseconds, n=7) as compared to non-treated myocytes (87 ± 3 milliseconds, n=5).

Small clusters of cells (2-3 cells per cluster) were patched to reduce input resistance and for better control of the current clamp, especially when we dial in a small current to set the membrane voltage to -80 mV (Figure 10A & B). Reduction of input resistance significantly eliminates shunt error associated with a gigaohm seal. Kopljar et al also showed ≈50% reduction in field potential duration in human induced pluripotent stem cell derived cardiomyocytes with 24 hour treatment of panobinostat (Kopljar et al.,

2016).

Reduction in gap junction coupling by HDAC inhibition

Although it’s widely accepted that HDAC inhibition enhances gap junction (GJ) mediated intracellular coupling in cancer cells (Dovzhanskiy et al., 2012), we found that they reduce GJ conductance (gj) in NMVMs. We observed dose dependent reduction in gj coupling by panobinostat treatment (Control: 33.43 ± 1.39 nS (n=20), 100 nM panobinostat: 19.56 ± 3.73 nS (n=7); 500 nM panobinostat: 5.13 ± 1.00 nS (n=6), 1 μM panobinostat: 3.7 ± 1.05 nS (n=5), p<0.05) (Figure 1 & Table 9 ). Concomitant reduction 73

in GJ coupling and altered distribution can desynchronize conduction leading to source to sink issues followed by ectopic foci and reentrant arrhythmias (Kleber & Saffitz, 2014).

Regulation of ion channel, calcium handling and gap junction genes by HDAC inhibition

More than a dozen genes involved in depolarization, repolarization, and trafficking of ion channels have been implicated in long QT syndrome. The alterations in LQT and GJ gene expression can deter homeostasis leading to cardiac toxicities, providing us with possible off-target effects of HDAC inhibition. Therefore, we investigated the effect of panobinostat on these genes to determine their role in the arrythmogenic liability of panobinostat. The genes included were: Cacna1c (CaV1.2; LQT8), Cacna1g (Cav3.1),

Kcnd2 (KV4.2), Kcnd3 (KV4.3), Kcnh2 (KV11.1, hERG; LQT2), Kcnj2 (Kir2.1; LQT7),

Kcnq1(KV7.1; LQT1), Kcne1 (MinK; LQT5), Kcne2 (MIRP1; LQT6), Kcna5 (Kv1.5/3.1)

Scn5a (NaV1.5; LQT3), Scn4b (NaV4; LQT10), Ank2 (ankyrin-B; LQT4), Slc8a (NCX),

Cdh2 (N-cadherin, Ncad), Gja1 (connexin43, Cx43), Kcnip2 (Kv4.3), and Serca2a

(Figure 13 A, B & C). We observed a statistically significant reduction in mRNA expression of Kcne2, Gja1 and increased mRNA expression of Kcne1, Scn4b, and

Serca2a with treatment of 100 nM panobinostat for 24 hours in NMVMs as compared to control (Figure 13 A,B & C). Out of these genes, abundance of Kcne2 (≈ <0.03%, 3.9 times 10-4), Kcne1 (≈ <0.6, 0.00632) and Scn4b (≈ <0.1%, 0.0019) was very low in control samples relative to Gapdh expression (Figure 13D). Considering both abundance and relative expression, significant downregulation of Gja1 and increased Serca2a expression were the major findings of this experiment. 74

Reduction in Cx43 and Nav1.5 protein expression induced by HDAC inhibition

We recorded a dose dependent reduction in INa and GJ coupling with increasing doses of panobinostat with concomitant reduction in Gja1 mRNA expression but no change in

Scn5a levels. Thus, we performed western blot analyses in NMVMs to determine any alterations in major ion channels and gap junctions induced by HDAC inhibition at the protein level. Protein expression analysis of cultured NMVMs revealed a dose dependent decrease of both Nav1.5 and Cx43 with panobinostat (p<0.05, n=3) (Figure 14 A, B & C).

Further, we also did westerns on heart homogenates of mice treated with panobinostat 30 mg/kg intraperitoneally with five days ON and two days OFF for two weeks to see if panobinostat treatment reduced protein expression of Cx43 and Nav1.5 with (Figure 14 D

& E). Acetyl-α-tubulin and acetyl – histone 3 were used as cytoplasmic and nuclear acetylation markers respectively. α–tubulin was used as a loading control.

75

Discussion The HDAC inhibitor class of drugs has tremendous potential in the field of cancer pharmacology due to their involvement in key regulatory and gene expression pathways.

These drugs have been extensively studied and approved for clinical indications, however, they have unintended cardiac side effects. Therefore, this study is focused on identifying molecular and electrophysiological effects of HDAC inhibitors in cardiac myocytes. Preclinical testing according to ICHS7A guidelines recommends screening based primarily on hERG dependent assay. hERG (IKr) is the major repolarizing current in human cardiomyocytes and its blockade is responsible for more than 95% of Long QT syndromes. Despite cardiac side effects of most pan-HDAC inhibitors, most of the

HDAC inhibitors have wide safety margins for hERG blockade (for example, the therapeutic dose for both Vorinostat and panobinostat therapeutic dose is >300 fold than their hERG IC50) (Kerr et al., 2010; Kopljar et al., 2016). Further, we also observed insignificant changes in steady state and transient outward K+ currents with HDAC inhibition (Figure 7). Thus, we think hERG is not responsible for cardiac side effects of

HDAC inhibitors and hence we focused our studies on other major cardiac ionic currents which can be altered by HDAC inhibition.

We have consistently seen concomitant reductions in GJ coupling and peak INa, without alterations in INa,Late and a slight shift of 2-3 mV during activation with no change in inactivation of INa with long term treatment of panobinostat (24 hours) (Figure 8). Acute studies with HDAC inhibitors showed no significant change in transient outward and steady state K+ currents (Figure 6). Recently, Kopljar et al also showed that onset of altered beating rate in human induced pluripotent stem cell cardiomyocytes was 76

approximately 12 hours that worsened with time (Kopljar et al., 2016). Thus, suggesting that HDAC inhibitors don’t act as direct blockers of ion channel but by inducing epigenetic alterations, trafficking, or act by altering post-translational modifications.

Two in vivo mice studies have previously shown safe administration of panobinostat at

10, 20, 30 mg/kg when injected intraperitoneally for two weeks (Catalano et al., 2012;

Vilas-Zornoza et al., 2012). These studies along with the intermittent dosing of panobinostat recommended in patients formed the basis of our treatment regime (Figure

3). In our experimental in vivo ECG monitoring we did not record any arrhythmic episodes till the 7th day of panobinostat injection (Table 8). Notably, half-life of panobinostat is approximately 37 hours which also indicates that this could be due to long term, accumulative cardiac side effects of panobinostat.

Faster breeding, ease of transgenics and effortless drug dosing make mice models a preferable choice for in vivo studies. Although, there are several mouse models for studying physiological consequences of there are significant differences between the mouse and the human heart. The mouse heart weighs ≈ 25 mg, which is 4% of human heart, and its heart rate is ≈ 600 beats per minute, which is 10 times faster than human heart. Excessive heart rate makes it difficult to identify VTs or VFs in mice and in some instances, these events might revert to sinus rhythm without even getting noticed.

Interestingly, there are drastic differences in ECG parameters within various breeds of mice as well (Wehrens et al., 2000). Ours is the only study to show ECG parameters of conscious C57BL/6J mice using DSI Telemetry (HR: 486.7± 1.08 beats per minute; RR:

126.3 ± 0.2 milliseconds; ST interval: 24.8 ± 0.1; QRS: 14.4 ± 0.01; PR interval 37.7 ± 77

0.15; & QT interval: 35.1 ± 0.1 milliseconds). Our values are close to those values reported by Klaus Willecke and colleagues in C57BL/6J mice anesthetized using Avertin injection intraperitoneally (Kirchhoff et al., 1998). We also showed dose dependent prolongation of QT and ST interval with panobinostat injection along with ventricular tachycardia episodes in mice which is consistent with prolonged QT intervals and arrhythmias seen in human subjects taking panobinostat. However, reduction in INa, no change in ICa,L and IK currents, and declined Gj coupling can’t explain the prolongation of

QT intervals observed in these mice. Diarrhea, is one of the uppermost side effects of

HDAC inhibitors and electrolyte disturbances (especially hypokalemia) are one of the risk factors of QT prolongation. Moreover, one of the metabolites of panobinostat 519-07

(also referred as BJB432 or M37.8), had an estimated IC50 value of 1.6 µM, which is 3 times less than hERG IC50 of panobinostat ( European Medicine Agency report,

FarydakTM). Thus, we suspect that depleted electrolytes or metabolites of panobinostat may attribute to unexplained prolonged QT intervals in mice and in patients.

Interestingly, depletion of electrolytes along with reduction of Cx43 with HDAC inhibition can modulate conduction velocity and cardiac conduction. George et al showed recently that wider perinexi due to reduction of extracellular Na+ and K+ increase conduction velocity in heterozygous Cx43 mouse (George et al., 2015; Veeraraghavan et al., 2014).

Neonatal mouse cardiomyocytes were preferred over adult cardiomyocytes because they are smaller and compact in size which makes them ideal for whole cell patch clamp experiments. Their cell capacitance is a fraction of that of the adult cardiomyocytes and 78

the most distinct advantage is the expression of rapid and slow components of delayed rectifiers responsible for repolarization (IKr and IKs) which are absent in adult mouse cardiomyocytes (Wang et al., 1996). Although neonatal mouse cells look premature and lack T-tubules, sodium current is the most prominent driving inward current for ventricular contractions in these cells. Their tetrodotoxin (TTX) sensitivity for INa is similar to that of adult ventricular cardiomyocytes (Nuss & Marban, 1994). Meier et al showed that TTX-sensitive sodium channel α-subunit isoforms Nav1.1, NaV1.2, Nav1.3,

Nav1.4 and Nav1.6 were detected in neonatal rats but at a significantly reduced level as compared to Nav1.5. This study used rat neonatal cardiomyocytes but most of our experiments are done on neonatal mouse cardiomyocytes. Haufe et al suggested that

Nav1.5 was the most predominant form of sodium channel subtype present at all stages of development. Their studies also revealed that NaV1.3 was the major neuronal sodium channel subtype being present at developmental stages which increases three fold from birth to 18 weeks. They found that neuronal sodium channel transcripts were 4.9% in isolated cardiomyocytes as compared to 23.6% in whole heart preparations of mRNA pool and since our experiments are based on isolated cells, contribution of neuronal sodium channel transcripts for INa would be negligible (Haufe et al., 2005). Moreover, none of these neuronal sodium channel subtypes have been linked to QT interval prolongation on ECG except mutations in NaV1.4 which have been linked to

‘’ disease (Haufe et al., 2007). We did not test for neuronal Nav subtypes but will consider testing them in future studies. Moreover, the strong correlation we found between reductions of peak INa (Figure 8) along with reduced protein 79

expression of Nav1.5 (Figure 14) is consistent with our interpretation that dose dependent reduction of INa is due to decreased Nav1.5 expression by HDAC inhibition. Dose dependent reduction of INa was observed without any change in capacitance in NMVMs

(Figure 12). Although we observed a general trend towards reduction of capacitance by panobinostat treatment, the differences were statistically non-significant. Generally, reduction in capacitance can be correlated with reduction in expression or disruption of transverse-tubules (invaginations on the surface of cardiac cell membrane); (Brette &

Orchard, 2003). Further, we also did not check for alterations in caveolin-3 (marker for t- tubules) by panobinostat treatment which can be considered in the future (Pavlović et al.,

2010).

The precise molecular mechanism of reduction in protein expression of Nav1.5 without significant downregulation of its mRNA transcript remains to yet be determined.

Similar to our observations, previously Gavillet et al has shown reduced Nav1.5 protein expression while Scn5a mRNA expression were unaltered in muscular dystrophic mice

(Gavillet et al., 2006). Some of the possible mechanisms could be due to, a) variability in the different transcripts which do not equally get translated to Nav1.5. In mice, two different types of RNA variants of Scn5a and three distinct alternative spliced transcripts have been detected (Rook et al., 2012) and; b) Surface expression and localization of

Nav1.5 have been found to be modulated by several interacting proteins like ankyrin G, syntrophin, MOG1, α-actinin-2, calmodulin and altered ubiquitinated mediated degradation by Nedd-4 (Detta et al., 2015). SAP97, glycerol-3-dehydrogenase 1 like gene

(GPD1-L), plakophillin-2, N-Cadherin, Cx43, tyrosine-phosphorylated β1, caveolin-3 all 80

have been linked with reductions in protein expression of Nav1.5 when downregulated

(Agullo-Pascual, Cerrone, et al., 2014; Chauhan et al., 2000; Detta et al., 2015; Jansen et al., 2012; Kang et al., 2012; London et al., 2007; Noorman et al., 2013; Sato et al., 2009;

Shy et al., 2013; Ziane et al., 2010). The effect of HDAC inhibition on these proteins are still unknown and will be considered for future studies.

Cardiac specific C-terminal deletion of Cx43 (Cx43D378stop) has been shown to reduce INa and conversely heterozygous deletion of Scn5a showed heterogeneous expression of Cx43, indicating the fact that altered expression and function of one affects the other (Agullo-Pascual, Lin, et al., 2014; Lübkemeier et al., 2013; van Veen et al.,

2005). Both Cx43 and Nav1.5 are a part of a macromolecular complex along with proteins mentioned earlier existing at the intercalated discs. Thus, reduced Cx43 and

Nav1.5 protein levels by panobinostat could perturb this macromolecular complex, thus modifying the synchronous conduction path which may induce reentrant arrhythmias.

Despite the magnitude of late sodium current (INa,Late) which constitutes approximate

0.1% of total INa, various pathological states like heart failure, ischemia, oxidative stress, congenital mutations in Scn5a causing LQT have been shown to increase the amplitude of INa,Late (Shryock et al., 2013). Increases in INa,late disturbs the delicate cytoplasmic balance of entry/exit of Na+ and Ca2+ ion inducing AP prolongation which facilitates

EADs and triggered activity (Kornyeyev et al., 2016). For example, heterozygous deletion of SCN5a (Nav1.5) F1486 LQT mutation caused AV block and polymorphic VT in a patient and was due to increased INa,Late which induced EADs when expressed in neonatal rat ventricular myocytes (Song et al., 2012; Yamamura et al., 2010). Recently, 81

novel homozygous mutations (R1309H) in voltage sensors of Nav1.5 DIII/S4 was found in patients which caused severe atrial and ventricular arrhythmias due to enhancement of

INa,Late (Wang et al., 2016). Pharmacological modulation of INa,Late is one of the new emerging targets in therapy (Banyasz et al., 2015). In our study, we report a significant finding that cardiac manifestations of HDAC inhibitors are not due to changes in INa,Late

(Figure 8G) and reductions in peak INa (Figure 8F) did not affect the inactivation kinetics or magnitude of INa,Late (Figure 8G).

Acetylation is second most studied post-translational modification after phosphorylation. The name ‘HDAC’ is a misnomer because recently more than 3600 acetylation sites on more than 1900 proteins have been found to be putative acetylation sites of deacetylases (Choudhary et al., 2009) which includes transcription factors, kinases, metabolic enzymes, cytoskeletal, and cell cycle proteins (Iyer et al., 2012).

Recently, Colussi et al identified that HDAC 3, 4, 5 and PCAF are associated with Cx43 and acetylated Cx43 has a role in lateralization and dissociation of Cx43 in dystrophic heart from intercalated discs (Colussi et al., 2011). Snyder and colleagues showed that acetylation and ubiquitination have opposite effects on identical lysines of epithelial sodium channel’s (ENac’s) to alter its degradation (Butler et al., 2015). It is unknown if

NaV1.5 or Cav1.2 is acetylated but bioinformatics analysis (PHOSIDA database) predicts two cytoplasmic acetylation sites near domain II and IV S4 domains of NaV1.5 with high probability (> 90%). We propose acetylation at these sites may account for +2-3 mV shift in activation of INa. In future studies, we will attempt to confirm these sites using immunoprecipitation and site-directed mutagenesis or mass spectrometry. 82

We conclude that the panobinostat induced reductions in peak INa and GJ coupling, along with a reduction in protein expression of both proteins, which can be a mechanism of arrhythmias in mice and, with careful extrapolation, may be a potential reason for cardiac toxicities in humans. Reduced coupling along with a reduction in excitability can cause asynchronous conduction and source-sink disturbances leading to reentrant arrhythmias. 83

Figure 4a: Electrocardiographic changes in mouse heart with panobinostat (pan- HDAC) inhibition. Panel (A) shows control ECG recorded at Day 0 before initiation of treatment. (B) & 84

(C) shows ventricular tachycardia event at day 12 with panobinostat 20 mg/kg and 30 mg/kg i.p injection respectively (sampling interval of 1 milliseconds).

85

Figure 4b: Electrocardiographic changes in mouse heart with panobinostat (pan- HDAC) inhibition. (D) Representative trace of extra-systole (premature ventricular contraction) in mice. Multiple instances of extrasystole were also seen in ventricles, summarized in Table 8. (E) ECG from the same mouse before and after injection of 30 mg/kg panobinostat at 12th day (indicating PR, ST and QT intervals along with bradycardia). All three mice injected with 30mg/kg panobinostat showed cardiac episodes of 2nd and 3rd degree AV block (F) and spontaneous AF. One mouse out of three died during the recording and showed 3rd degree AV block and died in less than 3 minutes (F). (G) ECG parameters are of all treatment groups on their last day normalized to their respective values before treatment. Statistically significant prolongation of the ST and the QT intervals with panobinostat treatment 10, 20, and 30 mg/kg. Panobinostat 30 mg/kg treatment lead to an excessively prolonged QT and ST intervals which was outside the defined parameter values of automated software used to analyze data. (H) Shows ECG parameters calculated manually and blindly indicating statistical significant prolongation of QT and ST intervals. Data normalized before and after treatment. *p < 0.05

86

Table 6: Table summarizing absolute and normalized ECG parameters of mice treated with panobinostat analyzed using Ponemah 5.2

Absolute values

ECG HR RR ST QRS PR QT QTcbψ Parameters (Beats/ (ms) (ms) (ms) (ms) (ms) (ms) (ms)/ minute) Treatment Control 126.37 ± 486.71 ± 24.87 ± 14.44 ± 37.74 ± 35.17 ± 99.67 ± (n=3) 0.26 1.08 0.11 0.02 0.16 0.11 0.31 128.02 10 mg/kg 112.76 ± 544.01 ± 32.76 ± 14.05 ± 36.73 ± 42.67 ± ± (n=3) 3.20 17.63 3.15* 0.42 1.88 3.40* 9.31 116.20 20 mg/kg 125.33 ± 533.24 ± 28.67 ± 15.36 ± 37.36 ± 39.68 ± ± (n=3) 29.33 96.29 1.63* 0.95 3.88 2.29* 12.61 30 mg/kg 127.20 ± 489.97 ± 34.76 ± 14.70 ± 42.41 ± 45.04 ± 126.5 ± (n=3) 12.59 38.20 4.75 1.19 5.62 4.84 8.28 Normalized ECG values to before and after treatment with various doses of panobinostat ECG ψ Parameters RR HR ST QRS PR QT QTcb (ms)/ (ms) Beats/m (ms) (ms) (ms) (ms) (ms) Treatment

Control 1.00 1.00 1.00 1.00 1.00 1.00 1.00 (n=3)

0.86 ± 1.18 ± 1.18 ± 0.95 ± 0.96 ± 1.11 ± 1.20 ± 10 mg/kg .08 0.11 0.04* 0.05 0.04 0.04* 0.05 (n=3)

1.01± 1.07± 1.44 ± 1.07 ± 1.01 ± 1.31 ± 1.32 ± 20 mg/kg 0.22 0.18 0.07* 0.07 0.10 0.06 0.06 (n=3)

1.04 ± 1.00 ± 1.32 ± 1.04 ± 1.12 ± 1.24 ± 1.23 ± 30 mg/kg 0.10 0.10 0.24 0.08 0.12 0.17* 0.17 (n=3)

Data shown in control row is Average ± SEM of all the recordings before initiation of treatment. Panobinostat was injected intraperitoneally for two consecutive weeks with 2 days OFF (rest period) at the end of the first five days. Ψ- QTcb is QT interval based on Bazette’s formula *p<0.05 87

Table 7: Table summarizing absolute and normalized ECG parameters of mice treated with panobinostat analyzed manually (Blind fold)

Absolute values for panobinostat 30 mg/kg i.p.

ECG Parameters PR QRS QT ST (ms)/ (ms) (ms) (ms) (ms) Treatment 45.35 ± 9.19 ± 28.81 ± 19.62 ± Control 0.70 0.30 0.47 0.49 30 mg/kg 47.71 ± 9.24 ± 43.10 ± 33.86 ± 1.66 0.22 1.12* 1.04* Normalized ECG values to before and after treatment with various doses of panobinostat ECG PR QRS QT ST Parameters/ (ms) (ms) (ms) (ms) Treatment

Control 1.00 1.00 1.00 1.00

30 mg/kg 1.09 ± 1.03 ± 1.52 ± 1.75 ± 0.17 0.10 0.14* 0.18* Data shown in control column is Average ± SEM of 68 ECG recordings calculated manually. 30 mg/kg treatment group is Average ± SEM of 16, 23, 24 ECG traces calculated manually in total from 3 mice treated with panobinostat 30 mg/kg i.p. Panobinostat was injected intraperitoneally for consecutive two weeks with 2 days OFF (rest period) after the first five days of treatment. RR and HR intervals were not calculated manually because Ponemah 5.2® is splendid in identifying R peaks of ECG Data was analyzed using Origin8.6 in a blind fold fashion. *p < 0.05 88

Table 8 : Summary of arrhythmic events at 14th and 7th day.

14th day Arrhythmia Summary

Ventricular panobinostat No of Mice AF Comments for Tachycardia PVC Treatment treated events some traces events Some traces 8 showed 10 mg/kg 3 44(3/3) 0 (2/3) prolonged T Wave -instances of 20 mg/kg 3 34(3/3) 4 7(3/3) multiple P waves -One mouse died at Day 5 so no 14 day data 1 -2 T-Wave 30 mg/kg 3 22(3/3) 2 (1/3) dispersions seen - instances of rapid electrical activity

7th day Arrhythmia Summary

2 10 mg/kg 3 12(3/3) 0 (1/3) 7 20 mg/kg 3 22(3/3) 2 (2/3) -one mouse died on Day 5 with severe bradycardia, 30 mg/kg 3 24 (3/3) 5 3(2/3) Prolonged QT and broad QRS -Extrasystole -10min long event of no QRS Parenthesis means (Number of animals with event / Number of animals implanted with transmitter)

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Figure 5: Dose dependent reduction in HDAC activity by panobinostat in neonatal mouse ventricular myocytes. Fluorescence based assay, NMVMs are cultured in 96 well plates. Fluor-de-lys® substrate is added to the wells which gets deacetylated by HDACs. At the end of 8 hours developer is added and fluorescence is measured with 365 nm excitation and 410 nm emission. Bi-exponential curve fitting revealed two Ki’s: KI1) 56 nM – serves as high affinity high capacity binding site KI2) 8.9 μM - which acts as low affinity low capacity binding site.

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Figure 6: Transient and Steady State K+ currents before and after acute treatment of panobinostat 1 µM in neonatal mouse ventricular myocytes. No significant changes in transient outward (Φ) and steady state (ø) K+ currents were recorded when 1μM panobinostat was added to the dish. Before and after recordings are displayed in (D) and (E) respectively. Family of traces are shown in (A) and (B). C – Voltage clamp protocol that was used to record these currents.

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Figure 7: Transient and Steady State K+ currents recorded after 24 hour treatment with 100 nM panobinostat in neonatal mouse ventricular myocytes. No significant changes in transient outward (Φ) and steady state (ø) K+ currents were recorded when NMVMs treated for 24 hours with 100 nM panobinostat were voltage clamped according to protocol (C). Current voltage relationships for transient outward and steady state K+ currents are displayed in (D) and (E) respectively. Family of traces are shown in (A) and (B). (C) – Voltage clamp protocol used to clamp these currents. 94

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Figure 8: Dose dependent reduction in sodium current (INa) with various doses of panobinostat in neonatal mouse ventricular myocytes for 24 hours. Families of traces for control (A), 100 nM panobinostat (B), and panobinostat 500 nM (C) are shown when NMVMs were treated with respective panobinostat concentration for 24 hours. These were voltage clamped for INa according to protocol as shown in (D). (E) τh is faster with 500 nM panobinostat treatment at +5 & +10 mV. (F) Shows I-V relationship indicating reduction in peak INa with 100 nM panobinostat (-59%) and 500 nM panobinostat (-55%) as compared to control.(*p<0.05). (G) Late sodium currents INa,Late also did not alter with 100 nM and 500 nM panobinostat. H) Boltzmann charge- voltage fit of normalized INa showing a minor 2mV shift in activation by 100 nM panobinostat treatment of NMVMs for 24 hours. 96

97

Figure 9: I-V relationship of L-type calcium currents in neonatal mouse ventricular myocytes with various doses of panobinostat. Families of traces for control (A), 100 nM panobinostat (B), 500 nM panobinostat (C), and 1 μM panobinostat are shown when NMVMs were treated with respective panobinostat concentrations. These were voltage clamped for ICa,L as shown in (D). (E) Voltage protocol used to clamp ICa,L Data shown as Average ± SEM (F) Shows I-V relationship indicating no change in peak ICa,L with any of the doses of panobinostat treatment of NMVMs for 24 hours.

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Figure 10: Shortening of action potential duration by treatment of neonatal mouse ventricular myocytes with 100 nM panobinostat for 24 hours. NMVMs were current clamped and APDs were elicited with 100 µsec, 100 pA stimulus. In figure, A) trace shows reduction in action potential duration with 100 nM panobinostat treatment (red) of NMVMs as compared to control (black) (representative AP ensemble average of 10 APs). B) Shows reduced APD50, APD75 and APD90 with 100 nM panobinostat of NMVMs (*p<0.005, n=5 for control, n=7 for panobinostat 100 nM treatment). APD50, APD75, APD90 is the time taken by the cell to achieve 50%, 75%, 90% repolarization.

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Figure 11: Dose dependent decrease in Gj conductance (nS) between neonatal mouse ventricular myocytes treated with panobinostat. We observed dose dependent reduction in Gj coupling by panobinostat treatment of NMVMs. (Control: 33.43 ± 1.39 nS (n=20), 100 nM panobinostat: 19.56 ± 3.73 nS (n=7); 500 nM panobinostat: 5.13 ± 1.00 nS (n=6), 1 μM panobinostat: 3.7 ± 1.05 nS (n=5), *p<0.05, **p<0.005) (Table 5). Data shown as Average ± SEM. [Xian Zhang generated some observations for this figure]

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Figure 12: Non-significant change in capacitance (pF) of neonatal mouse ventricular myocytes with increasing doses of panobinostat. No significant change in input capacitance (Cin) was seen with various doses of panobinostat treatment in NMVMs. Cin values for control (40.67 ± 5.29 pF, n=25), 100 nM panobinostat (40.92 ± 5.13 pF, n=29), 500 nM panobinostat (26.65 ± 4.12 pF, n=16) and 1 μM panobinostat (32.9 ± 4.21 pF, n=9). Data shown as Average ± SEM

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Table 9 : Summary of alterations in calcium, sodium currents and Gj coupling with different doses of panobinostat.

[Panobinostat] Control 100 500 1000 (nM) -3.56 ± Peak I (pA/pF) -3.00 ± 0.35 -3.49 ± 0.48 -3.34 ± 0.5 Ca,L 0.68

% change 0% +16.3% +13.3% +18.66%

-122.46 ± -146.7 ± Peak I (pA) -141 ± 22.24 -123 ± 20.3 Ca,L 19.41 26.5

% change 0% +19.6% +15.5% +0.8%

-247.16 ± -146.5 ± -135.92 ± Peak I (pA/pF) n.d Na 42.81 26.00 20.39

% change 0% -59.27% -55.05% --

-5287.5 -2757.5 -1992.61 ± ± Peak I (pA) ± n.d Na 718.51 448.74* 187.40*

% change 0% -52.1% 37.6% --

Cin (pF) 40.67 ± 5.29 40.92 ±5.13 26.65 ± 4.12 32.9 ± 4.21

% change 0% +0.06% -34.4% -19.1%

19.56 ± G (nS) 33.43 ± 1.39 5.13 ± 1.00* 3.7 ± 1.05* j 3.73*

% change 0% -41.48% -84.14% -88.54% n.d – not determined, *p<0.05 compared to control

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Figure 13: Changes in mRNA expression of various LQT genes caused by 100 nM panobinostat treatment for 24 hours. The relative mRNA expression levels were calculated by taking the ratio of mRNA expression of individual murine gene using RT-PCR from NMVMs treated with 100 104

nM panobinostat to that of untreated myocytes. A) Statistically significant reduction in mRNA expression of Kcne2 and increase in Kcne1. These genes encode for subunits MiRP1 & MinK for IKr (Kcnh2). (B&C) Reduction in expression of Cx43 (Gja1) without any change in N-cadherin (Cdh2). Increased mRNA expression of Scn4b (C), Serca2a (B) with treatment of 100 nM panobinostat for 24 hours in NMVMs as compared to control. (D) Relative mRNA abundance of genes in control samples relative to Gapdh expression. Each experiment was performed in triplicates with two internal controls (Gapdh & Rpl13a). *p < 0.05.

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Figure 14: Dose dependent reduction in Cx43, Nav1.5 levels by panobinostat in NMVMs & mouse treated with panobinostat 30 mg/kg intraperitoneally for two weeks. A representative blot of lysed ventricular myocytes treated with 50, 100, 500 nM panobinostat for 24 hours (A). (B & C) shows statistics of protein densitometry analysis from scans of three western blot experiments which reveal dose dependent reduction in NaV1.5 (B) and Cx43 (C) with panobinostat treatment (*p<0.05). (D) Representative blot of ventricular lysate of heart from adult mouse treated with panobinostat 30 mg/kg i.p. according to dose regime shown in Figure 3. (E) Shows protein densitometry analysis of three individual experiments showing reduction of Cx43 and NaV1.5 with panobinostat injection. Scans were normalized with individual α – tubulin as loading control. Acetyl-α-tubulin and acetyl – histone 3 is used as cytoplasmic and nuclear acetylation marker respectively.

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CHAPTER 3: FUNCTIONAL ALTERATIONS OF HDAC INHIBITION IN MOUSE ATRIUM.

# Data in this chapter includes work done by Dakshesh Patel 115

Introduction Atrial fibrillation (AF) is the most common of sustained arrhythmia in humans. The prevalence of AF is 1-2% in population which increases to 10% at the age of 80 years and rises to 18% at age 85 (Camm et al., 2012). Undoubtedly, it is the major cause of morbidity and mortality throughout the world (January et al., 2014). The pathophysiological mechanisms of AF are complex and it is caused mainly because of existence of comorbidities like aging, heart failure, obesity, hypertension and ischemic heart disease. Numerous structural, electrical and mechanicals factors are responsible for the pathogenesis of AF. Initiation of AF requires a trigger while sustenance of AF needs a substrate (Wakili et al., 2011). A trigger can be due to an altered expression or function of ion channels, which can lead to ectopic firing or impulse reentry through the atrial tissue (Iwasaki et al., 2011). Disturbed intracellular loading of Ca2+ or Na+ ions in atrial cells can also contribute to the induction of ‘Early’ or ‘Delayed’ After Depolarization

(EAD/DAD) causing AF. AF can be ‘paroxysmal’ which is self-terminating, or

‘persistent/permanent’ which is not self-terminating and requires medical intervention. In most cases, ‘paroxysmal or transient’ AF progresses to ‘permanent/ persistent’ AF

(Wakili et al., 2011). Mainly, this is due to structural remodeling induced by functional reentry substrates over prolonged periods. Structural remodeling primarily consists of tissue fibrosis due to excessive deposition of collagen in atrial interstitial tissue as a reparative response to repetitive functional reentry substrates. This reparative fibrosis involves replacement of dead cardiomyocytes with fibroblasts, which in turn couple with working atrial tissue to interfere in its electrical continuity leading to heterogeneous conduction (Burstein & Nattel, 2008). This creates a long term positive feedback where 116

‘AF begets AF’ (Wijffels et al., 1995). AF can exist independently, ‘lone-AF’, or along with an underlying pathology which may be heart failure, ischemic heart disease, cardiomyopathy, hypertension, obesity or congestive heart failure. Angiotensin II is a well-known profibrotic secretory molecule which leads to deposition of collagen in atrial tissue by activation of transforming growth factor β – SMAD signaling pathway (Weber et al., 1993).

The occurrence of AF is very common in life-threatening cancers, as well as in patients with a history of cancer (O’Neal et al., 2015) especially after cancer surgery (Farmakis et al., 2014). The underlying mechanisms which lead to increased prevalence of AF during cancer are unknown. AF manifestations in cancer can occur due to multiple factors, such as; a) multiple comorbidities like hypertension, electrolyte disturbances, age or metabolic disorders (Guzzetti, 2002); b) due to neoplasms/tumors adjacent to the heart for example lungs or esophagus (Farmakis et al., 2014); c) drug induced during treatment of cancer

(Suter & Ewer, 2013). Numerous cytotoxic drugs like cisplatin, doxorubicin, ondansetron, 5-flourouracil, gemcitabine, bisphosphonates etc have been shown to cause

AF in cancer patients (Kaakeh et al., 2012; Suter & Ewer, 2013; Van Der Hooft et al.,

2004). In the previous chapter, I discussed the role of histone deacetylase (HDAC) inhibitors in inducing adverse cardiac toxicities in ventricles. Interestingly, although not fatal, AF is the most common type of arrhythmia caused by HDAC inhibition in patients

(Subramanian et al., 2010). Clinically approved pan-HDAC inhibitors like romidepsin

(IstodaxTM), belinostat (BelinodaqTM), panobinostat (FarydakTM), (approved for cutaneous and peripheral T-cell lymphoma and multiple myeloma, alone or in 117

combination) have caused AF in patients during their respective clinical trials (Ellis et al.,

2008; Piekarz et al., 2006; Sandor et al., 2002; Steele et al., 2008). The underlying mechanism of AF induced by HDAC inhibition is relatively unknown. Thus, in this chapter I will discuss our preliminary observations and possible electrophysiological and molecular mechanisms that lead to an AF phenotype shown by HDAC inhibition in heart.

118

Materials and Methods In vivo ECG monitoring

All procedures and experiments on mice were done according to the protocols approved by institution’s Institutional Animal Care and Use Committee (IACUC). Please refer methods section (in vivo ECG monitoring) of Chapter 2 for detailed procedure for DSI

Telemetry surgery and data analysis.

Primary atrial culture

Newborn C57BL/6J mice [Charles River laboratories] were anesthetized with isoflurane and the hearts were excised in accordance with procedures approved by the institution's

Committee for the Humane Use of Animals. The excised hearts were separated into atria and ventricles and kept in DMS8 (in mM: 116 NaCl, 5.4 KCl, 1.0 NaH2PO4, and 5.5 dextrose). Detailed procedure is listed in chapter 2, Material and Methods section and in our previously published paper (Xu et al., 2013).

Whole cell patch clamping

Single whole cell patch electrode voltage clamp experiments were performed on neonatal mouse ventricular myocytes (NMVMs) using conventional procedures with an Axon

Instruments Axopatch 1D or 200B patch clamp amplifier, Digidata 1320A or 1440 A/D converter, and pClamp8.2 or 10.1 software (Molecular Devices).

Transient (peak) and steady state outward potassium (IK,to and IK,ss) currents were recorded during 1 sec voltage steps from a holding potential (Vh) of -100 mV to +60 mV in 10 mV increments. 119

Real-time PCR

Cellular RNA was extracted with a Qiagen RNeasy® mini kit, quantified by UV absorption, and 500 ng reverse-transcribed with Superscript®III™ (Qiagen). The RT-

PCR was carried out on 384 well plates using LightCycler® 480 Real-Time PCR System

(Roche). 1 μl of the 1:5 diluted cDNA reaction, 1 μl of 50 nM custom forward and reverse RT-PCR primer mixes, 5 μl of enzyme and SYBR Green dye mix, and 3 μl of water was added in a total volume of 10 μl (LightCycler® 480 SYBR Green I Master

Superscript®) in duplicates. All results were normalized to GAPDH and control samples. cT values were determined by the apparatus and the quality of the PCR product was confirmed by analyzing the melt-curve (Tm). RT-PCR primers for murine Cacna1c,

Cacna1g, Kcnd2, Kcnd3, Kcnh2, Kcnj2, Kcnq1, Kcne1, Kcne2, Scn5a, Scn4b, Ank2,

Slc8a, Cdh2, and Gja1 were designed to span exon-intron regions of the gene of interest.

All forward and reverse primers were purchased from realtimeprimers.com or Life

Technologies. The murine ion channel forward and reverse primer sequences are listed in

Table 10 below:

Statistics

Averaged values are presented as the Mean ± SEM. Statistical analyses were performed with the Normality and one-way ANOVA tests using the Bonferroni method in Origin

8.6. Data with p < 0.05 was considered significant unless specified. REST 2009® was used to analyze RT-PCR data, using two internal controls Gapdh and Rpl13a as internal controls.

120

Table 10: Primer sequences of murine genes used for real time PCR

Gene Forward Primer Sequence Reverse Primer Sequence

Gapdh 5'- TGCCACTCAGAAGACTGTGG-3' 5'-AGGAATGGGAGTTGCTGTTG-3'

Gja1 5'-GAGAGCCCGAACTCTCCTTT-3' 5'-TGGAGTAGGCTTGGACCTTG-3'

Kcne1 5'-AGACACACTGAGGCTCCAAG-3' 5'-CAGTCTCTGTCCTGGCATCT-3'

Kcnh2 5'-CAGGCTGACATCTGCCTACA-3' 5'-GAGGCTCTCCAAAGATGTCG-3'

Kcnd3 5'-CATCCCTGCATCTTTCTGGT-3' 5'-CCCTGCGTTTATCTGCTCTC-3'

Kcnd2 5'-CAGAACTGGGCTTCTTGCTC-3' 5'-ACCCAAAAATCTTCCCTGCT-3'

Kcnq1 5'-CAGCAGTGTAGGGCTTCTTCC-3' 5'-GGGTTGGAAGTGTTTCGTGT-3'

Kcnj2 5'-TTGCTTCGGCTCATTCTCTT-3' 5'-AGAGATGGATGCTTCCGAGA-3'

Kcne2 5'-AATTTGACCCAGACACTGGA-3' 5'-ACCACGATGAACGAGAACAT-3'

Cacna1c 5'-CAGCTCATGCCAACATGAAT-3' 5'-TCTTGGGTTTCCCATACTGC-3'

Cacna1g 5'-CCTCACCACCTTCAACATCC-3' 5'-TGAGCGTCCTGTGTGAACTC-3'

Scn5a 5'-CTTCACCAACAGCTGGAACA-3' 5'-GACATCATGAGGGCGAAGAG-3'

Scn4b 5'-TCCTCTTGAGTGACCTGGAG-3' 5'-CCAGGATGATGAGAGTCACC-3'

Ank2 5'-CAGAAACCACAAACCCACTC-3' 5'-AGCACGGATGCTCAAAATAG-3'

Cdh2 5'-TATGTGATGACGGTCACTGC -3' 5'-AAAGGCCATAAGTGGGATT-3'

Slc8a 5'-TGAATCTTGCACGCTCATTA-3' 5'-CCAGGCACATCCAAAGTATC-3'

Tgfβ1 5'-TGGAGCAACATGTGGAACTC-3' 5'-TGCCGTACAACTCCAGTGAC-3'

Tgfβr1 5'-ATTGCTGGTCCAGTCTGCTT-3' 5'-TCCAGACCCTGATGTTGTCA-3'

Tgfβr2 5'-GTGGGAACGGCAAGATACAT-3' 5'-TTTCATGCTCTCCACACAGG-3' 121

Results Electrocardiographic changes in atria of conscious mice treated with pan-HDAC inhibitor

Apart from the ventricular electrical morphogenic changes after panobinostat administration, we recorded non-sustained atrial fibrillations in every mouse injected with panobinostat. Figure 15A shows representative trace of regular sinus rhythm of a mouse before panobinostat injections followed by Figure 15 B&C showing atrial fibrillation events in mice treated with 20 & 30 mg/kg respectively). The erratic high frequency fibrillation of atria can be seen between consecutive RR intervals typical of

AF. Interestingly, these rapid electrical activity during PR intervals were first seen at the

5th or 6th day of injection. Due to rapid onset of AF in these mice by panobinostat we propose that the underlying mechanism causing AF is primarily electrical disturbances rather than structural/mechanical remodeling.

Transient and steady-state cardiac atrial ionic K+ currents during HDAC inhibition.

Electrical remodeling is one of the fundamental mechanisms of AF pathophysiology. The repolarization in atrial action potential is predominantly determined by ultra-rapid delayed rectifier (IKur) in most species and acetylcholine activated inward rectifier current

+ (IK,ACh). Both of these K currents have been previously shown to be implicated in lone

AF induced due to electrical remodeling (Balana et al., 2003; Brandt et al., 2000; Ehrlich,

2008) . Thus, we studied changes in steady state and transient outward K+ currents in primary atrial culture treated with panobinostat for 24 hours. We recorded statistically significant increase in steady state and transient outward K+ currents (Figure 16) (24 122

hours) treated with panobinostat 100nM. These currents were recorded in presence of 30

µM TTX, and 1 µM nifedipine to isolate K+ currents specifically and were clamped as shown in Figure 16C & D. The results indicate an increase in K+ currents with pan-

HDAC inhibition which can cause shortening of action potential duration thereby inducing multiple rotors ultimately causing AF.

Regulation of ion channel, calcium handling and gap junction genes by HDAC inhibition in atrial culture

The majority of ion channels, calcium handling proteins and GJ proteins have been implicated directly or indirectly in arrhythmias. More than 10 genes involved in depolarization, repolarization and trafficking of ion channels have been implicated in long QT syndrome as well (Amin et al., 2013). These alterations in the LQT and GJ gene expression can deter homeostasis leading to cardiac toxicities providing us with possible off-target effects of HDAC inhibition. Therefore, we investigated the effect of panobinostat on these genes to determine their role in the arrythmogenic liability of panobinostat. The genes included were: Cacna1c (CaV1.2; LQT8), Cacna1g (CaV3.1),

Kcnd2 (KV4.2), Kcnd3 (KV4.3), Kcnh2 (KV11.1, hERG; LQT2), Kcnj2 (Kir2.1; LQT7),

Kcnq1(KV7.1; LQT1), Kcne1 (MinK; LQT5), Kcne2 (MIRP1; LQT6), Kcna5 (Kv1.5/3.1)

Scn5a (NaV1.5; LQT3), Scn4b (NaVβ4; LQT10), Ank2 (ankyrin-B; LQT4), Slc8a (NCX),

Cdh2 (N-cadherin, Ncad), Gja1 (connexin43, Cx43), Kcnip2 (Kv4.3), and Serca2a

(Table 5). In addition to these genes, we also probed for transforming growth factor β1

(Tgfβ1), transforming growth factor receptor β1 (Tgfβr1) and transforming growth factor receptor β2 (Tgfβr2) to determine if pan-HDAC inhibition regulates growth factor 123

signaling. We observed statistically significant reduction in mRNA expression of Kcne2,

Kcnh2, Cacn1c, Cacn1g, Gja1, Serca2a, Slc8a1, Tgfβr2 and increased mRNA expression of Kcne1, Kcnq1, Ank2, Kcna5 and Gja5 with no significant changes in Scn4b, Scn5a,

Gjc1 after treatment of 100 nM panobinostat for 24 hours in primary atrial cultured cells as compared to control (Figure 17 A, B & C). Out of these genes abundance of Kcne1 (≈

<0.034%, 3.4 times 10-4), Kcne2 (≈ <0.01%, 0.000163), Scn4b (≈ <0.001%, 1.5 Χ 10-7) is very low in control relative to Gapdh expression (Figure 17). Considering both abundance and relative expression, significant downregulation of Gja1 and Serca2a and increased mRNA expression of Tgfβr2 and Kcna5 are the major findings of this experiment.

124

Discussion Atrial fibrillation is the most common form of sustained arrhythmia which can exist independently or secondary to other comorbidities like heart failure, hypertension, myocarditis, ischemia, aging and various other diseased states (Nattel, 2002). Limited literature is available regarding prognosis of AF in cancer except post cancer surgery

(Farmakis et al., 2014). However, ‘new-onset’ AF has been found in cancer patients treated with various anti-malignant drugs including HDAC inhibitors (Suter & Ewer,

2013). All the clinically used HDAC inhibitors approved in the market for various oncological indications have caused AF like phenotypes in patients leading to dropping out of patients from clinical trials or termination of the study (Ellis et al., 2008; Piekarz et al., 2009; Steele et al., 2008). Electrical, structural and neuro hormonal remodeling are considered as the fundamental mechanisms for pathophysiology of AF, although their involvement in AF associated with cancer or after cancer surgery is unknown. Electrical remodeling encompasses alteration in ion channel expression and function, while structural remodeling involves activation of pro-fibrotic signals, especially TGF-β and the renin angiotensin system eventually leading to fibrosis of atria (Burstein & Nattel,

2008).

In our preliminary studies, each mouse injected with panobinostat showed incidences of non-sustained AF which initiated at 5th or 6th day after injection (Figure 15). Due to the rapid onset of AF in mice by pan-HDAC inhibition, we propose AF susceptibility is due to electrical changes and not due to any collagen deposition (data not shown but we have performed collagen staining in atrial sections using picrosirius red). 125

Thus, we directed most of our focus on identifying factors responsible for AF by electrical remodeling mechanisms (including both, ectopic foci or functional reentry).

Atrial action potential (AP) has a lower resting potential than ventricular AP and is shorter in duration mainly because of its triangulated shape along with slow activation and magnitude of L-type Ca2+ current (Nattel et al., 2008). Repolarization in atria is predominated by ultra-rapid delayed rectifier currents (IKur) as compared to ventricles where it is IKr & IKs (Schotten et al., 2011). The density of IK1 is 5-10 fold lower in atrial cells compared to ventricles making them more susceptible to achieve the threshold for depolarization. The selective atria specific expression of IKur (encoded by murine gene

Kcna5) and IK,ACh (encoded by murine gene Kcnj3 & Kcnj4 ) makes them specific targets for treatment of atrial arrhythmias without effects on ventricles (Brandenburg et al., 2015;

Iwasaki et al., 2011). Due to regional heterogeneity and diversity of K channel subunits and their critical role in repolarization, they have been shown to play a significant role in maintenance and sustenance of AF. Several instances of gain of function mutations of K- channels are known which have caused AF via functional reentry. Gain of function mutations in α-subunits of IKs (Kcnq1) (Chen et al., 2003), IKr (Kcnh2) (Wakili et al.,

2011), IK1 (Kcnj2) (Hong et al., 2005) and Kcne2 (Yang et al., 2004) have been shown to cause lone AF. Loss of function mutations in ICa,L are also expected to shorten APD and promote AF (Antzelevitch et al., 2007). Similarly, patients suffering from Brugada syndrome with loss of function mutations in the sodium channel (Scn5a) have a short

APD and are also predisposed to AF, although they die predominantly due to ventricular fibrillation (Chen et al., 2007). In addition to the α-subunit of sodium channel, loss of 126

function mutations in β-subunits of sodium channel have also been associated with AF

(Scn1b, Scn2b, and Scn3b) (Watanabe et al., 2009). Our experiments show an increase in steady-state and transient outward K+ currents with pan-HDAC inhibition suggesting that faster repolarization can cause shorter APD inducing AF via functional reentry mechanisms (Figure 16). We don’t know the contribution of individual K+ channel subtypes to the total repolarizing steady-state and transient outward K+ currents. Hence, dissecting the effect of pan-HDAC inhibition on individual K+ current subtypes will be considered in the future. Our RT-PCR experiment also indicated a significant increase in expression of Kcna5 (IKur) at physiologically relevant levels (Figure 17 C & D). Kcne1

(encoding for subunit MinK) and Kcne2 (encoding for subunit Mirp1), which eventually form subunits of IKr & IKs are also increased. However, the abundance of these genes is insignificant relative to Gapdh. Interestingly, regulation of expression of these LQT genes can be modified at the epigenetic as well as the post-transcriptional levels.

MicroRNAs are short non-coding which can degrade newly transcribed mRNA post-transcriptionally by binding to their 3’- untranslated region and thus can affect their expression or function (Delcuve et al., 2012). The effect of HDAC inhibitors on was first identified in a breast cancer cell line (SKBr3). After exposure to dacinostat, these cells showed a 40% dysregulation of microRNAs out of which majority were significant downregulated (Scott et al., 2006). In this study, we did not study the effects of HDAC inhibitors on changes in expression of microRNAs, but could be considered in future experiments. 127

Reduction in gap junctional coupling can also slow conduction promoting reentry

(Verheule & Kaese, 2013). In adult mouse heart, Cx40 is majorly expressed in atria and ventricular conduction system, while in cultured atrial myocytes both Cx40 and Cx43 are equally expressed and contribute evenly to total atrial GJ conductance (Lin et al., 2010).

There are controversial reports about the role of Cx40 knock out mouse in AV conduction. Kirchoff et al (Hagendorff et al., 1999) and many others have reported significant prolongation of PR intervals in Cx40-/- mouse (Simon et al., 1998). On the other hand, another set of studies were not able to reproduce this result (Tamaddon et al.,

2000; VanderBrink et al., 2000). A later study which performed optical mapping of

Cx40-/- mouse hearts showed no effect on conduction velocity, but abolished the differences of conduction velocity between right and left atria (Leaf et al., 2008).

Interestingly, a study came out which emphasized the ratio of Cx40/Cx43 as an important determinant for propagation velocity over Cx40 and Cx43 per se (Beauchamp et al.,

2006). They showed an increase in conduction velocity of neonatal Cx40-/- knock out hearts in contrast to its adult mouse. Although, we observed increased mRNA expression of Cx40 by pan-HDAC inhibition, the ratio of Cx40/Cx43 was significantly increased

(fold change of 3.1 ± 0.65 with 100 nM panobinostat normalized to control, Gapdh) suggesting its role in reduction of conduction velocity. Thus, the expression levels of

Cx40 is inversely correlated with conduction velocity and relative levels of Cx40 and

Cx43 in atria can lead to novel coupling properties. In human heart, there are chamber specific differences in the distribution of connexins (Kanagaratnam et al., 2002). Cx43 is equally expressed in all chambers of heart while the order of Cx40 expression is; right 128

atria > left atria> right ventricle ≈ left ventricle (Vozzi et al., 1999). However, during our isolation procedure of neonatal pups, we did not separate left and right atria which should be considered before interpreting this data. Also, it is undetermined if increased mRNA expression of Cx40 will translate to higher protein concentration and can be evaluated in future.

The classical congenital LQTS initiated EADs which induced triggered arrhythmias can also predispose patients to AF (detailed mechanism discussed in Chapter 1) (Johnson et al., 2008). The first ever loss of function mutation was found in Kcne1 (IKr or IKs) which induced AF linked to an EAD mechanism (Ehrlich et al., 2005). Similarly, various instances like loss of function mutations in a) ultra-rapid delayed rectifier Kcna5 (IKur)

(via adrenaline triggered EADs) (Olson et al., 2006), b) adaptor protein Ankyrin-B (via disturbances in trafficking of calcium handling proteins induced DADs) (Mohler et al.,

2003) and gain of function mutation in sodium channel (Scn5a) have been shown to predispose AF via triggered arrhythmic mechanism (Makiyama et al., 2008). We also observed significant reductions in Serca2a, Slc8a1, Cacn1g and Cacn1c mRNA levels which signal towards altered calcium homeostasis in atria leading to AF (Figure 17A &

B). We did not test the changes in protein expression by pan-HDAC inhibition but preliminarily, RT-PCR data provides initial cues on changes in global expression of mRNA by HDAC inhibition. Also, alterations in trafficking scenarios and function

(current density) of individual ion channels as well as gap junctions should be considered in the future for better understanding of AF induced by HDAC inhibition in mice and humans. 129

Overall, we have found novel information, although preliminary, that pan-HDAC inhibitors can increase the steady-state and transient outward K+ currents in primary atrial culture along with deregulated expression of multiple ion channel, calcium handling and gap junctional genes. However, this information needs to be revalidated by dissecting individual K current subtypes’ function and surface expression. 130

Figure 15: Atrial fibrillation in mice on with pan-HDAC inhibitor, panobinostat. Non-sustained atrial fibrillations were seen in each mice injected with panobinostat 10, 20 and 30 mg/kg at the end of 2nd cycle (12th day). (A) Representative trace of regular sinus rhythm of mouse before treatment. (B & C) Representative ECG trace showing atrial fibrillation events in mice treated with 20 & 30 mg/kg respectively. The erratic high frequency fibrillation of atria can be seen between consecutive RR intervals typical of AF. (Sampling interval of 1 milliseconds) 131

132

Figure 16: Transient outward and steady state K+ currents of primary atrial culture treated with panobinostat for 24 hours. Family of traces of control (A) and 100 nM panobinostat (B) of transient outward (Φ) and steady state (ψ) K+ currents recorded when atrial cells were treated for 24 hours with 100 nM panobinostat for 24 hours. (C) Voltage clamp protocol used to clamp these currents. Current voltage relationships for transient outward and steady state K+ currents are displayed in (D) and (E) respectively. *p < 0.05

133

134

Figure 17: Changes in mRNA gene expression in primary neonatal atrial culture with panobinostat 100 nM treatment for 24 hours. The relative mRNA expression levels was calculated by taking ratio of mRNA expression of individual murine gene using RT-PCR from primary atrial myocytes treated with 100 135

nM panobinostat to that of untreated myocytes. A) Statistically significant reduction in mRNA expression of Cacn1c & Cacn1g (L & T type calcium channel) & Gja1 (Cx43) and increase in expression of Gja5 (Cx40). No significant change in mRNA expression of Scn4b & Scn5a with treatment of 100 nM panobinostat for 24 hours in atrial culture as compared to control (B) Reduction in expression of Serca2a, Slc8a1, Ank2, and Tgfβr2 without any change in N-cadherin. (C) Statistically significant reduction in mRNA expression of Kcne1 and increase in Kcne2. Expression of Kcna5 is increased (α-subunit (pore-forming) of ultra-rapid delayed rectifier (IKur)), which is a major current responsible for repolarization in atria. These genes encode subunits MiRP1 & MinK for IKr (Kcnh2), which are reduced as well. (D) Relative mRNA abundance of genes in control samples relative to Gapdh expression. Each experiment was performed in triplicate with two internal controls (Gapdh & Rpl13a). *p < 0.05.

136

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Xu, Q., Lin, X., Andrews, L., Patel, D., Lampe, P. D., & Veenstra, R. D. (2013). Histone 141

deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication. Front. Pharmacol., 4(April), 44. http://doi.org/10.3389/fphar.2013.00044

Yang, Y., Xia, M., Jin, Q., Bendahhou, S., Shi, J., Chen, Y., … Chen, Y. (2004). Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation. Am. J. Hum. Genet., 75(5), 899–905. http://doi.org/10.1086/425342

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CHAPTER 4: SUMMARY AND FUTURE STUDIES

Summary Over the last decade, extensive research in preclinical and clinical settings has identified the true potential of HDAC inhibitors in improving the quality of life of numerous cancer patients. Since October 2006, four HDAC inhibitors belonging to distinct structural classes of HDAC inhibitors have been approved by FDA for varied cancer indications

(details in previous chapters) (Yoon & Eom, 2016). Moreover, more than 600 clinical trials (out of which 135 are active) with newer, better, and safer HDAC inhibitors, individually or in combination, are currently ongoing for a variety of oncologic indications in the clinic. However, during preclinical and clinical studies various HDAC inhibitors showed severe cardiac toxicities like ventricular arrhythmias, atrial fibrillations, QTc prolongation, T–wave inversions, Torsade de pointes and death in certain cases (Catalano et al., 2012; Lynch et al., 2012; Kopljar et al., 2016; Shah et al.,

2006; Xu et al., 2013). The two most potent pan-HDAC inhibitors, romidepsin

(IstodaxTM) and panobinostat (FarydakTM) carry black box warning for severe cardiac abnormalities on their prescribing label and physicians are advised to avoid these drugs with underlying cardiac comorbidities or with drugs that prolong QT intervals (Fallis, A;

Novartis, 2015). The underlying mechanisms for HDAC inhibitor induced cardiac toxicities are unknown. These off-target effects of HDAC inhibitors are considered as a roadblock to their success in anti-cancer therapy. Thus, in this study, our focus was to illuminate the electrophysiological and molecular mechanisms which cause serious 143

cardiac side-effects of HDAC inhibitors. Below I will enunciate our results and conclusions regarding individual aspects of our investigations.

HDAC inhibitors cause ventricular tachycardias and atrial fibrillation in mice We found that intraperitoneal injection of panobinostat in C57BL/6J mice (two weeks recovery after implantation of ETAF10 transmitter) for two weeks with 2 days OFF (no injection) caused dose dependent statistically significant prolongation of ST and QT intervals. These mice had several episodes of ventricular tachycardia, premature ventricular contractions and non-sustained atrial fibrillations. Towards the end of the dosing regime, we did observe severe bradycardia along with second degree AV block in mice injected with panobinostat 30 mg/kg, followed by death in one instance.

Interestingly, each mouse injected with panobinostat showed signs of AF after 5 days of treatment. The treatment regimen was based on the intermittent dosing of panobinostat approval by FDA for patients of multiple myeloma (dose of 20 mg orally on 1st, 3rd and

5th day of the first two weeks of a 3 week cycle, for 8 weeks with or without food) and by previous studies of panobinostat in the field (Catalano et al., 2012; Novartis, 2015; Vilas-

Zornoza et al., 2012). Recently, consistent with our results, Spence et al showed that irrespective of structural class, all three pan-HDAC inhibitors vorinostat, mocetinostat and panobinostat delayed QTc intervals in dogs (Spence et al., 2016). We conclude from this experiment that pan-HDAC inhibitors increases the vulnerability to arrhythmias in mice.

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HDAC inhibitors’ induced cardiotoxicity is hERG (Ikr) independent

The delayed rectifier (IKr, also known as hERG) is one of the primary currents responsible for repolarization in humans and in neonatal mouse ventricular cardiomyocytes along with IKs (Wang et al., 1996). The majority of congenital and drug induced long QT syndromes caused by blockade of this current (Amin et al., 2013; Recanatini et al., 2005).

As a result, new chemical entities are subjected to extensive hERG binding or blocking studies to determine drug safety testing for regulatory approvals. Interestingly, the concentration at which most pan-HDAC inhibitors exhibit cardiac toxicity is much lower

(<300 fold for vorinostat and panobinostat) than their hERG binding IC50 reported in various studies (Kopljar et al., 2016; Spence et al., 2016). This is the prime finding of our study that both acute and long term treatment of panobinostat of neonatal mouse ventricular myocytes did not cause any change in steady state and transient outward K+ currents, which indicates that the HDAC inhibitor induced cardiotoxicity is independent of their hERG blockade. Based on acute K+ current experiments, we found that HDAC inhibitors do not act as direct blockers of K+ channels and can alter the repolarization via other transcriptional mechanisms which is consistent with the observations made by

Spence et al. Moreover, Kopljar et al also demonstrated that the onset of cardiotoxic effects of HDAC inhibitors in human induced pluripotent stem cells were started to be seen only after 12 hours, thereby, endorsing the idea of long term activity of HDAC inhibitors via transcriptional machinery, consistent with our experiments (Kopljar et al.,

2016).

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No change in L-type calcium current by HDAC inhibition – panobinostat Slow activation of calcium currents maintains a balance of inward and outward currents maintaining the ‘plateau’ phase of action potential. This slow entry of calcium is critical for excitation-contraction coupling in cardiomyocytes. In contrast to other pan-HDAC inhibitors tested in lab, which exhibited a two-fold isoproterenol independent stimulation of peak ICa,L (data not shown), panobinostat dose-dependently did not alter any ICa,L.

Reduction in peak INa and gap junctional coupling can cause reentrant as well as triggered arrhythmia with no change in INa,late Excitability and gap junctional (GJ) coupling are the two major determinants of conduction velocity. In contrast to IK and ICa.L which showed no significant change, we found that pan-HDAC inhibition dose-dependently reduced peak INa (≈50%), and GJ conductance (gj ≥ ≈ 50%) in neonatal mouse ventricular cardiomyocytes with no noticeable change in INa,Late. Significant reduction of both peak INa and gj can cause conduction slowing, disturbances in the source-sink relationship, and anisotropic conduction leading to enhanced vulnerability to reentry based arrhythmias. Reductions in gj can also enhance triggered arrhythmias by limiting the electrotonic inhibition in cardiomyocytes, thereby further increasing susceptibility to reentrant arrhythmias.

Stimulation of isolated ventricular cardiomyocytes treated with panobinostat showed ≈

50% reduction in APD90 compared to non-treated cardiomyocytes which can increase the propensity for delayed after depolarizations causing triggered arrhythmias.

Reduced protein and mRNA expression of Cx43 and Nav1.5 In correlation with our electrophysiological data, we observed reduction in protein expression of Cx43 and Nav1.5 in both NMVMs and ventricular lysates of mice treated 146

with panobinostat. The expression results were closely related to the HDAC inhibitory activity of panobinostat as indicated by acetyl-H3 and acetyl-α-tubulin, markers for nuclear and cytoplasmic acetylation respectively.

Atrial fibrillation can be caused by enhanced K currents and augmented expression of Kcna5 (IKur) Structural and electrical remodeling are fundamental mechanisms for pathophysiology of atrial fibrillation (AF). We found that the onset of AF phenotype was recorded in each mouse injected with panobinostat at the 5th day of injection. This rapid onset of AF invalidates any kind of structural remodeling induced by panobinostat. However, in our electrophysiological studies we observed statistically significant augmentation of steady- state and transient outward K+ currents, indicating some sort of electrical remodeling.

Repolarization in atria is predominated by the ultra-rapid delayed rectifier current (IKur) as compared to ventricles where Ikr and Iks are more prominent (Schotten et al., 2011).

Several instances of gain of function mutations of K-channels are known to have caused

AF via functional reentry mechanisms. Although these results are preliminary, they definitely point towards crucial K-channel mediated electrical remodeling induced by pan-HDAC inhibition.

Altered mRNA expression of ion-channel and calcium handling genes in atria and ventricles by pan-HDAC inhibition It is already considered that HDAC inhibitors alter 2-20% of genes in malignant cell lines

(Katoch et al., 2013). Thus, in our gene expression study for LQT genes, calcium handling and ion channel genes in atrial and ventricular culture treated with panobinostat, we found significant dysregulation of multitude of genes especially calcium handling 147

genes. For example, expression of sarcoplasmic reticulum Ca2+ATPase (Serca2a) and sodium calcium exchanger (Slc8a1) were significantly down-regulated in atria while in ventricles Serca2a was reduced while Slc8a1 expression levels unchanged. Similarly, we saw marked reduction in expression of L & T type calcium channels in atria but we didn’t observe any such change in ventricles. However, the physiological relevance of fold changes in gene expression should be based on the actual abundance of these genes in the cell. Interestingly, similar to our results, HDAC1 and HDAC2 double knock out mouse also did not show any significant change in expression of Serca2, ryanodine receptor

2(Ryr2) and Slc8a1 (Montgomery et al., 2007). Consistent with our results, Spence et al recently demonstrated HDAC mediated transcriptional dysregulation of genes involved in protein trafficking, scaffolding, adaptor proteins for ion channel surface expression and regulatory subunits of ion channels affect repolarization (Spence et al., 2016).

Overall, we conclude that a) reduction in magnitude of sodium current density and gap junctional coupling, b) unaltered sodium late current and c) unchanged repolarizing currents are the predominant reasons to account for cardiac side effects of

HDAC inhibitors during preclinical and clinical trials. Although, with these results, we cannot explain Long QT syndrome accrued by HDAC inhibitors in patients which could be due to electrolyte disturbances (hypokalemia; diarrhea is the most common side effect of HDAC inhibitors) or a metabolite of panobinostat with a higher proclivity for hERG blockade. Management of heart rhythm disorders and arrhythmias is always challenging in cancer patients especially due to comorbidities, age, and co-administration of multiple drugs for primary etiology along with chemotherapeutic agents. Biochemical 148

cardiotoxicity is less understood than electrophysiological cardiotoxicity. Careful extrapolation of our results, although in mice (neonatal and adult) can help guide the design of HDAC inhibitors with a better safety index. HDAC inhibitors currently in market are safe due to careful dose and dosage adjustments. The information gathered by this study can also form a basis for evaluating inclusion and exclusion criterion of patients enrolling in clinical trials and will enhance the vigilance of HDAC inhibitor ensued cardiotoxicity.

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Future Studies Identify Post-translational modification (acetylation) of ion-channels and Cx43 using immunoprecipitation and Mass Spectrometry Acetylation is one of the most widely studied post-translational modifications after phosphorylation in cardiac physiology. Classically, histones are considered the primary target of HDACs. However, recently, more than 1700 non-histone protein targets including transcription factors like p53, c-Myc and Nf-κB, signal transducers such as

STAT3, Smad7, cytoskeleton proteins like paxillin, cortactin, α-tubulin and molecular chaperones like heat shock protein 90 (HSP90) have been mentioned in the literature

(Choudhary et al., 2009; Singh et al., 2010). Acetylated proteins can have altered cellular localization, stability, protein-protein interactions and function.

In 2011, Colussi et al has already shown that Cx43 is acetylated in muscular dystrophic mouse heart. PCAF (a histone acetyl transferase) and HDAC 3, 4, 5, and are responsible for acetylation/deacetylation of Cx43 in these mice. This group has also shown the acetylation mediated ‘lateralization’ and dissociation of Cx43 gap junctions in dystrophic heart from intercalated discs (Colussi et al., 2011). Snyder and colleagues showed that acetylation and ubiquitination have opposite effects on identical lysine residues of the epithelial sodium channel (ENac) to alter its degradation (Butler et al., 2015). It is unknown if Nav1.5 or Cav1.2 is acetylated but bioinformatics analysis (PHOSIDA database) predicts two cytoplasmic acetylation sites near domain II and IV S4 domains of Nav1.5 with high probability (> 90%). We propose acetylation at these sites may account for +2-3 mV shift in activation of INa we observed in our experiments. 150

We detected reduction in protein expression of Nav1.5 with no change in mRNA expression underscoring the probability of regulation of Nav1.5 via post-translational mechanisms. In future studies, we will consider confirming these sites using immunoprecipitation and site-directed mutagenesis or mass spectrometry. In addition, we would also like to tease out individual HDACs and HATs involved in the post- translational regulation of Nav1.5. In 2013, Foster et al examined the cardiac acetyl-lysine proteome of guinea pig heart by affinity-peptide based enrichment using anti-acetyl- lysine antibody and subjecting the peptides to mass-spectroscopy. Although they found that calcium handling proteins like Serca2a and Ryr2 are acetylated, they were unsuccessful in identifying any plasma membrane bound proteins including ion-channels and Cx43. Moreover, the study had severe limitations due to non-specificity of anti-acetyl lysine antibody and the sample used was wild type guinea pig heart model (Foster et al.,

2013). In future studies, we want to investigate the effect of lysine acetylation in a heart model previously treated with HDAC inhibitor (preferably a pan-HDAC inhibitor) to better evaluate the probability of lysine acetylation of non-histone proteins.

Phosphorylation and dephosphorization by kinases and phosphatases maintain a dynamic balance in modulation of ion channels and gap junctions (Hulme et al., 2004;

Jeyaraman et al., 2012). Multiple phosphorylation sites are found on the C-terminal domains of Cx43 which have been shown to be implicated in distinct signaling cascades constitutively as well as in pathophysiological states (Jeyaraman et al., 2012).

Phosphorylation of Class II HDACs by a protein kinase D (PKD) and calcium/calmodulin dependent kinase (CAMK) regulate their transport from nucleus to 151

cytoplasm (McKinsey, 2012). In our study, we have focused most of our experiments on identifying functional and molecular mechanistic alterations of ion channels and gap junctions by HDAC inhibition. In the future, we would like to investigate the role of

HDAC inhibition on different kinases (e.g PKC, CAMKII, PKA), proteins that assist trafficking of ion-channels (MOG1, Caveolin-3, GPD1-L), adaptor proteins (ANK2,

SAP97) for anchorage of ion channels within plasma membrane, and proteins responsible for ubiquitination mediated proteasome degradation. Moreover, preliminarily we found significant downregulation in expression of Serca2a, Ryr2 and Slc8a1 by HDAC inhibition in primary mouse ventricular culture. Consistent with our results, HDAC1 and

HDAC2 dual knock out mouse also had drastic reduction gene expression of these proteins (Montgomery et al., 2007). Foster et al demonstrated that these proteins along with other calcium handling proteins are constitutively acetylated in guinea pig heart

(Foster et al., 2013). Phosphorylation by CAMKII regulates the shuttling of Class II

HDACs from nucleus to cytoplasm and CAMKII is known to form stable complexes with voltage gated calcium channel β1 & β2 subunits in turn facilitating L-type calcium current (Abiria & Colbran, 2010; Bers & Morotti, 2014). Interestingly, all other pan-

HDAC inhibitors except panobinostat tested in our lab showed stimulation of L-type calcium current. In consideration of the above mentioned observations, in the future we would like to determine the effect of HDAC inhibition on basal intracellular calcium levels in cardiomyocytes using FURA-2 AM (we performed these experiments but were unsuccessful due to slow shutter speed and low 340 nM signal). Efficient and synchronous exchange of calcium ions between cytoplasm and sarcoplasmic reticulum 152

during systole and diastole is essential for the smooth and effective contraction of ventricles. Any dysregulation in the levels of calcium milieu can lead to EADs or DADs and eventually to ventricular arrhythmias.

Alteration in conduction velocity by HDAC inhibition using multi-electrode array Excitability and GJ coupling are the determining factors of conduction velocity. Any reduction in these two parameters can translate to reduced propagation velocity. Although we demonstrated drastic reductions in the magnitude of sodium current density and gj conductance, we couldn’t determine if this translated to reduced conduction velocity in vitro culture or in vivo. We would like to use a multi-electrode array system

(Multichannel Systems) to determine the alterations in conduction velocity by pan-

HDAC inhibition. This array consists of 60 or more electrodes arranged in a particular fashion. Cardiomyocytes can be cultured on these electrodes which can be individually stimulated to determine conduction velocity. For ex vivo studies, optical imaging of mouse heart using voltage sensitive dyes, treated with a pan-HDAC inhibitor, can be done by mounting it on a Langendorff apparatus to study the propagation and conduction abnormalities.

Identify the role of various K-channel subtypes in atrial fibrillation induced by HDAC inhibition Repolarization of the atrial action potential is predominantly determined by ultra-rapid delayed rectifier (IKur) and acetylcholine activated inward rectifier current (IK,ACh) in most species. Several instances of gain of function mutations of K-channels are known to have caused AF via functional reentry. Gain of function mutation in α-subunit of IKs (Kcnq1)

(Chen et al., 2003), IKr (Kcnh2) (Wakili et al., 2011), IK1 (Kcnj2) (Hong et al., 2005) and 153

Kcne2 (Yang et al., 2004) have been shown to cause lone AF. In our in experiments, we observed augmentation of the magnitude of transient outward and steady state K+ currents with pan-HDAC inhibition in neonatal mouse atrial cultures. In the future, we would like to dissect out individual K+ current subtypes affected by HDAC inhibition. In addition, we saw increased mRNA expression of the gene encoding IKur (Kcna5). In the future, we would be interested in finding if its increased gene expression translates to higher surface protein expression and function.

Recovery after withdrawal of pan-HDAC inhibitor and Electrolytes During the panobinostat dosing, beginning on day 5, we saw occurrences of atrial fibrillation and ventricular tachycardia in mice implanted with transmitters. In our in vivo

ECG monitoring - telemetry experiments, mice were sacrificed at the end of the study.

However, we would like to continue monitoring the mice for ECG normalization and recovery after discontinuation of panobinostat injections. Moreover, in this project we were not able to explain QT interval prolongation seen in mice and patients with pan-

HDAC inhibition. Electrolyte disturbances can be one of the reasons for prolongation of

QT intervals especially because diarrhea is one of the severe side effects of all pan-

HDAC inhibitors approved in the market (for panobinostat, there was >40% incidence of hypophosphatemia, hypokalemia, hyponatremia and high creatinine). In the future, we want to analyze electrolyte levels (potassium, sodium and creatinine) in the mouse blood plasma to check if it correlates with longer QT intervals. Also, we want to check for a known metabolite of panobinostat (BJB432) which has 3 times lower hERG IC50 than panobinostat alone (EMEA,2015 – safety assessment report, Farydak®) 154

Functional and molecular alterations in ion-channels and gap junctions in human induced pluripotent stem cells Repolarization in human ventricular myocytes is mainly dependent on delayed rectifiers

IKr and IKs while in adult mice repolarization is dictated by Ito and IK1 encoded by Kv4.2,

Kv4.3, Kv1.4 and Kv2.1 (London, 2001; Nerbonne, 2004). Human induced pluripotent stem cells (hiPSCs) are created by reprogramming human fibroblasts by retroviral expression of reprogramming factors like sox7, oct4, nanog and lin28. High purity is ensured by selection based on blasticidin resistance of contracting cell clusters in culture

(Ma et al., 2011). Stem cell derived cardiomyocytes express both cardiac specific genes and proteins. They also retain atrial, ventricular, and nodal cell like action potentials and generate triggered arrhythmias by mechanisms of early and delayed after-depolarization. hiPSCs have a physiological range of QT intervals and their cell capacitance is similar to isolated mammalian cardiomyocytes (Satin et al., 2004). In addition, they synchronously contract and have intact sarcoplasmic reticulum Ca2+ release mechanisms (Germanguz et al., 2011; Peng et al., 2010). Importantly, these cells provide a new approach for studying human cardiac diseases in a dish and deliver new in vitro platforms for novel drug discovery especially for determining the arrhythmogenic potential of new molecules, especially cancer drugs because it’s unethical to test cancer drugs in healthy volunteers.

Thus, from a translational perspective, we want to investigate the electrophysiological and molecular alterations ensued by pan-HDAC inhibition in hiPSCs. Moreover, we want to translate our observations in neonatal mouse ventricular culture to hiPSCs.

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Class selective HDAC inhibition and cardiotoxicity All four HDAC inhibitor approved by the FDA for various oncological indications are pan-HDAC inhibitors. They inhibit all classes of HDACs at therapeutic concentrations used in patients. In our HDAC activity assay experiments, we found two apparent Ki’s

(high capacity high affinity and low capacity, low affinity sites) for almost all the HDAC inhibitors tested in lab, (for example vorinostat ; 300 nM & 2 μM , Romidepsin ; 5.4 nM

& 1.75 μM, panobinostat; 56 nM & 8.6 μM ). We hypothesize that these two apparent affinities correlate with affinities for inhibition of different classes of HDACs.

Interestingly, only pan-HDAC inhibitors have caused serious cardiac toxicities during clinical trials. Entinostat (Class I selective HDAC inhibitor) currently, in Phase 3 clinical trials in combination with exemestane (an aromatase inhibitor) for the treatment of breast cancer, is considered safer in context of cardiac toxicities relative to pan-HDAC inhibitors. The advent of new class selective HDAC inhibitors for example, Entinostat (a

Class I HDAC selective), Rocilinostat (a HDAC6 selective), TMP269 (a Class IIa HDAC selective) and many more, has provided us with new tools to dissect out the role of individual HDACs implicated in the modulation of ion channels and GJ proteins. In the future, we would like to test these class selective inhibitors and compare the results with pan-HDAC inhibition to identify the distinct role of class specific HDACs in cardiomyocytes.

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CHAPTER 5: APPENDIX

# Journal publications 161

Publication # 1

Title: Atrial fibrillation-associated Connexin40 mutants make hemichannels and synergistically form gap junction channels with novel properties

Authors: Patel, D., Gemel, J., Xu, Q., Simon, A. R., Lin, X., Matiukas, A., Veenstra, R.

D

Journal : FEBS Lett., 588(8), 1458–1464 (2014)

My contribution:

- I performed the single channel conductance recording of G38D and M163V

- I also did the hemi channel experiments of Cx40, Cx40-G38D, Cx40-M163V, and

Cx40 - G38D & M163V and identified that they close with calcium 2mM. Apart

from calcium, we tried Carbenoxolone to block hemichannels

- Some of the experiments for Vj-dependent gating for G38D, M163V and

combination of G38D + M163V were done during my rotation in this lab

- I also tried Propidium iodide and Yopro-2 experiments to identify if opening of

hemi-channels in calcium free solution

162

163

Publication # 2

Title: Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

Authors: Patel, D., Zhang, X., & Veenstra, R. D.

Journal : FEBS Lett., 588(8), 1372–1378 (2014)

My contribution:

- Worked on western blots to identify variability of Cx43 expression in HEK cells

Procured from different research laboratories

- I worked on creating a shRNA knock down of Cx43 in HEK cells and performed

western blots to confirm the knock down

164

165

Publication # 3

Title: Histone deacetylase inhibition reduces cardiac connexin43 expression and gap junction communication.

Authors: Xu, Q., Lin, X., Andrews, L., Patel, D., Lampe, P. D., & Veenstra, R. D.

Journal : Front. Pharmacol., 4(April), 44 (2013)

My contribution:

- Performed western blots to show reduction in Cx40 expression in neonatal mouse

atrial culture when treated with 1μM vorinostat for 24 hours.

166

167

Publication # 4

Title: Degradation of a connexin40 mutant linked to atrial fibrillation is accelerated.

Authors: Gemel, J., Simon, A. R., Patel, D., Xu, Q., Matiukas, A., Veenstra, R. D., &

Beyer, E. C

Journal : J. Mol. Cell. Cardiol., 74, 330–339 (2014)

My contribution:

- Performed some of the experiments for Vj dependent gating of V85I & L229M

mutant during my rotation in this laboratory

168

169