View of Shsp Families 22

UNIVERSITY OF CINCINNATI

Date: 26-Jan-2010

I, Jiang Qian , hereby submit this original work as part of the requirements for the degree of: Doctor of Philosophy in Molecular, Cellular & Biochemical Pharmacology It is entitled: The Role of Small Heat Shock Protein 20 and Its Phosphorylation in the

Regulation of Cardiac Function and Ischemia/Reperfusion Injury

Student Signature: Jiang Qian

This work and its defense approved by: Committee Chair: Evangelia Kranias, PhD Evangelia Kranias, PhD

Guochang Fan, PhD Guochang Fan, PhD

Walter Jones, PhD Walter Jones, PhD

Hongsheng Wang, PhD Hongsheng Wang, PhD

Muhammad Ashraf, PhD Muhammad Ashraf, PhD

Jo El Schultz, PhD Jo El Schultz, PhD

4/26/2010 366

The Role of Small Heat Shock Protein 20 and Its Phosphorylation in the Regulation of Cardiac Function and Ischemia/Reperfusion Injury

A dissertation submitted to the

Division of Research and Advanced Studies of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY (Ph.D.)

2010

Jiang Qian

M.D. Soochow University Medical College, 1995 M.S. Soochow University Medical College, 1998

Committee Chairperson: Dr. Evangelia G. Kranias

Abstract

The small heat shock protein (sHsp) with apparent molecular mass of 20 kD

(Hsp20) is one of 10 members of the sHsp family. Interestingly, Hsp20 is the only member within this family that contains a consensus peptide motif (RRAS) for protein kinase A (PKA)/protein kinase G (PKG)-dependent phosphorylation at Ser16. Recent studies have shown that enhanced myocardial function was associated with increased expression levels of Hsp20 and its phosphorylation. To further elucidate the possible mechanisms underlying the inotropic effects of Hsp20 and its phosphorylation, as well as their possible roles in ischemia/reperfusion-induced cardiac injury, the present study employed in vitro adenoviral-gene transfer and in vivo transgenic approaches.

Three transgenic mouse models were generated, including overexpressed wild-type

Hsp20, non-phosporylatable Hsp20S16A and constitutively phosphorylated Hsp20S16D.

Our study firstly revealed that acute overexpression of wild-type Hsp20 by adenoviral infection augmented cardiac myocyte contractility, which was further confirmed in Hsp20-transgenic murine hearts (10-fold overexpression). This hypercontractility was associated with increased activation of phospholamban (PLN), evidenced by 2-fold higher expression of Ser16/Thr17-phosphorylated PLN in

Hsp20-transgenic hearts related to non-transgenic controls. Furthermore, co- immunoprecipitation experiments indicated that Hsp20 was associated with type 1 phosphatase (PP1), suggesting Hsp20 may regulate PP1 activity in the mouse heart.

Indeed, PP1 activity was significantly reduced in Hsp20-transgenic hearts, compared to non-transgenic hearts. These results imply that Hsp20 positively regulate cardiac

function via inhibition of PP1 activity, and its downstream target, PLN phosphorylation.

Secondly, to further assess the functional significance of phospho-Ser16

Hsp20 in vivo and its possible roles in regulation of I/R-induced apoptosis and autophagy, we generated a transgenic mouse model with cardiac-specific expression of a non-phosphorylatable Hsp20, in which Ser16 was replaced with alanine to block its phosphorylation (Hsp20S16A). Our findings indicate that increased Hsp20S16A expression in the heart failed to protect hearts against ex vivo and in vivo I/R injury, accompanied by impaired autophagy and increased apoptosis. Accordingly, pretreatment of Hsp20S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20S16A hearts.

Thus, Hsp20 and its Ser16 phosphorylation may be involved in the regulation of I/R- induced cardiac autophagy and cell death.

Finally, we generated the Hsp20S16D transgenic mouse model, in which

Ser16 is replaced with aspartic acid (D), to further explore the in vivo role of phospho-

Ser16 Hsp20. Surprisingly, we observed that contractile parameters were significantly depressed in Hsp20S16D cardiomyocytes, compared with non-TGs. In vivo contractile function was also significantly impaired in TGs, compared with their non-transgenic littermates. In addition, TG mice developed left ventricular (LV) fibrosis at 6 weeks of age, without evidence of LV hypertrophy or dilation, and their life span was markedly shortened (mean age at death: 9 months). Further electron microscopy examination of the hearts revealed that double-membrane autophagosomes were more

prominent in TGs. This was associated with increased lysosomal activity, as evidenced by immunostaining with lysosomal-associated membrane protein-

1(LAMP-1) antibody, suggesting that autophagosome accumulation was not due to diminished activity of distal lysosomal pathways. Our data indicate that long-term augmentation of cardiac Hsp20 phosphorylation impairs cardiac function, accentuates pathological remodeling, and increases autophagic activity, leading to premature death. Therefore, it is of great interest to investigate the mechanisms underlying the cardiac dysfunction and remodeling in Hsp20S16D transgenic mice.

Collectively, these studies demonstrate that Hsp20 may be a key regulator of

Ca2+-cycling through modulation of PP1-PLN activity, and phosphorylation of Hsp20 is important in the regulation of ischemia/reperfusion-induced cardiac autophagy and cell death.

Acknowledgements

My deepest gratitude goes first and foremost to Dr. Evangelia G. Kranias, my thesis advisor, for her constant encouragement, guidance and support. She has walked me through all the stages of the research work and writing of this thesis. Without her consistent and illuminating instruction, this thesis could not have reached its present form.

I would like to express my heartfelt gratitude to my thesis co-advisor, Dr. Guo-Chang Fan, whose help, stimulating suggestions and encouragement helped me during all my research time in the lab.

I am deeply indebted to the rest of my thesis committee: Dr. Muhammad Ashraf, Dr. W. Keith Jones, Dr. Jo El Schultz and Dr. Hong-Sheng Wang, for their encouragement, insightful comments and criticisms.

I also owe my sincere gratitude to my fellow lab mates: Dr. Kobra Haghighi, Dr. Guoxiang Chu, Dr. Stela Florea, Dr. Tracy Pritchard, Dr. Wenfeng Cai, Dr. Wen Zhao, Dr. Xiaohong Wang, Chi Keung Lam, Sarah Figueria and Andrea Collins, for their help, support and valuable hints. I want to express my gratitude to Dr. Bryan Mitton for his help in generating the Hsp20 knockout target, and Dr. Qunying Yuan for her help in cardiomyocyte isolation techniques.

Last my thanks would go to my beloved parents and parents-in-law, for their constant encouragement and love, which I have relied on throughout these years. Especially, I would like to give my sincere thanks to my husband Yan, whose patient love enabled me to complete this work, and my babies Andy and Sophie, who help me understand the purpose, meaning, and integrity of life.

Table of Contents

Abstract 1

Acknowledgements 4

Table of Contents 6

List of Abbreviation 13

List of Tables and Figures 16

Chapter I Introduction 20

Section I. Heat shock proteins (Hsps) 20

I.1 Stress response and heat shock proteins 20

I.2 Small Heat Shock Proteins (sHsps) 22

I.2.1 Overview of sHsp families 22

I.2.2 HSPB1(Hsp25/27) 25

I.2.3 B-crystallin/CRYAB 26

I.2.4 Heat shock protein 20 (Hsp20/HSPB6) 28

I.2.4.1 Identification of Hsp20 28

I.2.4.2 Structure of Hsp20 29

I.2.4.3 Phosphorylation of Hsp20 31

I.2.4.4 Chaperone activity of Hsp20 34

I.2.4.5 Tissue distribution of Hsp20 34

I.2.4.6 Non-cardiac functional role of Hsp20 36

I.2.4.6.1 Smooth muscle relaxation 36

I.2.4.6.2 Platelet aggregation 42

I.2.4.6.3 Prevention of A fibril formation and toxicity 44

Section II. Hsps and cardiac function 47

II.1 Excitation-contraction coupling and cardiac function 47

II.2 Hsps and cardiac function 51

II.3 Hsp20 regulating ex vivo cardiac function 52

Section III. Hsps and cardiac disease 53

III.1 Hsp20 and atherosclerosis 53

III.2 Hsp20 and desmin-related cardiomyopathy 54

Section IV. Hsp20 and cardiac protection 55

IV.1 Cardiac ischemia/reperfusion injury 55

IV.2 Myocardial cell death in ischemia/reperfusion injury 58 IV.2.1 Necrosis and Apoptosis 58

IV.2.2 Autophagy 59 IV.3 Hsps and cardiac protection 60

Section V. Dissertation focus and hypothesis 61

Chapter II Materials and Methods 64

Section I. Infection of cardiomyocytes with recombinant adenoviruses 64 I.1 Recombinant adenovirusal constructs 64

I.2 Myocyte preparation and infection of myocytes 66 Section II. Generation of targeted mice 67

Section III. Isolation of mouse tail DNA and PCR conditions 68

Section IV. Quantitative immunoblotting 71

WT IV.1 Quantitative immunoblotting of total Hsp20 levels in Hsp20 and 71 Hsp20S16A transgenic mouse hearts

IV.2 Quantitative immunoblotting of other small heat shock proteins and SR 72 Ca2+-handling proteins in transgenic mouse

IV.3 Generation of a pSer16-Hsp20 antibody 73 IV.4 Quantitative immunoblotting of phospho-Ser16-Hsp20 levels in ischemia/reperfusion injured mouse hearts and failing human hearts 74

Section V. Morphological studies 74 Section VI. Verification of S16A-Hsp20 expression in transgenic mouse by 2- dimentional gel electrophoresis and mass spectrometry 75

VI.1 Two-dimensional gel electrophoresis 76

VI.2 Two-dimensional gel image analysis 77

VI.3 In-gel digestion of proteins for mass spectrometry 78

VI.4 Protein identification via MALDI-TOF and LC-MS/MS 79 Section VII. Isolation of myocyte and measurement of contractile parameters and Ca2+ transients 80

Section VIII. In vivo assessment of cardiac function using echocardiography 83 Section IX. Pressure-volume loop analyses 84

Section X. Ex vivo ischemia/reperfusion studies 85 X.1 Basal cardiac function measurement 85

X.2 Ischemia/reperfusion protocol 86 Section XI. In vivo Ischemia/reperfusion Study 86

Section XII. Cardiac injury and apoptosis analysis 87 Section XIII. Autophagy activity analysis 88

Section XIV. Oligomerization study by sucrose gradient electrophoresis 89

Section XV. Immunofluorescence staining 89

Section XVI. Immunoprecipitation 91

Section XVII. Protein phosphatase assays 92

Section XVIII. Generation of Hsp20 knockout mice 92

XVIII.1 Gene targeting constructs 92

XVIII.1.1 Strategy 92

XVIII.1.2 Generation of homology arms 97

XVIII.2 Screening of positively targeted embryonic stem cells 101

XVIII.2.1 DNA extraction 101

XVIII.2.2 PCR screenings for the Neo gene 103

XVIII.2.3 PCR screening for the short homology arm 103

XVIII.2.4 Southern Blotting 103

XVIII.2.4.1 Digest the DNA with EcoR1 104

XVIII.2.4.2 Run the digest on an agarose gel 107 XVIII.2.4.3 Denature the DNA 107

XVIII.2.4.4 Transfer the denatured DNA to the membrane 107

XVIII.2.4.5 Probe the membrane with labeled ssDNA 107 S16D Section XIX. Generation of Hsp20 transgenic mice 110 Section XX. Statistical analysis 111 Chapter III Results 112 Section I. Hsp20 and its phosphorylation at Serine 16 regulates cardiac function 112 I.1. Background and rationale 112

I.2. The effects of acute overexpression of Hsp20 on cardiomyocyte 114 contractility

I.3. Generation and characterization of Hsp20 transgenic mice 119

I.3.1 Genotyping of transgenic mice 119

I.3.2 Expression levels of Hsp20 and other sHsp in transgenic hearts 119

I.3.3 Morphological and histological studies of Hsp20 transgenic hearts 121

I.3.4 Assessment of Ca2+ cycling proteins levels in sarcolemma 125

I.3.5 Overexpression of Hsp20 enhanced contractility and Ca2+ kinetics in 128 cardiac myocytes 1.3.5.1 Hsp20 TG increased myocyte contractility 128

1.3.5.2 The Ca2+ kinetics in Hsp20 cardiac myocyte 131

I.3.6 Enhanced global cardiac contractile function in Hsp20 transgenic 135 mice

I.3.7 Phosphatase activity in Hsp20 hearts 137

I.3.8 Hsp20 associated with PP1 139

I.4. Generation of Hsp20S16A transgenic mice 140

I.4.1 Increased level of phosphorylated Ser16-Hsp20 in sustained 140 isoproterenol-treated rat cardiomyocytes I.4.2 Increased level of phosphorylated Ser16-Hsp20 in ischemia/reperfused 140 and failing hearts

I.4.3 Transgenic mice with cardiac-overexpression of Hsp20S16A 144

Section II. Hsp20 and its phosphorylation at Ser16 protects against cardiac 150 ischemia/reperfusion injury 150 II.1 Background and rationale

II.2 Impaired functional recovery in Hsp20S16A hearts during 152 ex vivo ischemia/reperfusion injury II.3 Increased necrosis and apoptosis in Hsp20S16A hearts upon ex vivo 155 ischemia/reperfusion after in vivo ischemia/reperfusion injury

II.4 Increased apoptosis and infarct size in Hsp20S16A hearts 158

II.5 Inactivation of autophagy in Hsp20S16A hearts upon ex vivo 161 ischemia/reperfusion II.6 Pretreatment of Hsp20S16A hearts with rapamycin improved functional 163 recovery in response to ischemia/reperfusion II.7 Effects of mutant S16A on Hsp20 oligomerization patterns following 166 ischemia/reperfusion 168 Section III. Generation of Hsp20 knock-out mice

III.1 Background and rationale 168

III.2 Generation of targeting constructs 170

III.3 Screening of the targeted embryonic stem cells 175

Section IV. Generation and characterization of Hsp20S16D transgenic mice 177

IV.1 Background and rationale 177

IV.2 Generation of Hsp20S16D transgenic mice 178

IV.3 Overexpression of Hsp20S16D reduced myocyte contractility 184 IV.4 Overexpression of Hsp20S16D depressed ex vivo and in vivo cardiac 187 function IV.5 Overexpression of Hsp20S16D induced interstitial fibrosis in the heart 190

IV.6 Overexpression of Hsp20S16D maintains myofilament arrangement 192

IV.7 Overexpression of Hsp20S16D increases autophagy activity 194

IV.8 Survival rate of Hsp20S16D transgenic mice 198

Chapter IV Discussion 199

Dissertation Summary 199

Section I. Hsp20 regulates cardiac function 202

I.1 The balance of protein kinase and phosphatase fine-tunes cardiac function 202 I.2 Hsp20 is a prominent regulator of SR Ca2+ cycling and contractile function 204 in cardiomyocytes I.3 Future study 212

I.4 Summary 214 Section II. Hsp20 and its phosphorylation at Ser16 protects against cardiac 216 ischemia/reperfusion injury II.1 Apoptosis, necrosis and autophagy in cardiac ischemia/reperfusion injury 216 II.2 Hsp20 and its phosphorylation at Ser16 protects against cardiac 220 ischemia/reperfusion injury II.3 Study limitations 227

II.4 Future Studies 230

II.5 Summary 232

Section III. Generation of Hsp20 KO mice 233

III.1 Design of Hsp20 gene targeting construct 233

III.2 Future studies 236

III.3 Summary 237

Section IV. Generation of Hsp20S16D transgenic mice 239 IV.1 Chronic expression of Hsp20S16D resulted in cardiac dysfunction and 239 remodeling IV.2 Future studies 242

IV.3 Summary 242

Conclusion of the dissertation 243

Reference 244

Appendix 291

List of Abbreviation Abbreviation Whole name 2-D Two dimensional BC -B crystallin A amyloid- peptide AD Alzheimer’s disease Ad.GFP Adenovirus containing GFP cDNA Ad. Hsp20 Adenovirus containing Hsp20 cDNA in sense orientation ALS Amyotrophic lateral sclerosis AMPK Adenosine monophosphate-activated protein kinase APP Amyloid precursor protein ATG Autophagy related gene BW Body weight Ca2+ Calcium

[Ca]i Ca concentration cAMP Cyclic adenosine monophosphate CaMKII Ca-camoldulin-kinase II CMV cytomegalovirus CRE Cre recombinase CSQ Calsequestrin cGMP Cyclic guanosine monophosphate DKO Double knockout EM Electron microscopy ES Embryonic stem ET Ejection times FS Fractional shortening G6PD Glucose-6-phosphate dehydrogenase GFP Green fluorescent protein HW Heart weight H&E Hematoxylin and eosin

HRC Histidine-rich Ca-binding protein Hsp Heat shock protein I/R Ischemia/reperfusion 2+ Ica L-type Ca channel current Iso Isoproterenol IP3 receptor 1,4,5-triphosphate receptors KO Knockout

LAMP-1 Lysosomal-associated membrane protein-1 LV Left ventricle LVEDD Left ventricular end diastolic dimension LVESD Left ventricular end systolic dimension LW Lung weight I-1 Inhibitor 1

INa Na channel current NCX Na-Ca exchanger NO Nitric oxide NPs Natriuretic peptides PCD Programmed cell death PCR Polymerase chain reaction PKA Protein kinase A PKC Protein kinase C PKG Protein kinase G PLN Phospholamban PP1 Protein phosphatase 1 PP2 Protein phosphatase 2 ROS Reactive oxygen species RyR2 Cardiac ryanodine receptor SM Smooth muscle SR Sarcoplasmic reticulum SERCA2a Cardiac sarcoplasmic reticulum Ca2+ ATPase T50, T80 and T90 Time to 50, 80 and 90% of decay of Ca transients

TG Transgenic TK Thymidine kinase TL Tibia length TLRs Toll-like receptors TNF-alpha Tumor necrosis factor-alpha TnI Troponin I TUNEL Terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling WT Wild type

List of Tables and Figures Table 1. Nomenclature and classification 23 Figure 1. Alignment of amino acid sequences of mouse, rat, human Hsp20 30 Figure 2. Predicted phosphorylation sites of Hsp20 33 Figure 3. Mechanisms of Hsp20 in regulation of smooth muscle relaxation 41 Figure 4. Mechanisms of Hsp20 inhibiting platelet aggregation 43 Figure 5. Sarcoplasmic reticulum Ca2+ cycling 49 Figure 6. Diagram of recombinant adenoviral vectors 65 Figure 7. Diagram of transgenic Hsp20WT and Hsp20S16A mouse constructs 69 Table 2. The sequence of primers for genotyping and the predicted length of PCR 70 products. Table 3. PCR cycling parameters for genotyping 70 Table 4. Gene targeting procedures 94 Figure 8. Generation of the Hsp20 gene targeting vector 95 Figure 9. The pKO-NTKV-1902 scramble vector 96 Table 5. PCR conditions for generation and initial screening of the Hsp20 targeting 99 homology arms Figure 10. The TOPO 2.1 cloning vector 100 Figure 11. PCR screening strategy of ES cells for homologous recombination 102 Figure 12. Southern blot strategy 105 Figure 13. Sensitivity and specificity of Southern blot probes 106 Table 6. The sequence of primers for generation of Southern blot probe 109 Table 7. PCR cycling parameters for generation of Southern blot probe 109 Figure 14. Diagram of transgenic Hsp20S16D mouse constructs 110 Figure 15. Infection efficiency of adenoviral viruses and expression levels of Hsp20 in 116 Ad.GFP, Ad. Hsp20 infected cardiac myocytes Figure 16. Basal effects of Hsp20 expression on myocyte contractility 117 Figure 17. Basal effects of Hsp20 expression on myocyte Ca2+ transients 118 Figure 18. Generation and characterization of cardiac-specific Hsp20 TG mice 120 Figure 19. Histological study of Hsp20 TG hearts 122

Figure 20. Heart weight/body weight ratio and lung weight/body weight ratio of Hsp20 123 TG mice Figure 21. Ultrastructure of cardiomyocytes from wild type and Hsp20 transgenic heart 124 by electron microscopy Figure 22. SR Ca2+ regulatory proteins in Hsp20 TG hearts 126 Figure 23. Phosphorylation of SR Ca2+ regulatory proteins in Hsp20 TG hearts 127 Figure 24. Mechanics of isolated wild type and Hsp20 TG cardiomyocytes under basal 129 conditions

Figure 25. Mechanics of isolated wild type and Hsp20 TG cardiomyocytes in responses 130 to isoproterenol Figure 26. Isolated mouse cardiac myocytes were stimulated at 0.5Hz to obtain the Ca2+ 132 transient Figure 27. Caffeine induced Ca2+ release in isolated mouse cardiac myocytes under 134 0.5Hz stimulation, with or without isoproterenol-treatment (100mmol/L) Figure 28. Enhanced in vivo cardiac function of Hsp20 transgenic mice 136 Figure 29. PP1 activity in the homogenate isolated from the LV myocardium of wild 138 type and Hsp20 transgenic hearts Figure 30. Protein interaction of Hsp20 with PP1 139 Figure 31. Phospho-S16-Hsp20 levels in 30min isoproterenol-treated rat cardiomyocytes 142 Figure 32. Phosphorylation of Hsp20 in ischemia/reperfusion injured wild-type mouse 143 hearts and failing human hearts Figure 33. Generation of Hsp20S16A transgenic mouse models 145 Figure 34. Basal functions of non-transgenic and Hsp20S16A hearts. 146 Figure 35. 2D and Mass-SPEC results of wild type and Hsp20S16A hearts 147

Figure 36. Characterization of Hsp20S16A hearts 149 Figure 37. Overexpression of Hsp20S16A increased susceptibility to ex vivo 154 ischemia/reperfusion injury Figure 38. Hsp20S16A overexpression increased IR-induced necrosis and apoptosis. 157 Figure 39. Evaluation of apoptosis in non-transgenic and Hsp20S16A transgenic 159 myocardium subjected to in vivo 30min ischemia, followed by 24h

reperfusion Figure 40. Infarct areas were increased in Hsp20S16A TG hearts after prolonged 160 ischemia/reperfusion injury

Figure 41. Autophagy is enhanced in Hsp20WT hearts and attenuated in Hsp20S16A hearts 162 in response to ischemia/reperfusion injury Figure 42. Pretreatment of autophagy inducer rapamycin in Hsp20S16A hearts led to 164 increased level of autophagy Figure 43. Pretreatment of autophagy inducer rapamycin in Hsp20S16A hearts led to 165 increased functional recovery Figure 44. Sucrose-gradient centrifugation analysis of Hsp20 proteins 167 Figure 45. Generation of Hsp20 targeting construct 172 Figure 46. Sequencing of short arm construct 173 Figure 47. Sequencing of long arm construct 174 Figure 48. Representative Southern blot results of ES cells for homologous 176 recombination Table 8. Phosphorylation sites of sHsp and identification of protein kinases 177 Figure 49. Generation of Hsp20S16D TG mouse models 179 Figure 50. Confirmation of Hsp20S16D expression in transgenic hearts 181 Figure 51. Characterization of Hsp20S16D TG mouse model 183

Figure 52. Basal contractility of isolated myocytes from WT and Hsp20S16D TG hearts 185 Figure 53. Mechanics of isolated wild type and Hsp20S16D TG cardiomyocytes in 186 responses to isoproterenol Figure 54. Ex vivo cardiac function of wild type and Hsp20S16D TG mice by Langendorff 188 perfusion Figure 55. In vivo function of Hsp20S16D hearts assessed by echocardiogram 189 Figure 56. Representative sections of Masson’s Trichrome stained cardiac sections from 191 12-weeks old transgenic and wild type mice Figure 57. Electron microscopy showing the normal arrangement of myofilaments in 193 wild type and Hsp20S16D cardiomyocytes Figure 58. Electron microscopy of autophagosomes in wild type and Hsp20S16D myocytes 196

Figure 59. Increased abundance of lysosomal markers in Hsp20S16D ventricle 197

Figure 60. Premature death in Hsp20S16D transgenic mice 198 Figure 61. Proposed scheme for phosphorylation of Ser16-Hsp20 protecting against 222 cardiac ischemia/reperfusion injury Figure 62. Schematic model demonstrating normal autophagic flux and the induction of 231 autophagosome formation when turnover is blocked

Figure 63. Proposed Tamoxifen-inducible disruption of the Hsp20 gene 238

Chapter I

Introduction

Section I. Heat shock proteins (Hsps)

I.1 Stress response and heat shock proteins

First described in Drosophila by Ritossa in 1962, the heat shock response was characterized by a puffing pattern in the salivary glands of Drosophila exposed to above-normal temperatures.1, 2 Chromosomal puffs were later demonstrated to be active sites of increased transcription and translation of proteins identified as “heat shock proteins” (Hsps).3 Subsequently, it was determined that heat shock proteins can be induced by a number of stressors in addition to heat, such as catecholamines, heavy metals, arsenite, hypoxia, free radicals, and osmotic stress. Thus, the heat shock proteins are often referred to as stress proteins. Cells stressed by a mild insult are more resistant to a subsequent stress, and this response, termed stress tolerance, is mediated , at least in part, by the heat shock proteins.4

The stress response is ubiquitous and highly conserved.4 It is observed in all organisms from bacteria to plants and animals. Although certain features of the response vary from organism to organism, many are universal, or nearly so.

Furthermore, Hsps are present in all organisms at normal temperatures and play vital roles in normal cell function.2, 5

The predominant function of Hsps is the folding and unfolding of protein substrates.2They act like “chaperones”, making sure that the cell’s proteins are folded in the correct shape, which is essential for their function. They also shuttle proteins from one compartment to another inside the cell, and transport old proteins tothe proteasome (“garbage disposals” inside the cell).6Hsps also play a role in protein degradation: some of the essential components of the cytoplasmic ubiquitin- dependent degradative pathway are Hsps.5, 7 These functions of Hsps are essential in every living cell. They are required for preventing and/or repairing the damage resulting from stress.5

Hsps belong to multigene families that range in molecular size from 10 to

150 kDa and are found in all major cellular compartments.3 The Hsps are named according to their molecular weights as follows: Hsp27, Hsp70, and Hsp90, and the corresponding heat shock protein genes are: hsp27, hsp70, and hsp90.8 The distinction between constitutively expressed (e.g., Hsp70 and Hsp90) or cognate members of the Hsp family and their inducible isoforms (Hsp70i and Hsp90, respectively) is arbitrary, since accumulating evidence, in physiologically relevant in vivo systems, now indicates that such relationships depend on cell- and tissue- restricted expression.

Traditionally, Hsps are grouped into five major families according to molecular weights.9They were designated Hsp90 (heat shock protein of apparent molecular weight 90 kDa), Hsp70 (70-kDa Hsps), Hsp60 (60-kDa Hsps), Hsp40 or

DnaJ (40-kDa Hsps), and the small heat shockproteins(sHsps)9

I.2 Small heat shock proteins (sHsps)

I.2.1 Overview of sHsp families

sHsps are a large family of proteins with monomeric molecular weight of

12-43 kDa, present within the cell as large oligomeric complexes, ranging in size from 200-800 kDa. The structural homology between members of the sHsp family is quite low. sHsps share an evolutionarily conserved sequence of 80-100 amino acids, called "-crystallin domain” (core region), which consists of two -sheets held together by extensive hydrophobic interactions.10, 11 This core region contributes to sHsp resistance to thermal denaturation and has been highly conserved throughout the superfamily's evolution.11-14 In contrast, the NH2- and

COOH-terminal extensions outside the core region vary in length, sequence, and hydrophobic index, and it is likely that these differences provide the basis for sHsp structural and functional diversity.11 Earlier studies have established a critical role for the NH2-terminal region in the assembly of sHsps into high molecular masses, while the COOH-terminal sequence seems to be primarily involved with activity.

sHsp secondary structure is dominated by -strands with limited -helical content, and -sheets within the -crystallin domain mediate dimmer formation.10, 11

Crystallization of two sHsps has contributed significantly to the description of oligomerization, quaternary structure, subunit exchange, and chaperone activity.11-14

At least 10 mammalian sHsps, termed HspB1–10, have been identified in recent genome surveys of mice, rats, and human, but their function has remained

poorly understood. 15-20 The present classification seeks to distinguish two functional categories: Class I sHsps (HspB1, CRYAB, HspB6/Hsp20, and HspB8) that are ubiquitously expressed, and Class II sHsps (HspB2, HspB3, HspB7, CRYAA,

HspB9, and ODF1), which display tissue-restricted patterns of expression. Of particular interest, the majority of Class I members are predominantly heat- inducible, suggesting that their function can be elicited under pathophysiological conditions. In parallel, available evidence suggests that Class II members are not stress-inducible and are predominantly distributed in striated muscle (Class IIm) and testis (Class IIt), indicating that their tissue-specific expression is regulated by separate mechanism(s).

Table 1. Nomenclature and classification

HUGO Alternative Heat-inducible Class Localization

HSPB1 Hsp27 (h and r), +15 and-16 Class I Ubiquitous Hsp25 (m)

CRYAB B-crystallin, HSPB5 +17 Class I Ubiquitous

HSPB6 Hsp20, p20 –18 Class I Ubiquitous

HSPB8 Hsp22, H11 kinase + 19 Class I Ubiquitous

HSPB2 MKBP – 20 Class IIm Cardiac and skeletal muscle

HSPB3 HspL27 – 18 Class IIm Cardiac and skeletal muscle

HSPB7 cvHsp ? Class IIm Cardiac and skeletal muscle

CRYAA A-crystallin, HSPB4 –17 Class IIe Lens of the eye

HSPB9 None ? Class IIt Testis

ODF1 HSPB10 ? Class IIt Testis

Currently accepted nomenclature from the HUGO Human Gene Nomenclature Database (http://www.gene.ucl.ac.uk/cgi-bin/nomenclature/searchgenes.pl),21 alternative names, and proposed classification. Class I = ubiquitous expression, Class II = tissue-restricted expression: cardiac and skeletal muscle, Class IIm; lens of the eye, Class IIe; testis, Class IIt. Refer to text for specific descriptions. ( +: heat inducible; -: not heat inducible)

I.2.2 HspB1 (Hsp25/27)

Among the best characterized members of the sHsp family, HspB1

(Hsp25/27) is the prototypical Class I protein, which is expressed constitutively and ubiquitously in most cell types and intact organisms.22-30 Of interest, HspB1 also highly expressed in cardiac and skeletal muscle.18,31 The HspB1 gene expression can be induced by various stressors, such as heavy metals, oxidative stress, hormones, hypoxia, and ischemia in diverse cells and tissues. 23, 28, 32, 33 The mechanisms of activation and function of HspB1 have been thoroughly reviewed elsewhere.34, 35

Phosphorylation of HspB1 by PKC and MAPKs has been implicated in its cardioprotective effects,36-39 which are partially mediated through actin polymerization as cap-binding protein with the cytoskeletal thin-filament proteins.40,41 Reciprocal effects on redox state, the balance between pro-oxidant and antioxidant conditions, have been implicated with HspB1 expression both in cultured cells and the intact organisms.42,43 It is increasingly apparent that a complex pattern of interactions among other sHsps, the cytoskeletal, and the antioxidative mechanisms may be responsible for HspB1’s functional versatility. To date, the formal question as to whether or not HSPB1 serves an essential or redundant role for normal development and ontogeny has not been answered in the published literature.

I.2.3 B-crystallin/CRYAB

Crystallins are the predominant proteins in the mammalian eye lens, where their high concentration contributes to lens refractive power.44,45 The largest of these,

-crystallin, belongs to the family of small heat shock proteins (sHsps).46 In the lens, it is a dynamic multimeric complex composed of two types of homologous subunits,

A-crystallin and B-crystallin(-BC), each of which has molecular mass of about

20 kDa47, 48This complex not only contributes to lens refractive power but also assists in maintaining transparency.49-52The -crystallins, particularly -BC, are also found individually in nonlenticular tissues. Whether present as hetero- or homo-oligomers, however, they have the capacity to respond to several different environmental stress situations, such as heat shock, to provide protection at the cellular level.53-57 Additionally, -BC has been shown to be associated with neurological disorders such as Alzheimer disease, Alexander disease, and amyotrophic lateral sclerosis (ALS) and to protect against apoptosis.10, 58-70

-BC comprises up to 3% of cardiac homogenates, representing the most abundant sHsp in heart.27, 64, 71 Transgenic mice overexpressing -BC are protected against cardiac ischemia–reperfusion injury.72,73 Isolated perfused hearts from the

B-crystallin /HspB2 KO mouse displayed normal contractility; however, when compared with WT, after I/R, KO mouse hearts exhibited a twofold reduction in contractile recovery as well as increased necrosis and apoptosis. Additionally, when compared with WT, KO mouse hearts exhibited 43% less reduced glutathione, which is known to protect from I/R-induced damage74Thus, neither -BC nor

HspB2 is essential for myocardial development and function under non-stressful condition, but one or both are required for maximal functional recovery and protection from I/R-induced necrosis and apoptosis.

A recent study revealed distinct roles for -BC and HspB2 in I/R injury, with -BC protecting mechanical properties and HspB2 protecting energy reserve.75

Isolated hearts of wild type mice (WT), mice lacking both -BC/HspB2 (double knockout, DKO) , WT mice overexpressing mouse-BC protein (mCryABTg), and mice with no HspB2 generated by crossing DKO with mCryABTg

(DKO/mCryABTg) were stressed with either ischemia/reperfusion or inotropic stimulation. Contractile performance and energetics were measured using 31P NMR spectroscopy. Ischemia/reperfusion caused severe diastolic dysfunction in DKO hearts. Recovery of ATP and PCr during reperfusion was impaired only in

DKO/mCryABTg. During inotropic stimulation, DKO/mCryABTg showed blunted systolic and diastolic function and revealed massive energy wasting on acute stress.

Thus, -BC and HspB2 proteins play nonredundant roles in the hearts: -BC in structural remodeling and HspB2 in maintaining energetic balance.

The missense mutation in the -BC gene, Arg120Gly (R120G), gives rise to the inherited, adult onset, desmin-related myopathy, a neuromuscular disorder where desmin, an intermediate filament protein, aggregates with B-crystallin67,76

The mutation disrupts -BC structure, chaperone activity and intermediate filament interaction, an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic

activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase,77demonstrating the functional importance of residue R120.

This was the first sHsp mutation shown to cause inherited human muscle disease, but two additional dominant negative -BC mutations have since been linked to myofibrillar myopathy, but not cardiomyopathy.59 The -BC C-terminus is truncated by 13 residues in one case and 25 in another, a region important for sHsp solubilization, chaperone activity and oligomer formation. Additionally, other posttranslational modifications such as phosphorylation, O-linked glycosylation, and acetylation have been reported for -BC.

I.2.4 Heat shock protein 20 (Hsp20/HspB6)

I.2.4.1 Identification of Hsp20

In 1994, Kato et al.78 purified and identified another sHsp, Hsp20, later designated HspB6, in many tissues such as myocardium, liver, lung, kidney, and brain.18, 23 Upon further analysis, Hsp20 was observed to have sequences homologous to both B-crystallin and Hsp27, and translocated from the cytosol to the insoluble fraction under stressful conditions. Although Hsp20 does not exhibit the ‘classical’ heat shock response,18 other signal transduction pathways appear to mediate its biological roles. Under stressful conditions, phosphorylation of Hsp20 increases myocyte shortening rate and Ca2+ uptake79,80, inhibition of apoptosis,81 and/or migration of the protein to the nucleus.82

I.2.4.2 Structure of Hsp20

The primary structure of Hsp20 is highly homologous to that of A- and

B-crystallin and to a smaller extent to the primary structure of small heat shock proteins with molecular mass 25/27 kD (Hsp25/27).83 Recently published data indicate that the -crystallin domain of Hsp20 is folded in a manner similar to that of the protein with molecular mass 23 kD (Hsp23), which is co-chaperone of the heat shock protein with molecular mass 90 kD (Hsp90).83, 84 As in all members of the family of small heat shock proteins, the -crystallin domain that is composed of several -sheets is located in the C-terminal part of Hsp20 (Fig. 1, 2a).83

The less conservative N-terminal domain, as well as the variable C-terminal end contain small quantities of -helices and do not have highly ordered structure.83

Hsp20 can be phosphorylated by different protein kinases. One site of phosphorylation is located in the very N-terminal part (Ser 16) and the second (or other) sites of phosphorylation are located in the central or C-terminal parts of

Hsp20 (Ser 157), which may also contain putative sites for other protein kinases

(Fig. 2a). 85

In the earliest studies, it was found that Hsp20 is co-purified with B- crystallin and Hsp27 and forms high molecular mass oligomers with these proteins.78 According to the literature, isolated Hsp20 forms low (43-67 kD) and high (200-470 kD) molecular mass complexes.78 The recombinant human Hsp20 predominantly forms dimers and partial proteolysis of the C-terminal end or the

drop of pH to 5.5 leads to the aggregation of Hsp20, in the form of unordered

aggregates with high molecular mass.86

Homo Sapiens MEIPVPVQPSWLRRASAPLPGLSAPGRLFDQRFGEGLLEAELAALCPTTLAPYYLRAPSV 60 Rattus Norvegicus MEIPVPVQPSWLRRASAPLPGFSAPGRLFDQRFGEGLLEAELASLCPAAIAPYYLRAPSV 60 Mus Musculus MEIPVPVQPSWLRRASAPLPGFSAPGRLFDQRFGEGLLEAELASLCPAAIAPYYLRAPSV 60 *** *****************:*:*******************:***:::****** : Homo Sapiens ALPVAQVPTDPGHFSVLLDVKHFSPEEIAVKVVGEHVEVHARHEERPDEHGFVAREFH 120 Rattus Norvegicus ALPTAQVPTDPGYFSVLLDVKHFSPEEISVKVVGDHVEVHARHEERPDEHGFIAREFH 120 Mus Musculus ALPTAQVSTDSGYFSVLLDVKHFLPEEISVKVVDDHVEVHARHEERPDEHGFIAREFH 120 ::. . . ****:***************:*****:*****************:*****

Homo Sapiens RRYRLPPGVDPAAVTSALSPEGVLSI QAAPASAQA---PPPAAAK 160 Rattus Norvegicus RRYRLPPGVDPAAVTSALSPEGVLSI QATPASAQASLPSPPAAK 162 Mus Musculus RRYRLPPGVDPAAVTSALSPEGVLSI QATPASAQAQLPSPPAAK 162 **************************** *.*.** *.*.***

Figure 1. Alignment of amino acid sequences of the mouse, rat, and human Hsp20. Consensus patterns predicted for a cAMP-dependent protein kinase phosphorylation site (RRAS) are bolded. Sequences corresponding to heat shock protein conserved domain are bolded and underlined. Identical (*) amino acid residues and conserved (:) or semiconserved (.) substitutions are indicated below the aligned sequences.

I.2.4.3 Phosphorylation of Hsp20

Early investigations of Hsp20 showed that this protein can be phosphorylated in vivo87,88, 89 The pI value of the unphosphorylated protein is close to 6.3.90 In addition to this form, different tissues contain Hsp20 forms with pI 6.0,

5.9, and 5.7.90-92 These data indicate that the forms with pI 6.0 and/or 5.9 represent singly phosphorylated Hsp20, whereas a form with pI 5.7 represents the doubly phosphorylated Hsp20 protein. Special attempts have been undertaken to determine the sites of Hsp20 phosphorylation. In rat skeletal muscle, a form with pI 6.0 represents Hsp20 phosphorylated at Ser157. This site seems to be phosphorylated by phosphatidylinositol 3-kinase (or protein kinases activated by this kinase).93

Surprisingly, human and bovine Hsp20 lack Ser157 (in these proteins Ser157 is replaced by Pro). However, bovine tissues contain a phosphorylated form of Hsp20 with pI 6.0.87,88In this case, the form with pI 6.0 in bovine tissues contains a single phosphorylated site that is located in the peptide restricted by residues 120-137

(potentially Thr133, Ser134, or Ser137, based on the corresponding amino acid residues in human Hsp20).91 This site is phosphorylated in response to stimulation by phorbol esters that activate protein kinase C. However, this peptide does not contain the consensus sequence recognized by protein kinase C itself. Therefore, it seems probable that this site is phosphorylated not by protein kinase C itself, but by protein kinases that are activated by protein kinase C. The Hsp20 form with pI 5.9 contains Ser16 phosphorylated by cyclic nucleotide dependent protein kinases or potentially another unidentified site91. Hsp20 with pI 5.7 also contains phosphorylated Ser16 and another phosphorylation site located in the peptide

restricted by residues 120-137 (probably Thr133, Ser134, Ser137, based on human

Hsp20).91 Thus, Hsp20 can be phosphorylated at different sites (see Fig. 2b). It is suggested that under normal conditions, Hsp20 is either unphosphorylated or contains a single phosphorylated site in the central or C-terminal part of the molecule (Ser157 of rat Hsp20 or peptide restricted by residues 120-137 in bovine tissues.90 Upon activation of cyclic nucleotide dependent protein kinases, Ser16 also becomes phosphorylated. Phosphorylation affects the interaction of Hsp20 with other proteins, as well as the functions performed by this protein in the cell.88

Figure 2. Predicted phosphorylation sites of Hsp20. (A) Scheme of the structure of small heat shock protein with molecular mass 20 kD. Highly conservative -crystallin domain and variable N- and C-terminal ends are labeled. The position of Ser16 phosphorylated by cAMP- or cGMP- dependent protein kinases (PKA and PKG, respectively) and Ser157 phosphorylated by phosphatidylinositol 3-kinase (PI3K) or by protein kinases activated by this enzyme are marked by P. The peptide containing the sites phosphorylated by protein kinase C (PKC) or by protein kinases activated by protein kinase C is also indicated on the scheme. (B) Scheme of possible location of the sites phosphorylated in Hsp20 having different pI. Protein kinases directly or indirectly involved in phosphorylation of specific sites are indicated in parenthesis.

I.2.4.4 Chaperone activity of Hsp20

For a long time it was considered that Hsp20 has a very low chaperone activity in vitro.94 Recently, published data contradict this assumption. It was shown that the recombinant human Hsp20 has chaperone activity that is comparable with that of commercial -crystallin.86 These data agree with earlier published data of van de Klundert et al.82 These authors have shown that overexpression of either

Hsp20 or B-crystallin confers thermal resistance in Chinese hamster ovary cells.

However, B-crystallin enhancesthe recovery of co-expressed firefly luciferase, whereas Hsp20 was ineffective.82 This fact may indicate that the molecular mechanisms of protective action of Hsp20 might be different from that of B- crystallin.

I.2.4.5 Tissue distribution of Hsp20

Hsp20 is expressed in practically all organs and tissues.85 The Hsp20 content is especially high in slow skeletal muscle (m. soleus), diaphragm, heart, and smooth muscles where its quantity is 1,200-13,000 ng/mg cell protein.78 In the heart,

Hsp20 concentration is lower than B-crystallin; 27, 64, 71 however, in the bladder and rectum, which are mostly composed of smooth muscle, concentration of Hsp20 is larger than B-crystallin.71, 78 The lowest Hsp20 content is detected in liver and different regions of the brain, where its content is 3-6 ng/mg cell proteins78 It is worthwhile to mention that the Hsp20 content varies in different blood vessels. For instance, smooth muscle cells of the left interior mammary artery contain the largest quantity of small heat shock proteins (B-crystallin, Hsp20, Hsp27), whereas the

smooth muscle cells from the umbilical vein contain the lowest quantity of these proteins.95 Furthermore, it has been shown that in rats the level of Hsp20 in m. soleus is increased from 100 up to 8,000 ng/mg immediately after birth, whereas during the same period the Hsp20 content increased from 150 up to 700-900 ng/mg in extensor digitorum longus.96 A similar trend for regulation of Hsp20 expression was observed in different organs of pigs during their ontogenesis. As a rule, the

Hsp20 content is increased both in heart and skeletal muscle during ontogenesis.23

Different experimental conditions strongly affect the expression of Hsp20.

Indeed, it has been shown that spinal cord transaction97 and hind limb suspension induced atrophy98or denervation96 are accompanied by a significant decrease in

Hsp20 content in slow muscles or in muscle with antigravity function. At the same time, spinal cord transaction97 or denervation96 have small effects on Hsp20 content in flexor muscles. It was concluded that Hsp20 was predominantly expressed in muscle fibers expressing slow isoforms of myosin heavy chains97. Several attempts were undertaken to localize Hsp20 within different muscles. Using immunofluorescence microscopy, Pipkin et al.80 have shown that Hsp20 was colocalized with actin filaments. Moreover, anti-Hsp20 antibodies stained cell membranes, probably at the sites of actin filament fixation (dense bodies). Confocal microscopy of pig carotid artery indicates that Hsp20 was present throughout the cytoplasm, although some focal regions of the cytoplasm were found to contain more Hsp20 than the remaining cytoplasm.99 Thus, at the present time, there is no straightforward data indicating that Hsp20 is directly bound to the contractile apparatus.

Under rest, small heat shock proteins are majorly diffuse in the cell. In response to different unfavorable conditions (i.e. myofibril damage induced by long contraction), the small heat shock proteins are translocated to the contractile apparatus and participate in its protection and/or reparation.100 Such translocation was detected for B-crystallin and small heat shock protein with molecular mass of

27 kD (Hsp27). However, stimulation of smooth muscle by different agonists88, 99 or poisoning of smooth muscle by arsenite101 was not accompanied by translocation of

Hsp20 from cytoplasm to contractile apparatus. At the same time, proteasomal inhibition in cardiac myoblast cell line H9C2 was accompanied by translocation of

Hsp20 to stress fibers102 and cyclic nucleotide dependent phosphorylation of Hsp20 seems to increase its affinity to actin filaments in smooth muscle and cardiomyocytes.81, 89Thus, at present the exact intracellular location of Hsp20 remains ambiguous.

I.2.4.6 Non-cardiac functional role of Hsp20

I.2.4.6.1 Smooth muscle relaxation

Smooth muscle (SM) is an integral part of most mammalian organ systems, participating in many physiological processes such as the maintenance of vascular tone and pressure, uterine contractions resulting in parturition, and intestinal motility related to the digestive process.103 Contraction and relaxation of smooth muscle is a tightly regulated process involving numerous endogenous substances and their intracellular second messengers.103, 104

The intracellular second messengers involved in smooth muscle relaxation are the cyclic nucleotides, cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), generated by the activity of adenylate and guanylate cyclases, respectively103, 104. These cyclic nucleotides exert their intracellular effects by activating a specific protein kinase: cAMP-dependent protein kinase (PKA) or cGMP-dependent protein kinase (PKG), correspondingly.103, 104

Cyclic AMP is the dominant mediator of smooth muscle relaxation stimulated by -adrenergic agonists, whereas cGMP-mediated relaxation is triggered by nitric oxide (NO) and natriuretic peptides (NPs).105 Classically, cAMP was the principal second messenger involved in smooth muscle relaxation; hence several -adrenergic agonists were developed and used to alter SM tone for the management of a number of pathologic conditions in humans.105

In early investigations,87, 88, 91 PKA and PKG phosphorylate Ser16 of Hsp20 and this event is accompanied by relaxation of carotid artery smooth muscle.

Phosphorylation of Hsp20 does not affect formation of active actomyosin complex, but somehow disturbs the interaction of actomyosin complex with the structural elements providing its fixation to the cell membrane106. Brophy et al.107, 108 also reported that Hsp20 co-immunoprecipitates with actin and -actinin in vitro and phosphorylation of Ser16 decreases this interaction of Hsp20 with actin and - actinin. During contraction, unphosphorylated Hsp20 is bound to actin filaments and

-actinin. This stabilizes actin filaments and their fixation to dense bodies interacting with the cell membrane. After phosphorylation, Hsp20 dissociates from

polymerized actin and -actinin, which inhibits transmission of force and results in relaxation.107, 108

Special investigations deal with physiologic release of NO and the phosphorylation of HSP20 in smooth muscle relaxation.108, 109 It is well known that under certain conditions, mechanical stress of endothelia induces activation of NO synthase. Entering the medium, NO activates guanylate cyclase and increases the intracellular level of cGMP that activates cGMP-dependent protein kinase. This enzyme phosphorylates Hsp20, thus contributing to vasorelaxation.108, 109

Development of new methods has provided novel data on the role of Hsp20 in regulation of smooth muscle contraction. Brophy’s laboratory110 used a synthetic peptide consisting of 24 residues, which contains 11 residues of a protein transduction domain and 13 residues of Hsp20 sequence surrounding Ser16. They found that addition of this peptide containing phosphorylated Ser16 resulted in relaxation of pig carotid artery contracted by serotonin, while a scrambled peptide was ineffective in induction of relaxation.110 Transfection of rat mesangial cells

(specialized cells around blood vessels in the kidneys) with intact Hsp20 gene further defined the physiologic function of Hsp20 in smooth muscle.111 In these experiments, overexpression of Hsp20 decreased the contractile activity of mesangial cells. Although an attempt to determine the intracellular localization of

Hsp20 was not very successful, overexpression of Hsp20 resulted in disappearance of actin stress fibers in the central part of the cell. Therefore, the authors again suggested that Hsp20 affects different actin-binding proteins (-actinin108 or

Cofilin112) or actin polymerization and in this way prevents the development of contraction.

However, a principally different viewpoint prevails in publications from other laboratories. Rembold et al.89 also found that phosphorylation of Ser16 of

Hsp20 indeed induced relaxation of vessel smooth muscle. However, according to these authors phosphorylation of Hsp20 was accompanied by decrease in oxygen consumption, thus indicating that Hsp20 somehow affected formation of the active actomyosin complex. Moreover, these authors suggested that phosphorylation increased the affinity of Hsp20 for actin filaments. It is postulated that the amino acid residues 110-121 of Hsp20 had a high degree of sequence homology with a region of cardiac and skeletal TnI , termed the TnI inhibitory region (skeletal TnI104-

89 115 and cardiac TnI136-147). Thus, according to a hypothesis of Rembold et al. , phosphorylation of Hsp20 led to translocation of Hsp20 from cytoplasm to actin filaments and to inhibition of normal functioning of myosin cross-bridges. More detailed analysis of relaxation induced by forskolin and nitroglycerine resulted in detection of two phases.89 The first rapid phase of relaxation was accompanied by dephosphorylation of myosin light chains. In the later stages of relaxation, phosphorylation of myosin light chains returned practically to its high level, but the muscle remained relaxed. This unusual behavior was probably due to phosphorylation of Hsp20 that was able to switch-off actin filaments and prevent formation of active actomyosin complex. The heat shock (heating of coronary artery rings up to 43-46°C for 4 h) was accompanied by an increase in Hsp20 expression and by significant increase in its Ser16 phosphorylation without increases in cAMP

or cGMP, suggesting an alternate mechanism, perhaps phosphatase inhibition, for the increase in Ser16-Hsp20 phosphorylation79, 113.

Thus, the literature unequivocally indicates that phosphorylation of Hsp20 by cyclic nucleotide dependent protein kinases correlates with smooth muscle relaxation (Figure 3.), although the detailed molecular mechanism of this process remains enigmatic. All authors were in agreement that relaxation correlating with

Hsp20 phosphorylation was based on the ability of Hsp20 to affect actin filaments.

However, two alternative hypotheses were put forward. According to Brophy and coauthors,107 Hsp20 had no effect on switching-on actin filaments, but affected fixing of actin filaments to the cell membranes and cytoskeleton. Rembold et al.89 found that under certain conditions Hsp20 was tightly bound to actin and was switching off actin filaments, similar to troponin I. Both of the above hypotheses postulated that Hsp20 was an actin-binding protein. According to Brophy,107 phosphorylated Hsp20 predominantly interacted with monomeric actin, whereas unphosphorylated Hsp20 predominantly interacted with polymeric actin. In contrast to this viewpoint, Rembold et al.89 postulated that phosphorylated Hsp20 interacted more tightly with polymeric actin and (unlike unphosphorylated Hsp20) was able to switch-off actin filaments.

Figure 3. Mechanisms of Hsp20 in regulation of smooth muscle relaxation. In response to a variety of stimuli, endothelial cells (ECs) produce nitric oxide (NO), which is a gaseous molecule that diffuses across the cell membrane, activating guanylyl cyclase (GC) in neighboring smooth muscle cells (SMC). Sodium nitroprusside (SNP) is a pharmacologic agent that activates guanylyl cyclase. This leads to increases in cGMP and activation of PKG. Prostaglandins (PGs) and forskolin (FSK) lead to relaxation via another cyclic nucleotide-dependent signaling pathway, activation of adenylyl cyclase (AC), and increases in cAMP. This leads to activation of PKA. PKA and PKG phosphorylate HSP20. HSP20 may directly interact with elements of contractile machinery, leading to vasorelaxation. Phosphodiesterases (PDEs) convert cAMP and cGMP to AMP and GMP, respectively.

I.2.4.6.2 Platelet aggregation

The platelet is a specialized adhesive cell that plays a key role in the normal hemostatic process through its ability to rapidly adhere to subendothelial matrix proteins (platelet adhesion) and to other activated platelets (platelet aggregation) at sites of vascular injury.114 Platelet aggregation is part of the sequential events leading to the thrombus formation after vascular injury or in subsequent reocclusion, interfering successful thrombolytic therapy. The final common mechanism in platelet aggregation is the cross-linking of adjacent activated platelets by the binding of fibrinogen to the GP complex IIb/IIIa.114 Kazawa et al.115 identified two forms of Hsp20, an aggregated form (Hsp20-L) and a dissociated form (Hsp20-S), in crude extracts of muscles. The latter has a dimeric structure with covalent binding via a disulfide bond between subunits. Hsp20-S, but not Hsp20-L, possesses strong inhibitory properties to platelet aggregation in vitro and ex vivo.116 Moreover, in order to further define antiplatelet actions produced by Hsps, the same group evaluated the effects of various combined peptides isolated from Hsp20 and B- crystallin on platelet aggregation.117 These results indicated that 9 amino-acid sequences could inhibit platelet activation induced by thrombin via PAR-1 receptor and GPIb/V/IX- vWF axis.117 This novel peptide of thrombin inhibitor could be a useful tool for the creation of new compounds of antithrombotic therapy.

A. mHsp20 MEIPVPVQPSWLRRASAPLPGFSA--PGRLFDQRFGEGLLEA

B. Hsp20

Figure 4. Mechanisms of Hsp20 inhibiting platelet aggregation. (A) Amino acid sequence in Hsp20 which plays a role in inhibiting platelet aggregation. (B) Schematic of Hsp20 showing its inhibition of platelet aggregation induced by thrombin in vitro using human platelets 115. However, mechanisms underlying the inhibition of platelet aggregation remain to be clarified.

I.2.4.6.3 Prevention of A fibril formation and toxicity

Alzheimer’s disease (AD), a neurodegenerative disorder,is the leading cause of dementia in the aging population. It is characterized by two primary histopathological lesions: senile or amyloid plaques and neurofibrillary tangles. 118, 119

Amyloid plaques are found outside the neurons, while neurofibrillary tangles are found inside the neurons. 118, 119 Amyloid plaques are mostly made up of a protein called amyloid- peptide (A) which is itself part of a much larger protein called APP

(amyloid precursor protein). 120 It is hypothesized that A contributes to the neurodegeneration associated with AD via the toxicity of the peptide in aggregated form.121,122

A aggregates readily, both in vitro and in vivo, into fibrils, protofibrils, and low molecular weight intermediates/oligomers, and its toxicity is linked to its aggregation state123-126 The toxic A structures include a nonfibrillar species of 17–

42 kDa,125, 127protofibrils species with hydrodynamic radii on the order of 9–367 nm,128, 129 and fibril species. Some investigators suggested that the smaller oligomeric

A species were more toxic than fibril and protofibril forms.127, 130 Many believe that one strategy for preventing neurodegeneration associated with AD is the prevention of aggregation of A into its toxic oligomeric or fibril forms.

Several inhibitors of A aggregation have been developed to prevent A toxicity.131-134 Some of these inhibitors are small peptides with mimic sequence of

A, which is believed to be essential for aggregation and fibril formation.133 At nearly equimolar concentrations of these agents and A, peptide inhibitors have

proven useful in preventing both Aaggregation and toxicity.133, 135 The application of molecular chaperones including -crystallins and other small heat shock proteins has been explored recently.

Initial studies demonstrated that increased immunoreactivity for B-crystallin and Hsp27in AD brains.136, 137 More recent immunohistochemical studies have found

B-crystallin, Hsp27, Hsp20 and Hsp22 associated with deposits of A.138, 139

However, the biological relevance of this association is unclear. Studies examining the interaction of sHsps with A in vitro have produced inconsistent, and sometimes conflicting, results. For example, it has been reported that human Hsp27 inhibited A

1–42 fibril formation,140 but B-crystallin was actually found to increase toxicity despite reduced fibril formation.66 Recently, it was found that Hsp20 isolated from the erythrocyte parasite Babesia bovis, can preventA aggregation and toxicity at very low concentrations of Hsp20 to A (1:1000)141 Furthermore, Hsp20 did not simply slow the rate of A aggregation, but actually changed the fibril formation pathway.142

When Hsp20 was mixed with A, they formed large non-toxic multimeric complexes.142 Two other small heat shock proteins, carrot Hsp17.7 and human Hsp27, were also able to prevent A aggregation, but they failed to attenuate A toxicity.142

While all three sHsps have a well-conserved -crystallin domain, they have limited sequence homology outside the -crystallin domain (less than 20% outside of the - crystallin domain).142 These sequence differences could result in different binding affinity between the sHsp and A, binding affinities for different A oligomeric species, and/or different modes of multimeric assembly associated with sHsp

aggregation prevention. Given that Hsp20 may have a unique mechanism of interaction with A, which results in attenuation of both A aggregation and toxicity, understanding this interaction may provide insights into the design of new molecules for A toxicity prevention associated with Alzheimer’s disease.

Section II Hsp and Cardiac function

II.1 Excitation-contraction coupling and cardiac function

In adult mammalian hearts, excitation-contraction coupling is the key determinant of cardiac function, processing from the electrical excitation to the contraction of myocytes.143-145 During a cardiac action potential, upon the depolarization of sarcolemma, Ca2+ enters the cell through the L-type Ca2+ channel,

2+ as an inward Ca current (Ica-L), which activates the sarcoplasmic reticulum (SR)

Ca2+ release channel, ryanodine receptor (RyR2), triggering Ca2+ release from the

SR. This process is referred to as Ca2+-induced Ca2+ release.143-145 The combination of Ca2+ influx and release raises the free intracellular Ca2+ concentration ([Ca2+]i) from 150nM to 1M, allowing Ca2+ to bind to the myofilament protein troponin C

(TnC), which then initiates contraction.143, 145, 146 When Ca2+ binds to the TnC, this induces a conformational change in the regulatory complex such that troponin-I

(TnI) exposes a site on the actin molecule that is able to bind to the myosin ATPase located on the myosin head. This binding results in ATP hydrolysis that supplies energy for a conformational change to occur in the actin-myosin complex. The result of these changes is a movement ("ratcheting") between the myosin heads and the actin, such that the actin and myosin filaments slide past each other thereby shortening the sarcomere length. Ratcheting cycles occur as long as the cytosolic

Ca2+ remains elevated.147

For relaxation to occur, Ca2+ must be removed from the cytosol, allowing

Ca2+ to dissociate from troponin C.148 Four separate Ca2+ handling systems

participate in the removal of Ca2+: (1) SR Ca2+-ATPase (SERCA2a), (2) sarcolemmal Na-Ca exchanger, (3) sarcolemmal Ca2+-ATPase and (4) mitochondrial Ca2+ uniport.143, 145, 146, 149-151 Although the contribution of Na-Ca exchanger and SERCA2a to Ca2+ decline is species-dependent, the sarcolemmal

Ca2+-ATPase and mitochondrial Ca2+ uniport generally play a minor role in the Ca2+ decline and they only remove 1-2% of the Ca2+ from cytosol during relaxation.152,

153 Thus, the Ca2+ influx via L-type Ca2+ channel and Ca2+ efflux from SR is responsible for initiation of cardiac contraction, while the Na-Ca exchanger and

SERCA2a pump are the main systems responsible for removal of the Ca2+ to allow relaxation during diastole (Figure 5). The reduced intracellular Ca2+ induces a conformational change in the troponin complex leading, once again, to TnI inhibition of the actin binding site. At the end of the cycle, a new ATP binds to the myosin head, displacing the ADP, and the initial sarcomere length is restored.

Figure 5. Sarcoplasmic reticulum (SR) Ca2+ cycling. The sarcoplasmic reticulum acts as a Ca2+ source during contraction and a calcium sink during relaxation. Relaxation is mediated by the transport of Ca2+ into the SR lumen by a Ca2+-ATPase (SERCA2a), which is under reversible regulation by phospholamban (PLN). Ca2+ then binds to calsequestrin (CSQ) in the lumen of the SR. For the initiation of contraction, Ca2+ is released through the calcium channels or ryanodine receptors (RyR2), which are under regulation by junctin (JNC), triadin (TRD) and partially by a novel protein, the histidine rich calcium-binding protein (HRC). Thus, the SR is the major regulator of Ca2+-handling and contractility in muscle.

Enhancing the concentration of cytosolic Ca2+ increases the amount of ATP hydrolyzed and the force generated by the actin and myosin interactions as well as the velocity of shortening. Physiologically, cytosolic Ca2+ concentrations are influenced primarily by beta-adrenoceptor-coupled mechanisms.Beta-adrenergic stimulation, as occurs when sympathetic nerves are activated, increases cAMP which in turn activates protein kinase to increase Ca2+ entry into the cell through L- type Ca2+ channels. Activation of the IP3 signal transduction pathway also can stimulate the release of Ca2+ from the SR through IP3 receptors located on the SR.

Furthermore, activation of the cAMP-dependent protein kinase phosphorylates a protein (phospholamban) on the SR that normally inhibits Ca2+ uptake. This disinhibition of phospholamban leads to an increased rate of Ca2+ uptake by the

SR. Therefore, beta-adrenergic stimulation increases the force and shortening velocity of contraction (i.e., positive inotropy), and increases the rate of relaxation

(i.e., positive lusitropy).

Another potential regulatory mechanism for excitation-contraction coupling involves altering the sensitivity of TnC for Ca2+.143 There are investigational drugs that enhance TnC Ca2+ sensitivity and thereby exert a positive inotropic influence on the heart. One potential downside with these drugs, however, is that enhanced TnC binding to Ca2+ can reduce the rate of relaxation, thereby causing diastolic dysfunction.143

II.2 Hsps and cardiac function

There is limited evidence about the role of Hsp in maintaining cardiac contractility and regulating Ca2+ handling. Double knock-out -BC and HspB2 genes had almost no effect on mouse cardiac function,74 while deletion of inducible

Hsp70 genes leads to a cardiac phenotype characterized by impaired contractile function and altered Ca2+ handling.154 Kim154 et al. reported that in Hsp70 null myocytes alterations of the Ca2+ transient were characterized by a prolonged time to peak and time of Ca2+ decline. These alterations may explain the impaired contractility in the myocyte, as measured by edge detection, with diminished cell shortening and slowed rate of contraction and relaxation. These alterations were specific for the Hsp70 ablation because adenoviral-induced expression of Hsp70 reverted the contractile dysfunction. One of the possible mechanisms to explain the cardiac alterations observed here is a diminished SERCA2a expression. These investigators observed a discrete but consistent SERCA2a protein downregulation in the hearts of KO mice. The prolonged Ca2+ transient and the slowed cell relaxation support a diminished SERCA2a activity. It is well accepted that diminished

SERCA2a activity can lead to lower Ca2+ loading and an impaired contraction.

Their findings demonstrated that Ca2+ loading was diminished, with an increased time to peak in the Ca2+ transient, and a decreased caffeine-induced Ca2+ release. In addition, adenoviral SERCA2a expression reestablished Ca2+ transients and contractility of KO cardiomyocytes. The mechanism involved in the decrease of

SERCA2a activity/expression can be related to absence of the inhibitory effect of

Hsp70 on p38-MAPKs and/or Raf-1/ERK pathway activity. In the

immunoprecipitation experiment, they found that Hsp70 interacts directly with Raf-

1, possibly modulating its activity154

II.3 Hsp20 regulating ex vivo cardiac function

The functional relationship between Hsp20 and cytoskeletal proteins is not fully explored. In early investigations, Hsp20 is found predominantly in the cytosol.80, 82 Upon heat stress, Hsp20 remains largely soluble and a subpopulation of this protein migrates into the nucleus, while another part stays in the cytoplasm of neonatal cardiomyocyte.82 In cultured adult rat cardiomyocytes, immunoreactive

Hsp20 is present in transverse bands or Z bands together with B-crystallin and actin.80 Hsp20 staining is also found on the cell membranes.80 The localization of

Hsp20 to specific cytoskeletal domains suggests its role in regulating cytoskeletal dynamics, therefore participating in regulating cardiac function.

Pipkin et al80 first studied the function role of phosphorylated Hsp20 by adding the phosphopeptide analogues of Hsp20 (Hsp20 phospho-Serine 16 analogue

N-WLRRASphos APLPGLK) into transiently permeabilized rat cardiac myocytes, which led to increases in the rate of shortening and relaxation of the myocytes.

However, the nonphosphorylated peptide (N-WLRRAAAPLPGLK) did not change the shortening rate. By using adenoviral gene transfer of mouse Hsp20 into adult rat ventricular myocytes, Chu et al155 also reported that transfection of Hsp20 led to the increase of basal cell contraction. However, the in vivo role of Hsp20 on cardiac function and the underlying mechanisms remain unclear.

Section III Hsps and cardiac diseases

III.1 Hsps and atherosclerosis

Atherosclerosis, a chronic inflammatory disease of the arteries, is the main underlying cause of myocardial infarction and stroke. It is characterized by the accumulation of lipids and extracellular matrix in the intima of large and medium sized arteries.156, 157 It is associated with mononuclear cell infiltration, and smooth muscle proliferation.156 157

The potential relationship between serum Hsps, Hsp antibody concentrations and atherosclerosis was initially explored in the early 1990s.158, 159 Xu et a.160 first reported, based on a large prospective population survey, that serum antibodies against Hsp65 were significantly elevated in patients 40–79 years of age with carotid atherosclerosis compared with those without lesions. This increased antibody level was independent of age, sex and other established risk factors.160 A subsequent follow-up study161 confirmed that the Hsp65 antibody levels were highly consistent over a five-year observation period for any given individual, and remained elevated especially in subjects with progressive carotid atherosclerosis.

This study also showed that anti-Hsp65 antibody titers were significantly predictive of five-year mortality independent of age, sex and other cardiovascular risk factors.161 In contrast, an increased serum Hsp70 concentration is reported to be associated with a lower risk of cardiovascular disease.162 It has also been reported that the severity of coronary artery disease (number of diseased vessels) is inversely related to serum Hsp70 concentrations.162

The expression of Hsp60 and Hsp70 in atherosclerotic lesions was first reported by Kleindienst et al.163 and Berberian et al.158 Hsp60 expression was found to be highest in the shoulder regions and around the necrotic core of atherosclerotic plaques.164 Double immunofluorescence identified the majority of positively stained cells as macrophages, endothelial cells, and a few smooth muscle cells.165

Expression of Hsp70 was shown to be most concentrated in the center of thickened atheromatous plaques.158 In advanced atherosclerotic lesions, Hsp70 was found to be expressed by several cell types, including smooth muscle cells, dendritic cells and monocyte/macrophages, while in early atherosclerotic lesions only dendrtic cells expressed it.166 Hsp70 appears to have an athero-protective role, which may be mediated by its anti-inflammatory properties167 and its effect on the survival of smooth muscle and endothelial cells.168, 169

III.2 Hsps and desmin-related cardiomyopathy

Desmin-related myopathies (DRM) are inherited neuromuscular disorders, characterized by adult onset and delayed accumulation of aggregates of desmin, a protein belonging to the type III intermediate filament family, in the sarcoplasma of skeletal and cardiac muscles.170DRM has been described as an autosomal dominant myopathy, that resulted in weakness of the proximal and distal limb muscle

(including neck, velopharynx and trunk muscles), signs of cardiomyopathy and cataract in several generations of a French family.171Analysis of patient muscle biopsies showed electron dense granulofilamentous aggregates that were shown to be an accumulation of desmin.170 However, a defect in the desmin gene located in chromosome 2q35 was excluded in this family by linkage analysis and direct

sequencing172 In fact, Vicart et al67 identified an R120G missense mutation in B- crystallin, that co-segregates with the disease phenotype in this family. It was found that the R120G mutation disrupted B-crystallin structure, chaperone activity and intermediate filament interaction, demonstrating the functional importance of residue R120.173 This was the first sHsp mutation shown to cause inherited human muscle disease. Two additional dominant negative B-crystallin mutations have since been linked to myofibrillar myopathy, but not cardiomyopathy.174 These mutations led to truncated B-crystallin C-terminus by 13 residues in one patient and 25 residues in another. The C-terminus is a region important for sHsp solubilization, chaperone activity and oligomer formation.174

Section IV Hsps and cardiac protection

IVI.1 Cardiac ischemia/reperfusion injury

Myocardial infarction is a major cause of morbidity and mortality in

Western nations.175 Myocardial infarction occurs when the coronary artery supplying oxygen-rich blood to a region of the heart is blocked (e.g., due to coronary obstruction, surgical clamp, heart transplantation). The resulting condition of low oxygen and nutrient supply is termed ischemia (from Greek: “ischo,” to hold back, and “haima,” blood). Following a bout of ischemia, reperfusion (e.g., via vasodilator pharmaceuticals, angioplasty) must be achieved in order to prevent the irreversible destruction of affected tissues.176-178Paradoxically, reperfusion itself causes damage and subsequent cell death. This condition is referred to as ischemia/reperfusion (I/R) injury.177-179

Ischemia/reperfusion causes two distinct components of damage to the heart:

(1) ischemic injury which results from the initial loss of blood flow,180 and (2) reperfusion injury, which occurs upon the restoration of oxygenated blood flow.

During ischemia, with loss of oxygen, mitochondrial oxidative phosphorylation rapidly stops, with a resultant loss of the major source of ATP production for energy metabolism. A compensatory increase in anaerobic glycolysis for ATP production leads to the accumulation of hydrogen ions and lactate, resulting in intracellular acidosis and inhibition of glycolysis, as well as mitochrondrial fatty acid and residual energy metabolism.180 Impaired contraction with persistent electrical activity (excitation contraction uncoupling) develops in association with alterations in ion transport systems in the sarcolemma and organellar membranes.181-183

If ischemia persists, the myocardium will develop a severe ATP deficit, resulting in an irreversible injury and culminating in cell death.184, 185 Reperfusion of ischemic myocardium is therefore a prerequisite for cellular survival.186 However, reperfusion can result in further damage to the myocardium.176-179 The major mediators of reperfusion injury are oxygen radicals, Ca2+ loading, and neutrophils.184, 185

The oxygen radicals are generated by injured myocytes and endothelial cells in the ischemic zone as well as neutrophils that enter the ischemic zone, and become activated on reperfusion. When the occlusion of the coronary branch that

perfuses the ischemic myocardium is removed, the superoxide anion (O2 ) production increases as a result of the activation of various enzymatic complexes.187

The superoxide anion and other ROS strongly oxidize the myocardial fibers already damaged by the ischemia, thus favoring the apoptosis.188, 189 The increased free radicals also lead to changes in membrane permeability, membrane lipid bilayer disruption and functional modification of various cellular proteins.190 In addition,

ROS induced the cellular and mitochondrial overload of Ca2+.191 Consequently, a massive quantity of ROS was released from mitochondria. Ultimately, these may contribute to the activation of mPTP, to the reduced availability of NO· and to the activation of the transcription factor NFB, which all promote myocardial damages.177 The neutrophils accumulate in the microcirculation, release inflammatory mediators, and contribute to myocardium damage and microvascular obstruction, which leads to the no-reflow phenomenon in the reperfused myocardium.184, 185 In addition, damage to the cardiac conduction system can lead to a variety of arrhythmias including sudden cardiac death.192

Two common conditions of cardiac cell survival in the context of ischemia/reperfusion are myocardial hibernation and stunning. The concept of myocardial “hibernation” was introduced nearly two decades ago.193 Hihernating myocardium is ischemic myocardium supplied by a narrowed coronary artery in which ischemic cells remain viable but contraction is chronically depressed.194

Stunned myocardium is viable myocardium salvaged by coronary reperfusion that exhibits prolonged postischemic dysfunction.195 One of the crucial differences between these two concepts is that resting myocardial perfusion is normal or near normal in stunning, but is reduced in hibernation.196

IV.2 Myocardial cell death in ischemia/reperfusion injury

IV.2.1 Necrosis and Apoptosis

It has been established that following I/R, cell death occurs via both necrosis and apoptosis,197 two distinct modes of death. Necrosis (Greek for: death, causing to die) is best defined by light or electron microscopic detection of cell and organelle swelling or rupture of surface membranes with spillage of intracellular contents, which provokes an inflammatory response.198 Ischemia induces necrosis in a certain subset of cells, whereby the extent of necrotic cell loss is a function of the duration of the ischemic insult.199 Necrotic cells are mainly found in the central zone of the infarct.

In contrast, apoptosis (from Greek: “falling off,”figurative for the falling of leaves; also termed Type I programmed cell death (PCD) is a highly regulated, genetically determined mechanism that does not provoke an inflammatory response.200 Moreover, apoptosis requires energy in form of ATP for its successful completion.200 Apoptosis plays a role in pathophysiological conditions but is also essential in normal tissue homeostasis, allowing the organ or tissue to rid itself of cells which are dysfunctional or no longer needed.201 Apoptotic cell death is characterized by cell shrinkage, membrane blebbing, and nuclear condensation and degradation.202 The cell is eventually broken into small membrane-enclosed pieces (apoptotic bodies), which in vivo are removed by macrophages, or taken up by neighboring cells.203 This prevents the release of cellular compounds and thus ensures that an inflammatory response is not provoked.202-204 In I/R injury, apoptotic cell loss manifests itself during the reperfusion period due to the slowly orchestrated execution of the apoptotic cell death program, and is more apparent at the marginal zone of the infarct.177

The term oncosis is preferred by some investigators of cell death as an alternative to what others refer to as necrosis.205-207 Oncosis (Greek for swelling) is defined as a prelethal pathway leading to cell death accompanied by cellular swelling, organelle swelling, blebbing, and increased membrane permeability.206 The process of oncosis ultimately leads to depletion of cellular energy stores and failure of the ionic pumps in the plasma membrane. Oncosis may result from injury as well as from toxic agents that interfere with ATP generation or processes that cause uncontrolled cellular energy consumption.206 In this view, necrosis is the further degradation of the dead cell, whether it dies by oncosis or apoptosis.

IV.2.2 Autophagy

Autophagy, a lysosomal degradative pathway, is another process that has been suggested to play a role in ischemia-reperfusion injury.208, 209 Autophagy is a physiological mechanism that is used to remove damaged organelles, such as mitochondria or endoplasmic reticulum. Autophagy is also initiated by starvation

(ie. lack of nutrients).210 However, extensive autophagy can cause cell death.210

There are conflicting data as to whether the increased autophagy that occurs during ischemia and reperfusion is beneficial or detrimental. 208, 209, 211-213 There are studies showing that inhibition of autophagy during ischemia or anoxia is detrimental, suggesting a beneficial role for stimulation of autophagy during ischemia 211.

However, in contrast to these studies, a decrease in beclin1 (a protein that stimulates autophagy) reduces ischemia-reperfusion-mediated autophagy and myocyte death.212 Consistent with a beneficial role for autophagy in ischemia-reperfusion, in

HL-1 cells an increase in beclin1, which increased autophagy, was also shown to

decrease activation of BAX, and knockdown of beclin1 increased activation of BAX

213. Thus, the role of autophagy in cell death during ischemia-reperfusion is still somewhat unclear.

IV.3 Hsps and cardiac protection

In the heart, initial studies in cultured cardiac cells demonstrated a clear protective effect of over-expressing Hsp70 against subsequent challenge with elevated temperature or hypoxia.214 Subsequently, the results were extended by several groups which reported that hearts from transgenic mice over-expressing

Hsp70 demonstrated enhanced resistance to ischemia/reperfusion injury.215-217 The protective effect of Hsp70 was further confirmed by loss-of-function studies in

Hsp70 knockout mice. Interestingly, in cultured cardiac cells over-expression of B- crystallin or Hsp27 also has a potent protective effect, whereas over-expression of

Hsp90 produces a much more limited protection, and over-expression of hsp56 is not protective.106 ,214 Moreover, in other studies of the B-crystallin73 and Hsp27 218,

219transgenic mice, cardiac ischemia/reperfusion injury was reduced, whereas mice featuring the disruption of the B-crystallin and HspB2 genes exhibited increased mortality, compared with WT mice. Furthermore, the hearts of these knockout mice, exhibited increased necrosis, apoptosis, and only half the contractile recovery of

WT mouse hearts, when exposed to ischemia/reperfusion. The protective activity of

Hsps involves decreased oxidative stress,218 preservation of mitochondrial respiration and ATP production75, 77 and reduction of caspase-3 activity.220

Section V Dissertation focus and hypotheses

The underlying hypothesis of this dissertation is that Hsp20 and its phosphorylation at Ser16 regulate cardiac function and protects against ischemia/reperfusion injury. The dissertation tests this hypothesis by utilizing genetically-modified mouse models to determine the roles of Hsp20 and its phosphorylation in cardiovascular physiology and path physiology, specifically in cardiac contractility and after ischemia-reperfusion injury. This dissertation extends our knowledge on the in vivo roles of Hsp20 and its phosporylation in the heart, providing further support towards the development of Hsp20 as a therapeutic target for heart failure and ischemic heart disease.

Hypothesis 1: Hsp20 regulates cardiac function

PKA and type 1/2 protein phosphatases (PP1 and PP2) are the major counter- players in balancing phosphorylation and dephoshorylation processes in cardiomyocytes. It is well-recognized that Hsp20 is the only member of small heat- shock proteins that contains a conserved phosphorylation site for PKA/PKG, and is known to modulate smooth muscle relaxation, and increases cardiac muscle shortening. More interestingly, microcystin-affinity chromatography experiments confirmed that Hsp20 interacted with the catalytic subunit of Type 1 Protein

Phosphatase (PP-1C) in rabbit skeletal muscle. Whether Hsp20 can also interact with

PP1 activity and affect contractile function in the heart is not clear. To elucidate the role of Hsp20 in regulating contractility, cardiomyocytes from wild type and Hsp20 transgenic hearts were subjected to contractility and Ca2+ transient studies.

Phosphorylation of PKA target proteins in these hearts, such as phospholamban,

RyR2 and TnI was evaluated by Western Blots. The association of Hsp20 with PP1 was tested by immunoprecipitation and PP1 activity was assessed.

Hypothesis 2: Hsp20 and its phosphorylation protect the myocardium from ischemia-reperfusion injury.

The levels of cardiac Hsp20 and its phosphorylation were significantly increased compared with Hsp27 and B-crystallin in animal hearts after myocardial ischemia, exercise training, rapid right ventricular pacing, and pharmacological treatment by doxorubicin and chronic -adrenergic stimulation.221-223 More recently, a

P20L substitution in human Hsp20 was identified, which was associated with diminished phosphorylation at Ser16 and complete abrogation of the protective effects of Hsp20, suggesting an instrumental role of phosphorylated Hsp20 in cardioprotection.224 Indeed, the constitutively phosphorylated mutant of Hsp20

(Hsp20S16D) conferred protection against -agonist-induced apoptosis in cultured cardiomyocytes; conversely, the constitutively dephosphorylated mutant, namely

Hsp20S16A, displayed no anti-apoptotic properties, implying a mechanistic link between phosphorylated Hsp20 and its protection.81 In fact, our previous study has shown that the protective effect of Hsp20 against ischemia/reperfusion was associated with increased phosphorylation of Hsp20.225 To elucidate the in vivo role of phosphorylated Hsp20 in ischemia-reperfusion injury, wildtype mice and mice with cardiac-specific overexpression of Hsp20S16A were subjected to ex vivo and in vivo ischemia/reperfusion injury. Endpoints of cardioprotection were assessed (contractile functional recovery and myocardial infarct size). Apoptosis (by TUNEL staining,

Caspase 3 and DNA fragmentation), necrosis (by LDH release) and autophagy (by

Beclin-1 and LC3-II/LC3-I ratio) were assessed. Functional recovery was also evaluated in rapamycin (autophagy inducer) pre-treated Hsp20S16A hearts, to determine whether differences observed in ischemia/reperfusion injury and cardioprotection are due to alterations in autophagy activity.

Chapter II

Materials and Methods

Section I. Infection of cardiomyocytes with recombinant adenoviruses

I.1 Recombinant adenoviral constructs:

Recombinant adenoviruses were developed using the pAdTrack-

CMV/pAd.Easy-1 system. Briefly, Hsp20WT and Hsp20S16A cDNA in the sense orientation was cloned between the cytomegalovirus (CMV) enhancer/promoter and the SV40 polyadenylation signal sequence of the shuttle vector pAdTrack-CMV, which also carries a GFP expression cassette (Figure 6.). The resultant plasmids were linearized and co-transformed into E. coli BJ5183 cells with an adenoviral backbone plasmid, pAdEasy-1. Recombinant adenoviral vectors were generated through homologous recombination and selected for kanamycin resistance. The recombination was confirmed by multiple-restriction endonuclease analyses. Then the linearized recombinant vectors were transfected into HEK 293 cells.

Adenovirus-GFP, which contains GFP and –gal cDNAs controlled by separate

CMV promoter, was used as control. The recombinant viruses were harvested and were prepared as high-titer stocks and further purified by CsCl banding. Viral particle titration was performed by using plaque assay. Briefly, multiple dilutions of virus were plated out with proper amount of 293 cells. When evenly distributed, individual plaques were observed. The individual plaque number was counted and

the plaque forming units per mL (pfu) was obtained by multiplying the count by the dilution factor.

Figure 6. Diagram of recombinant adenoviral vectors. Mouse cardiac Hsp20 cDNA and its mutants of TCA-GCA (replaced Ser16 with Ala to block phosphorylation) were inserted, respectively, into a GFP-containing adenoviral vector backbone (E1/E3 deleted), namely Ad.Hsp20 and Ad.S16A, respectively. The mutated nucleotides were identified in bold and underlined. Hsp20WT and Hsp20S16A cDNA in sense orientation was cloned between cytomegalovirus (CMV) enhancer/promoter and SV40 polyadenylation (polyA) signal sequence of the shuttle vector pAdTrack-CMV. GFP: green fluorescent protein.

I.2 Myocyte preparation and infection of myocytes

Adult rat ventricular myocytes were obtained from Langendorff-perfused hearts of male Sprague-Dawley rats (~300g, Harlan Laboratory) at room temperature.226,227 Briefly, rats were anesthetized with sodium pentobarbital

(50mg/kg, ip) and heparinized (10,000U/kg, ip). The hearts were placed into perfusion chamber as rapidly as possible and cannulated through the aorta, perfused with modified Krebs-Henseleit buffer (KHB) (in mmol/L: NaCl: 118, KCl: 4.8,

HEPES: 25, K2PO4: 1.25, MgSO4: 1.25, glucose: 11, taurine: 5 and BDM: 10, pH7.4) for 5 minutes. During the perfusion, the buffer was saturated with oxygen.

Hearts were then perfused with an enzyme solution, which contained 0.7mg/mL collagenase type II (263U/mg), 0.2mg/mL hyaluronidase, 0.1% BSA and 25M

Ca2+, for 10 minutes. The enzyme solution for heart treatment is used repeatedly, which reduced the amount of enzymes needed for a single isolation. Subsequently, the Ca2+ concentration in the perfusion buffer was raised to 100M, and perfusion continued for 5 additional minutes. Finally, ventricular tissue was excised, minced into 1-2-mm2 fragments and carefully resuspended for 1 min with a large pipette

(for better convenience, the tip of the headpiece was obliquely cut; the edges needed to be very smooth). In case of incomplete separation, the suspension was filtered through two gauze layers in order to remove the remaining fragments. Cells were harvested and resuspended in 1.8mM Ca2+-KHB with 1% BSA, centrifuged briefly again, and resuspended in ACCT medium consisting of DMEM containing 2mg/mL

BSA, 2mM L-carnitine, 5mM creatine, 5mM taurine, 100IU/mL penicillin, and

100ug/mL streptomycin. Cells were then counted and plated on laminin-coated

glass coverslips or dishes. After 1-2 hours, the dishes were infected for 2 hours with adenoviruses in diluted media, at a multiplicity of infection of 500MOI, before addition of suitable volume of culture media. Transfection efficiency, determined by

GFP gene expression in the cultured cardiac myocytes under fluorescence microscopy, was consistently >95% by this method. Myocyte mechanics and Ca2+ kinetics were recorded after 24-hour incubation. To obtain intracellular Ca2+ signals, cells were incubated with the acetoxymethyl ester form of fura-2 (Fura-2 /AM;

2M). The above in vitro studies were performed with the assistance and guidance of Dr. Guo-Chang Fan and Dr. Qunying Yuan in the Kranias’ laboratory.

Section II. Generation of transgenic mice

We generated TG mice that carry the mouse cardiac Hsp20WT and

Hsp20S16A cDNA under the control of the -MHC mouse promoter (Figure 7), as described.225 A 0.5kb wild-type or mutant mouse Hsp20cDNA was ligated with the murine cardiac -myosin heavy chain gene promoter (5.5-kb) (Figure 7). In all studies, male mice between 12 to 16 weeks of age for the Hsp20S16A transgene were compared with age-matched transgenics overexpressing wild type Hsp20 and wild type controls.225 The care of all animals used in the present study was in accordance with the University of Cincinnati animal care guidelines. The Hsp20WT and

S16A Hsp20 transgenic constructs were generated by Dr. Guo-Chang Fan.

Section III. Isolation of mouse tail DNA and PCR conditions

At the time of weaning the mice, approximately 0.5cm of the mouse tails were clipped to identify the transgenic mice. Genomic DNA from tail biopsies was isolated by the proteinase K digestion method (Qiangen DNeasy blood and Tissue kit, Cat.No. 69504), according to manufacturer’s guide. For genotyping the

Hsp20WT and Hsp20S16A transgenic mice by PCR, a 20l PCR mix (containing 2l of template DNA, 0.2l of Taq DNA Polymerase (Roche), 2l of 10x buffer, 0.2l of 25mM dNTP mix, 0.125l of each primer, and 15.35l of dH2O) was used. The sequence of primers was listed in Table 2. PCR cycling parameters were presented in Table 3.

-MHCp mHsp20cDNA hGHpA 5.5 kb 0.5kb 0.6kb BamH BamH

ATGGAGATCCCCGTGCCTGTGCAGCCTTCTTGGCTGCGCCGTGCTTCAGCT……GCCAAGTAG Ser ATGGAGATCCCCGTGCCTGTGCAGCCTTCTTGGCTGCGCCGTGCTGCAGCT… ..GCCAAGTAG Ala

Figure 7. Diagram of transgenic Hsp20WT and Hsp20S16A mouse constructs. The mouse Hsp20 cDNA and mutant Hsp20 cDNA, in which Serine 16 encoded by codon TCA was mutated into GCA (encoding alanine), was driven by the -myosin heavy chain promoter ( -MHCp).

Table 2. The sequence of primers for genotyping and the predicted length of PCR products

Primers Sequence Product Length

Upper arm of-MHC P1 5-CACATAGAAGCCTAGCCCACAC-3 promoter 600 bp Lower arm of P2 5-GCTTGTCCTGTGCA-GCTGGGAC-3 mcHsp20

Upper arm of TSH-ß P3 5-TCCTCAAAGATGCTCATTAG-3 350bp Lower arm of TSH-ß P4 5-GTAACTCACTCATGCAAAGT-3

Table 3. PCR cycling parameters for genotyping

Segment Number of cycles Temperature Duration

1 1 95 C 1 minute

95 C 30 seconds

2 30 54 C 30 seconds

72 C 30 seconds

3 1 72 C 10 minutes

Section IV. Quantitative immunoblotting

IV.1: Quantitative immunoblotting of total Hsp20 levels in Hsp20WT and Hsp20S16A transgenic mouse hearts

To determine the total Hsp20 protein levels in transgenic hearts, immunoblotting was performed. 12-16 weeks old Hsp20WT, Hsp20S16A transgenic and non-transgenic (NTG) mouse hearts were homogenized in buffer containing:

10mM imidazole, 0.3M sucrose, 1mM DTT, 10mM Na-Metabisulfate, 0.3mM phenylmethylsulfonyl fluoride (PMSF), and 50mM sodium fluoride. In addition,

Phosphatase Inhibitor Cocktail Set I and II (Calbiochem Cat. No. 524624, 524625) and proteinase inhibitor tablets (Roche Cat. No. 04693124001) were added into homogenate buffer, according to the manufacturer’s guide. In a cold room (4 C), mouse hearts free of fat and auricles, previously stored at -80 C, were placed into the buffer, and homogenized in a blender. The final homogenate was aliquoted and stored at -80 C.

A total of 10μg cardiac homogenate was subjected at room temperature to

SDS-PAGE on a 13% gel in running buffer (25mM Tris, 191.8mM glycine, 1%

SDS), followed by a transfer to 0.1μm nitrocellulose membranes (BioRad

Laboratories). The transfer was performed at 4oC for 2.5 hours at 180mA in a blotting buffer containing 25mM Tris, 191.8mM glycine, and 20% methanol. After blocking in a solution of 5% non-fat dried milk dissolved in TBS (100mM Tris,

0.9% NaCl, pH 7.4) for 1 hour at room temperature, the membranes were incubated

overnight at 4oC with an anti-Hsp20 mouse monoclonal antibody (1:1000, RDI).

After washed three times in TBS, the membranes were incubated for 1-2 hours with a horseradish peroxidase-conjugated anti-mouse secondary antibody (1:5000,

Amersham Biosciences, Piscataway, NJ, USA), followed by 3 rounds of wash in

TBS at room temperature (20 minutes for each wash). Finally, the membranes were developed by enhanced chemiluminence Western blotting detection system

(Amersham Biosciences, Piscataway, NJ, USA). Calsequestrin is used as a loading control to check if samples are evenly loaded in the gel. To quantify the protein level, 5, 10 and 20g of protein pooled from wild type hearts was run in the same blot as a standard linear range. A linear correlation was obtained from this range and a coefficient of above 0.97 was defined as an acceptable linear relationship. Protein levels in different hearts were quantified using this linear relationship. The final results were normalized to those of non-transgenic hearts and expressed as fold of that in non-transgenic hearts.

IV.2 Quantitative immunoblotting of other small heat shock proteins and SR Ca2+ handling proteins in transgenic hearts

To examine the alteration of other small heat shock proteins and cardiac SR

Ca2+ handling proteins in Hsp20WT, Hsp20S16A transgenic and non-transgenic hearts, cardiac homogenates were subjected to quantitative immunoblotting, as described above. The specific primary antibodies were: monoclonal antibodies to B- crystallin (1:1000), Hsp27(1:1000), phospholamban (1:1000), RyR (1:1000), L-type

Ca2+ channel (1:500), Na-Ca exchanger (1:1000) (Affinity Bioreagents, Golden, CO,

USA); and polyclonal antibodies to phosphorylated RyR (Serine2809) (1:1000,

Badrilla, UK), CSQ (1:1000), (Affinity Bioreagents, Golden, CO, USA), and polyclonal antibody to SERCA2a (1:500, homemade). The HRP-conjugated anti- mouse or anti-rabbit secondary antibodies (1:5000) were from Amersham

Biosciences, Piscataway, NJ, USA)]. To quantify the protein levels, three appropriate amounts of protein pooled from non-transgenic hearts were run in the same blot as a standard linear range for each protein. A linear correlation was obtained from this range and a coefficient of above 0.97 was defined as an acceptable linear relationship. Protein levels in different hearts were quantified using this linear relationship. CSQ was used as an internal control. Proteins levels obtained from the linear standard relationship were normalized to CSQ levels, and the final results were normalized to those of non-transgenic hearts and expressed as fold of non-transgenic. All the other protein levels were quantified by using

AlphaEaseFC software (Alpha Innotech, San Leandro, CA, USA).

IV.3 Generation of a pSer16-Hsp20 Antibody

The pSer16-Hsp20 antibody was generated by Affinity Bioreagents (Golden,

CO) using the BioSpecificTM plan. Specifically, specific-pathogen-free-rabbits were immunized with the synthetic phosphopeptide SWLLRRA-S-(PO3)-APLPG conjugated to keyhole limpet hemocyanin, using glutaraldehyde as a cross-linker.

The adjuvant used during the immunizations was Freund's complete adjuvant for the primary injection and Freund's incomplete adjuvant for the following boosts. The phosphorylation state-specific antibody was then antigen affinity-purified from the

antisera through a two-step purification process utilizing the non-phosphorylated peptide followed by the phosphorylated peptide.

IV.4 Quantitative immunoblotting of phosphor-Ser16-Hsp20 levels in ischemia/reperfusion injured mouse hearts and failing human hearts

Human tissues from the anterior wall of 4 non-failing and 5 failing left ventricles were obtained from Dr. Roger Hajjar’s Laboratory (Mount Sinai School of

Medicine). The failing hearts were obtained at the time of cardiac transplantation.

Information about each patient was collected and linked to the subject number (but not the patient name) and documented in a spreadsheet. Once in the laboratory, the tissue was cut into pieces, frozen in liquid nitrogen, and stored at –80 C. All investigation conforms with the principles outlined in the Declaration of Helsinki228.

Mouse (wild type, 12-16w, male Fvb/n) hearts were subjected to ex vivo ischemia/reperfusion by Langendorff perfusion with a protocol of 45min ischemia/120min reperfusion, as described in the following methods. Total Hsp20 and pSer16-Hsp20 protein levels in heart homogenate from ischemia/reperfusion injured mouse hearts and human heart samples were measured by quantitative immunoblotting by using mouse monoclonal Hsp20 antibody and pSer16-Hsp20 polyclonal antibody, as described above.

Section V. Morphological studies

Gravimeteric analysis was performed by weighing anesthetized mice and/or their isolated heart and lungs on a Mettler Toledo, AT201 analytical scale. After hearts were excised, fat and connective tissues were dissected, and then the hearts were dried on Kimwipe paper and weighed. Histological examination of hematoxylin and eosin (H&E), and Masson’s trichrome stained atria and ventricles were performed by the Division of Comparative Pathology at the University of Cincinnati. Briefly, the heart tissue was fixed in 10% formalin, dehydrated through graded alcohols, and embedded in paraffin. Longitudinal sections (5 μm) of the heart (cut at 50-μm intervals) were stained with hematoxylin and eosin or subject to trichrome staining.

The sarcolemma was labeled with wheat germ agglutinin (WGA,

Invitrogen). WGA selectively binds to N-acetylglucosamine and N- acetylneuraminic. Oregon Green / 488 (Invitrogen)-conjugated WGA was dissolved in PBS (pH 7.4) to give a stock solution with a concentration of 1mg/ml.

The stock solution was divided into small aliquots and frozen at -18oC. An aliquot was thawed and further diluted with deionized water to give a working solution with a concentration of 200g/ml. Paraffin-embedded tissue was first deparaffinized and incubated with WGA working solution for 60 minutes. Immunofluorescent images were taken by a confocal microscope under a 40x water immersion objective lens.

Section VI. Verification of S16A-Hsp20 expression in transgenic mouse by two-dimensional gel electrophoresis and Mas-spectrometry

VI.1 Two-dimensional gel electrophoresis

Mouse left ventricular tissue was isolated and proteins were extracted for two-dimensional (2-D) gel electrophoresis by the Proteomics Lab of the Genome

Research Institute, University of Cincinnati226. First-dimensional separation was performed by using the IPGphorTM isoelectric focusing system (Amersham

Biosciences). For the broad-range analysis, 150 g of protein was diluted into rehydration buffer (8 M urea, 2% CHAPS, 20 mM DTT, 0.5% pharmalytes 3–10, and 0.01% bromophenol blue) and applied to ImmobilineTM Drystrips (pH range of

3–10 NL and 4–7 NL, Amersham Biosciences). For narrow-range analysis, 250 g of protein was diluted into rehydration buffer (8 M urea, 2% CHAPS, 20 mM DTT,

0.5% pharmalytes 3–10, and 0.01% bromophenol blue) and applied to

ImmobilineTM Drystrips (pH range of 4.0–5.0, 4.5–5.5, 5.0–6.0, 5.5–6.7, 6–9,

Amersham Biosciences). For the basic range (pH 6–9), 200 g of protein sample was diluted into DeStreak Rehydration Buffer (Amersham Biosciences) and applied to Immobiline™ DryStrips. All strips were rehydrated overnight at room temperature in a reswelling tray prior to being moved to ceramic strip holders and placed onto the IPGphor for isoelectric focusing. Isoelectric focusing (IEF) was performed from that point according to the manufacturer’s protocols, and IEF runs were stopped at 35,000–40,000 volt-hours for the broad range pH (3–10, 4–7, and

6–9) gels and at 50,000–60,000 volt-hours for the narrow range (4.0– 5.0, 4.5–5.5,

5.0–6.0, 5.5–6.7) gels. Upon completion of IEF, the IPG strips were removed from the ceramic holders and equilibrated in an SDS buffer (2% SDS; 6M urea; 30% glycerol; 50 mM Tris-HCl, pH 8.8; and 0.01% bromophenol blue) for two 15- minute intervals. The first 15- minute incubation SDS buffer contained 1% DTT, and the second contained 2.5% iodoacetamide. After equilibration, the IPG strips

were placed atop 12% SDS-polyacrylamide slab gels and embedded with a 0.5% agarose solution. Gels were run in the Investigator 2D running system (Genomic

Solutions, Inc., Ann Arbor, MI) containing running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS) at a regulated temperature of 15°C. The gels were run at

1500mW until the dye front reached the bottom of the gel. Twelve cardiac ventricle samples (non-transgenic, Hsp20WT Hsp20S16A, and Hsp20S16D transgenic hearts, n=3 for each group) were run in triplicate per each pH range used. The completed 2-DE gels were stained using SYPRO Ruby (Molecular Probes Inc., Eugene, OR). Briefly, each gel was fixed in 200 ml of 30% ethanol and 10% acetic acid for 1 h at room temperature on an orbital shaker platform. The fixative solution was replaced with

200 ml/gel of SYPRO Ruby staining solution. The gels were incubated overnight in staining solution, while rotating at room temperature. After staining was complete, the gels were washed twice in a solution of 10% ethanol and 5% acetic acid for 1 h.

SYPRO fluorescent dye-stained gels were scanned using a FLA-3000 (Fuji Medical

Systems, Stamford, CT) with a 475 nm fluorescent laser and a yellow 520 nm filter.

VI.2 Two-dimensional gel image analysis

Digitized 2-D gel images were analyzed for protein alterations, using the

Z3® 2-D ImageMaster 2D Elite software program and were annotated for mass spectrometry with the Melanie 3 (Swiss Bioinformatics Institute, Basel, Switzerland) software program. Replicate gels from tissue samples (3 per pH range) were matched with each other, and then against other samples from the same treatment set (wild-type or transgenic) to create an averaged image, or raw master gel (RMG).

The averaging of the replicate gel sets eliminates most of the gel-to-gel variation in

staining intensity. The RMGs for the wild-type set (one for each pH range) and the transgenic set (one for each pH range) were then matched, pairwise, against each other and analyzed to calculate the differential expression of specific protein spots.

Differential expression (DE) is calculated by Z3 as a ratio: the numerator is the volume and intensity of a selected spot present in the transgenic RMG image, and the denominator is the volume and intensity of the corresponding spot in the wild- type RMG image. In this study, a 2-fold increase or decrease in protein expression was used as the cutoff. A DE of 2.0 or greater represents an increase in expression of 2-fold or greater; whereas, a DE of 0.5 or less is equivalent to a 2-fold or greater decrease in expression in the transgenic heart. Some spots in the wild-type 2D-gel image could not be matched to a corresponding spot in the transgenic image, and vice versa; these unmatched spots were confirmed visually and designated

“exclusively detected” in transgenic or wild-type hearts. However, being able to detect a protein spot in only one sample may indicate either a very large difference in the protein’s expression level or exclusive expression in this sample compared with its control.

VI.3 In-gel digestion of proteins for mass spectrometry

Protein spots excised from Sypro Ruby stained gels were digested with trypsin according to Shevchenko and modified, as described here229. The gel pieces were distained by using 100 L of a 1:1 mixture of 50 mM ammonium bicarbonate: acetonitrile for 10 min, which was then dehydrated with 25 L acetronitrile.

Dehydrated gel pieces were treated with dithiothreitol (25 L, 10 mM) and then

with iodoacetic acid (25 L, 50 mM) to reduce and alkylate the cysteine residues.

Gel pieces were then twice-washed with 50 mM ammonium bicarbonate and dehydrated with 25 L acetonitrile. The acetonitrile was removed, and the samples were then briefly dried in a vacuum centrifuge. Gel pieces were covered with 10 L digestion buffer, containing 25 mM ammonium bicarbonate and 20 ng/L TPCK- modified trypsin. After 45 min at room temperature, the gel pieces had been re- swollen and an additional 10 L of 50 mM ammonium bicarbonate was added.

Digestion was allowed to proceed overnight at 37°C. The resulting peptides were extracted using 5% formic acid.

VI.4 Protein identification via MALDI-TOF and LC-MS/MS

For MALDI-TOF identification, the tryptic peptides were loaded onto C18

ZipTips (Millipore, Framingham, MA) for de-salting. MALDI-MS for peptide mass mapping was performed using a PerSeptive Biosystems Voyager Elite-DE

(Framingham, MA) operating in reflector mode. The extracted peptide solution was mixed 1:1 with a saturated solution of a-cyano-4- hydroxycinnamic acid (in 50% acetonitrile; 0.1% TFA), and 1 L was deposited on the sample plate and allowed to air-dry. In some cases, the peptides were also analyzed by capillary LCMS/ MS.

Digest solution (10 l) was preconcentrated and desalted on a protein trapping precolumn (CapTrap, Michrom BioResources, Aubun, CA) by using 20 L of 1%

TFA before being eluted onto a capillary column (0.3×150 mm PepMap C-18, 5 m,

300Å; LC Packings, San Francisco, CA). An Eldex MicroPro HPLC system (Eldex

Laboratories, Napa, CA) was used to deliver a step gradient at a flow rate of 5

L/min. The column was equilibrated in 10% Buffer B (0.1% formic acid, 80%

acetonitrile, 20% water) and then increased to 75% Buffer B over 35 min, followed by an increase to 95% Buffer B over 3 min and an additional 2 min. The percentage of Buffer B was then decreased to 10% for a 15-min equilibration period prior to injection of the next sample. Buffer A consisted of 0.1% formic acid, 2% acetonitrile, and 98% water. A microautosampler (Alcott 718 Al, Norcross, GA) was used for automated injections. An ion-trap mass spectrometer (LCQ, Finnigan

MAT, San Jose, CA) was used for peptide sequencing. The ion trap was programmed to perform data-dependent analysis, which consisted of a first full-scan

MS over the range of 300–1500 and a second scan, which was an MS/MS scan of the most abundant ion in the first scan. (13) The resulting spectra from MALDI-MS and LC-MS/MS were used to search the SWISSPROT and NCBI databases with

MASCOT.

Section VII. Isolation of myocyte and measurement of contractile parameters and Ca transients

In order to culture cardiomyocytes or to observe the contractile function of adult mouse cardiomyocytes, it is necessary to isolate high-yield and high-quality cardiomyocytes155. At first, the mouse was injected with 2.5% Avertin (150mg/ kg,

IP), and mouse hearts were excised and cannulated by the aorta onto a 20-gauge needle at room temperature in ice-cold Ca-free tyrode solution. The cannulated heart was mounted on a Langendorff perfusion apparatus and perfused with Ca2+-free

Tyrode solution (140mmol/L NaCl, 4mmol/L KCl, 1mmol/L MgCl2, 10 mmol/L glucose, and 5mmol/L HEPES, pH 7.4) at 37 C for 3minutes. The initial perfusion pressure was maintained at 40 mmHg by regulating the flow rate. Perfusion was

then switched to the same solution containing 1mg/mL type II collagenase

(276U/mg, Worthington), and the enzymatic digestion was terminated immediately, when the heart became flaccid (~8-10 minutes, with perfusion pressure lowered to

28 mmHg). All solutions were continuously bubbled with 100% oxygen. The heart was then quickly removed from the Langendorff system, and the atria and aorta were dissected away. The left ventricular tissue was minced into small portions in a dish containing Ca2+-free Tyrode solution. The single myocytes were gently dispersed from these portions by a wide tipped pipette, and further filtered through a

240-m screen. The cardiomyocyte suspension was sequentially washed in 25, 100,

200M and 1mM Ca2+-Tyrode solution, and resuspended in 1.8mM Ca2+-Tyrode solution for further analysis.

Cell shortening and Ca2+ transients were measured separately in cardiomyocytes at room temperature. To obtain intracellular Ca2+ signals, cells were incubated with the acetoxymethyl ester form of fura-2 (Fura-2/AM; 2M) in 1mM

Ca2+-Tyrode solution for 30minutes with slow and continuous rotation at room temperature. The cells were then resuspended in 1.8mM Ca2+-Tyrode solution.

300μL of 1.8mM Ca2+-Tyrode solution and 100μL of the myocyte suspension were placed in a Plexiglas chamber, which was positioned on the stage of an inverted epifluorescence microscope (Nikon Diaphot 200) at room temperature (22–23oC).

Myocyte contraction was field-stimulated by a Grass S5 stimulator (0.5Hz, square waves), and contractions were videotaped and digitized on a computer. Pulse duration was set at 4 milliseconds and voltage at 80mV. A video edge motion detector (Crescent Electronics) was used to measure myocyte length and cell

shortening, from which the percent fractional shortening (FS: (resting cell length- maximal cell shortening length) / resting cell length ×100) and maximal rates of contraction (+dL/dt) and relaxation (-dL/dt) were calculated.

For Ca2+ signal measurements, the cells were alternately excited at 340 and

380nm by a Delta Scan dual-beam spectrophotofluorometer (Photon Technology

International). Ca transients were recorded as the 340nm:380nm ratio of the resulting 510nm emissions. For caffeine-induced Ca2+ release, field stimulation was stopped before 10mM caffeine was rapidly (in 10 seconds) applied to the cells, and the following Ca2+ transients were recorded. Baseline and amplitude, estimated by the 340 nm:380 nm ratio, and the time to 50% decay of the Ca2+ signal were acquired. To examine the contribution of Na-Ca exchanger in the extrusion of intracellular Ca2+, cells were incubated with 5mM nickel before application of caffeine, and time to 50% decay of caffeine-induced Ca2+ transients was measured.

10-12 cells per heart were studied, and each animal was analyzed as a single n. Data were acquired using Felix® computer software (Photon Technology International,

Lawrenceville, New Jersey, USA) and analyzed using Ion Wizard® software (Ion

Optix, Corp.).

Section VIII. In vivo assessment of cardiac function using echocardiography

Echocardiography was performed to examine cardiac contractile function in a noninvasive manner, as described previously. Mice were moderately anesthetized with 2.5% Avertin (0.01 mL/g), and animals were allowed to breathe spontaneously.

The animal’s chest was shaved, its extremities were secured to the examining surface with paper tape, and the animal was placed on a warming pad (37°C;

Deltaphase isothermal pad, Braintree Scientific) to maintain normothermia. Two- dimensional targeted M-mode and color flow-directed pulsed wave Doppler studies were performed with a 9mHz imaging and a 5-7.5 MHz Doppler transducer

(Philips-2000). M-mode measurements of the left ventricular end diastolic dimension (LVEDD) and left ventricular end systolic dimension (LVESD), posterior and anterior wall thickness (PW and AW) and ejection time, measured at end diastole, were calculated from original tracings using a commercially available image analysis system (Freeland Medical), according to the leading-edge convention established by the American Society of Echocardiography,230 and by using the steepest continuous endocardiac echoes. The onset of the QRS complex and the peak of the posterior wall motion served as indicators of end diastole and end systole, respectively. Doppler measurements of peak and integrated aortic velocities, acceleration, and ejection times (ET) were made from ½ inch videotapes.

Calculated variables included: left ventricular fractional shortening p; normalized mean velocity of circumferential fiber shortening corrected for heart rateVcfc =

1/2 (FS/ET/(R-Rint) ), where ET was taken from heart rate-matched aortic Doppler spectra. A minimum of three beats were averaged for each measurement.

Section IX. Pressure-volume loop analyses

A standard 1.4 French pressure-conductance catheter with 4.5-mm signal electrode spacing (SPR-839; Millar Instruments, Houston, TX) was volume calibrated by submerging it into heparin-treated murine blood in a series of cylindrical holes with known diameters (1.5 to 4.0 mm) and 1 cm deep. This catheter was inserted into the right carotid artery of mice to measure baseline arterial pressure, then advanced retrograde into the LV to record closed chest hemodynamics with the ARIA Pressure Volume Conductance System (Millar

Instruments). The combination of pressure and volume signals allows accurate positioning of the catheter in the left ventricle. A transverse abdominal incision was performed to expose the inferior caval vein, without opening the thorax. During data acquisition ventilation was stopped to avoid influence from ventilation of the lungs on the pressure and volume signals. The acquisition protocol consisted of measurements of baseline cardiac function and inferior caval vein occlusions.

Pressure-volume (P-V) loops were analyzed with the PVAN 3.4 software package

(Millar Instruments) with the conversion of relative volume units (RVU) to absolute volume by applying the formula (slope of 3.213·RVU – intercept of 8.867) based on initial catheter calibration. The major hemodynamic parameters collected were maximum first derivative of change in systolic pressure rise with respect to time

(dP/dtmax), maximum first derivative of change in diastolic pressure fall with respect to time (dP/dtmin). On the basis of the loops collected during the transient occlusions of inferior caval vein, the end systolic pressure volume relationship

(ESPVR) was derived.

Section X. Ex vivo ischemia/reperfusion studies

X.1 Basal cardiac function measurements

Our methods for isolating and perfusing mouse hearts by the Langendorff method have been described previously225. Briefly, animals were injected intraperitoneally with heparin (10,000 U/kg) and anesthetized with pentobarbital

(10g/kg, IP). The thorax was rapidly opened and the heart excised and arrested in

Krebs-Henseleit buffer. A short perfusion cannula was inserted into the aortic root to initiate retrograde perfusion. The hearts were perfused with Krebs-Henseleit buffer (in mmol/L: NaCl 118, KCl 4.7, CaCl2 1.8, KH2PO4 1.2, MgSO4 1.2,

NaHCO3 24.0, and glucose 10.0) equilibrated with 95% O2 /5% CO2 to yield a pH of 7.4. A ventricular balloon, composed of polyvinyl chloride film and connected to a polyethylene tube, was inserted into the LV through the mitral valve from pulmonary vein. The balloon was connected to a pressure transducer (Micro-Med,

Ltd) for recording of LV pressures. The balloon was inflated with water to adjust the end-diastolic pressure (EDP) to 5-8 mm Hg, and the balloon volume was held constant for the duration of the experiment. Before treatment, hearts were perfused at constant pressure (65 cm H2O) for a 30-minute stabilization period. Contractility was assessed with a fluid-filled intraventricular balloon connected to a pressure transducer (Micro-Med, Ltd). The end-diastolic pressure (EDP) was set to 5 to 10 mm Hg. LV developed pressure (LVDP), EDP, maximal rates of contraction

(+dP/dt) and relaxation (-dP/dt) were collected online by use of a commercially available data acquisition system (PowerLab ADInstruments). Developed pressure

(the difference between systolic and diastolic pressures), ±dP/dt and EDP were used as indices of contractile and diastolic function, respectively. A bipolar electrode

(NuMed) was inserted into the right atrium, and atrial pacing was performed at 400 bpm with a Grass S-5 stimulator. At the end of the perfusion period, hearts were either frozen rapidly in liquid nitrogen and stored at -80°C or immersed in 1.5% paraformaldehyde.

X.2 Ischemia/reperfusion Protocol

Global ischemia was applied by stop perfusion completely and reperfusion was introduced by restoring the perfusion. Several ischemia/reperfusion protocols have been used in wild type FVB/N male mice, such as 30min ischemia/120min reperfusion, 45min ischemia/120min reperfusion, and 60min ischemia/120min reperfusion. In the current study, the protocol of 45min ischemia/120min reperfusion was used based on preliminary results, upon which the functional recovery was around 80%. However 30min ischemia/120min reperfusion resulted in a full functional recovery and less than 15% recovery was observed under 60min ischemia/120min reperfusion.

Section XI. In vivo Ischemia/reperfusion Study

Hsp20WT, Hsp20S16ATG and control mice were anesthetized with pentobarbital (90 mg/kg IP), intubated with PE 90 tubing, and ventilated using a mouse miniventilator

(Harvard Apparatus, Holliston, MA). After left thoracotomy and exposure of the heart, the left anterior descending coronary artery (LAD) was ligated with 6-0 polypropylene just proximal to its main branching point. The suture was tied over a 1- mm polyethylene tube (PE-10) that was left in place during the planned period of ischemia (30 minutes). Blood flow was then reestablished by removal of the tube. The

occurrence of reperfusion could be assessed by the observation of blood flow in epicardial coronary arteries through the operation via microscope and by monitoring of ECG (Digi-Med Sinus Rhythm Analyzer, Micro-Med, Inc., Louisville, KY). Mice were kept for 24 hour during reperfusion. Subsequently, the heart was removed for analysis. For each condition, at least 6 successfully operated animals were used, based on our previous studies.225, 231 Sham procedures were identical, with the exception of the actual tying of the polypropylene suture. Infarct sizing was performed as described in our previous studies231. The heart was perfused with 1% TTC (37°C, 60 mm Hg).

Then, the occluder was tied again and the heart was perfused with 5% phthalo blue.

Hearts were transversely cut into 5 to 6 sections, with 1 section made at the site of the ligature. Infarct sizes were determined and expressed as a percentage of the region at risk.

Section XII: Cardiac Injury and Apoptosis Analysis

Cardiac injury was assessed by measuring lactate dehydrogenase (LDH) release. Perfusion effluent was collected every 10 minutes of pre-ischemia and also during reperfusion, and stored at -80 C for measuring the total LDH released from the heart. An In Vitro Toxicology Assay Kit (Sigma) was used and LDH level was expressed as units per gram of wet heart weight. DNA fragmentation was analyzed using a Cell Death Detection ELISAplus kit (Roche), which measures the content of cytosolic mono- and oligo-nucleosomes (180 base pair nucleotides or multiples).

Results were normalized to the standard, provided in the kit, and expressed as a fold increase over control. For terminal dUTP nick end-labeling (TUNEL) assays, hearts were removed from the apparatus after I/R, and the atrial tissue was dissected away.

The ventricles were fixed in 10% buffered formalin and later embedded in paraffin according to standard procedures and 5 m-thick sections were obtained to perform

TUNEL assays using the DeadEnd™ Fluorometric TUNEL system (Promega,

Madison, WI), according to the manufacturer’s instructions. Green nuclei TUNEL- positive myocytes were determined by randomly counting 10 fields of the midventricular section and were expressed as a percentage of the total cardiomyocyte population. Myocyte cytoplasm was detected by -sarcomeric actin

(1:50 dilution; Sigma) labeling; nuclei were stained by DAPI (Invitrogen). Sections were analyzed with a fluorescence microscope.

Changes in the caspase 3 activity were studied using Western blot analysis.

The presence of caspase-3 activation was assessed by the observation of a 17-kDa subunit that was derived from the cleavage of 32-kDa proenzyme caspase-3.

Section XIII. Autophagy Activity Analysis

Autophagy levels in basal or post-ischemia/reperfusion heats were accessed by measuring the protein markers, which are incorporated into the completed autophagosome.232 The microtubuleassociated protein light chain 3 (LC3) is the only protein in higher eukaryotes that is known to remain associated with the completed autophagosome.233 As such, it is the most common marker used to follow autophagic induction. The increase in the levels of lipidated LC3, referred to as

LC3-II, or LC3-II/LC3-I (soluble fraction), by Western blot, indicate the induction of autophagy.232, 234 Levels of Beclin-1, another autophagy marker, were also

detected by Western blot. Beclin 1, an autophagy related protein, is a critical player in the formation of autophagosome.235

Section XIV. Oligomerization study by sucrose gradient

electrophoresis

Frozen heart samples were homogenized in 10 mmol/L HEPES-buffered saline (pH=7.4) containing phosphatase inhibitor and proteinase inhibitor cocktail sets (Calbiochem) and 0.5% Triton X-100 before centrifugation at 16000g. After protein quantification, 100 μg of protein was loaded on a 2 mL sucrose gradient (5% to 40%) and spun at 166,180g for 5 hours with a TLS 55 rotor. Fractions (100 μL) were collected from the top and mixed with Laemmli buffer directly before denaturation and analysis by 12% SDS-PAGE for immunoanalysis, as described218.

Section XV. Immunofluorescence Staining

We use immunofluorescence staining to study the cellular co-localization of

Hsp20 and actin/actinin in wild type and Hsp20S16A hearts. Double staining of

Hsp20 antibody (mouse monoclonal, RDI) and actin/actinin (rabbit polyclonal,

Sigma) antibody were performed. Anti-mouse secondary antibody was conjugated with Alexa Fluor®- 488 dye (green) from Invitrogen. The absorption maximum wavelength of Alexa Fluor®-488 occurs near 494 nm, and the emission maximum is near 519 nm. Anti-rabbit secondary antibody was conjugated with Alexa Fluor®-

594 (red). The absorption maximum wavelength of Alexa Fluor®-594 occurs near

590nm, and the emission maximum is near 617nm. If the two fluorescent signals are in the same location, then the proteins (Hsp20 and actin/actinin) are located in the

same cellular region. This is an indirect way of determining if two proteins interact; if they are located in the same region there is a chance they might bind each other.

1. Preparation of Slides

Cardiac myocytes were isolated from freshly dissected mouse hearts by collagenase perfusion under the Langendorff system, as described previously. Isolated cells were plated on glass slides and fixed by using the following procedures:

10 minutes with 10% formalin in PBS (keep wet)

5 minutes with ice cold methanol, allow to air dry

5 minutes with ice cold acetone, allow to air dry

2. Procedure for Double Staining

1) 1st Serum Blocking: The cells were then blocked by placing the slides in a solution containing normal goat serum, which was the same species as secondary antibody (Invitrogen). The proteins from serum bind to and block nonspecific sites on cellular proteins, where the antibody might bind. This helped to minimize background signals.

2) 1st Primary Antibody: Slides were incubated with 1st primary antibody at appropriate dilution in “antibody dilution buffer” overnight at 4°C. Notes: (1) Serial dilutions for Hsp20 were 1:1000, 1:2000, 1:5000, 1:10000. (2) Dilution for actin or actinin was 1:5000. (3) Not rinses between steps 1) and 2).

3) Rinse in PBS for 5x5min.

4) 2nd Primary Antibody: Slides were incubated in 2nd primary antibodies at

1:5000 dilution in antibody dilution buffer for 1 hour at room temperature 22-25°C.

5) Rinse in PBS 20 for 5x5 min.

6) Detection: ProLong® Gold Antifade Reagent (Invitrogen) was mounted and a coverslip (thin glass square) was placed over the cells and sealed with clear nail polish to preserve the samples. The slides were then examined under a fluorescent microscope where the fluorescent tag was excited with the proper wavelength and emitted a fluorescent signal. This signal would fade if the sample was continually excited, so it was important to minimize exposure to the wavelength. Digital photographs of the fluorescence were generally taken and kept as a record of the signal.

Section XVI. Immunoprecipitation

It is possible to use antibody-antigen precipitation (immuoprecipitation) to isolate a specific antigen from complex protein mixtures, such as cell or tissue lysates. Here we use this method to investigate the interactions between Hsp20 and PP1, or PP2.

Firstly, cardiac homogenate from wild-type, Hsp20WT TG hearts were lysed in extraction buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1mM

EGTA, 1% Triton, 2.5mM sodium pyrophosphate, 1mM beta-glycerophosphate, 1mM

Na2VO4, 1ug/ml leupeptin; supplemented with 1x protease inhibitor cocktail) using an end-over-end rotator at 4°C. Lysates were centrifuged at 13,000 x g for 10 minutes, and supernatants were collected. The cleared supernatant was then incubated overnight at 4oC with 10 μl (2 μg) of mouse monoclonal IgG against Hsp20 (RDI) or an equivalent amount of control IgG. A parallel negative control was conducted, which contained no added antibody. Following precipitation with 100 μl of protein G- agarose for 2 h, the beads were washed five times with lysis buffer. The final pellet

was subjected to SDS-PAGE and immunoblotting as described above. A reciprocal immunoprecipitation approach was performed by using the mouse monoclonal PP1 and PP2a antibody (Santa Cruz).

Section XVII. Protein phosphatase assays

Protein phosphatase activity was assayed with [32P]phosphorylase a as substrate, as previously described.236 Mouse ventricular tissue was homogenized at

4°C for 1 min in buffer containing 4 mM EDTA (pH 7.4) and 15 mM 2- mercaptoethanol with a Polytron PT-10 homogenizer. Homogenates were centrifuged at 14,000 g for 20 min at 4°C, and the supernatants were used for determination of phosphorylase phosphatase activity. The reaction mixture contained (in mM) 20 Tris·HCl (pH 7.4), 5 caffeine, 0.1 EDTA, and 15 2- mercaptoethanol and aliquots of the supernatants. The dephosphorylation reactions were initiated by adding [32P]phosphorylase a and carried out at 30°C for 10 min.

The reaction was terminated by addition of 50% trichloroacetic acid. The precipitated proteins were sedimented by centrifugation at 14,000 g for 5 min, and an aliquot of the supernatants was counted in a liquid scintillation counter. Protein concentration was measured according to the method of Bradford.237

Section XVIII. Generation of Hsp20 knockout mice

XVIII.1 Gene targeting constructs

XVIII.1.1. Strategy Hsp20-null mice were generated by targeted disruption of the entire Hsp20 gene locus through homologous recombination. The overall

procedures for gene targeting are listed in Table 4. Firstly, we screen the murine

Hsp20 cDNA with web resources (www.ncbi.nlm.nih.gov/genome/guide/mouse;

UCSC Genome Bioinformatics /genome.ucsc.edu) to find and purchase bacterial artificial chromosome (BAC) clones (on a C57BL/6 background) that contain mouse Hsp20 gene (bacpac.chori.org). Hsp20 gene is located on human chromosome 19, and on mouse chromosome 7. Genomic Hsp20 is composed of 3 exons and 2 introns, and spans a total of 1.3kb; its short length facilitates its total ablation (Figure 8). Total gene ablation, as opposed to ablation of selected exons, was selected in order to avoid cryptic expression of an Hsp20 protein fragment with biological activity. Replacement of the Hsp20 gene locus with the Neo gene was selected, because expression of the Neo gene renders cells resistant to the antibiotic genetecin (G418), which allows for the ES cell selection early in the targeting process. Furthermore, this DNA sequence could later act as a selection marker for genotyping of mice.

We have used the NTKV-1902 plasmid (Stratagene) as the backbone of our construct (Figure 8). This plasmid contains the Neomycin resistance gene (neo), which is driven by the pPhosphoglycerate Kinase (PGK) promoter, and is followed by the Bovine Growth Hormone (bgh) polyA tail. The Neo gene cassette is flanked by two polylinker regions for cloning of genomic DNA. This plasmid also encodes the

Thymidine Kinase gene (TK), which is driven by the c-myc Promoter (MC1) and followed by the thymidine kinase polyA tail. The TK gene can be used as a negative selection marker, since correct homologous recombination should exclude the TK gene from the genome; expression of the TK gene renders cells susceptible to the

antibiotic ganciclovir.

Table 4.

Figure 8. Generation of the Hsp20 gene targeting vector. (A) The mouse Hsp20 gene is located on Chromosome 7, possesses 3 exons, and is ~1.3kb in length. The lengths of the exons of Hsp20 are shown. (B) The targeting construct was designed to replace the entire Hsp20 coding region, including 338 base pairs of thepromoter region, by homologous recombination. The “short” and “long” homology arms were generated by amplification of genomic DNA flanking the Hsp20 gene, and were subcloned into the NTKV-1902 vector.

Figure 9. The pKO-NTKV-1902 scrambler vector. This plasmid possesses two distinct multiple cloning sites (MCS A and B), which were utilized to insert the homology arms for generating the Hsp20 gene targeting vector. These MCS sites flank the Neomycin resistance gene (Neo), which is driven by the PGK promoter, and renders cells resistant to the antibiotic genetecin. The short homology arm was cloned into MCS A using the NotI and SaI sites. Following this, the long homology arm was cloned into MCS B using the XhoI and HindIII sites. This vector also expresses thymidine kinase, driven by the MC1 promoter, which renders cells susceptible to the antibiotic ganciclovir.

To generate the construct, segments of genomic DNA flanking the Hsp20 gene were cloned into each of the two polylinker regions of the NTKV-1902 plasmid.

The short homology arm (5’ flanking region containing) was 2.05 kb in length, and was located 338 bp upstream of the “start” translation codon (ATG) of Hsp20. The long homology arm (3’ flanking region) was 3.25kb in length and was located 1201 bp downstream of the “stop” translation codon. Thus, the entire 1.3kb Hsp20 gene plus 1539 bp of flanking sequence was to be replaced with the Neo gene by homologous recombination (Figure 9).

XVIII.1.2. Generation of homology arms To generate the homology arms, primers were designed which possessed restriction enzyme sites 5’ to the homologous sequence. For the short homology arm, the forward primer was: 5’ TATG CCTG

CATG TGCC TGTG. The reverse primer sequence was: 5’CATG CCAC AATG

ATTG CCAC. For the long homology arm, the forward primer was: 5’ AAGC CGAT

CTGA CTCC ACTG. The reverse primer was: 5’ CTTC TTCA TCCA GGCG TTCC.

PCR conditions are given in Table 5. PCR was performed using the TaqPlus Long

PCR system (Stratagene). The PCR fragments obtained for the short and long homology arms were each subcloned into the pCR 2.1-TOPO vector. This vector is supplied as a linearized fragment, possessing single 3’-thymidine overhangs and covalently-bound Topoisomerase enzyme, allows for efficient ligation and cloning.

(Figure 10). The TOPO system was used because direct cloning of PCR fragments into the NTKV-1902 vector was not possible; this was likely due to low abundance of the PCR fragments and/or their relatively large sizes.

After sequence verification, the short and long homology arms were cloned stepwise into the NTKV-1902 vector by releasing them from the TOPO 2.1 vector by restriction enzyme digestion, followed by overnight ligation into the NTKV vector.

For cloning details, see Table 5. Fragments cloned into the NTKV vector were amplified and sequenced to ensure their integrity. Once complete, the targeting construct was opened with NotI and submitted to UC Gene Targeted Mouse Facility for electroporation into embryonic stem cell line238. After selection with G418 and gancyclovir, DNA was isolated from surviving clones, digested with EcoR1, for further screening by PCR and Southern blot analysis following standard protocols.

Table 5.

Figure 10. The TOPO 2.1 cloning vector (Invitrogen) Since the PCR products of the short and long arms were difficult to directly clone into the NTKV-1902 vector, the TOPO 2.1 cloning system was used. (A) The TOPO 2.1 vector possesses two single 3’-thymidine overhangs, to which the topoisomerase enzyme is covalently linked. The thymidine overhangs are flanked by multiple cloning sites (MCS) for subsequent restriction enzyme analysis of PCR fragment insertion. (B) Since Taq-generated PCR products possess 3’ adenosine overhangs, the PCR fragment can be inserted into the TOPO vector by base-pairing with the thymidine overhangs of the plasmids. Topoisomerase catalyzes the covalent ligation of the PCR fragment into the vector following base-pairing.

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XVIII.2 Screening of positively targeted embryonic stem-cells

XVIII.2.1. DNA extraction. After electroporation of the targeting construct and selection of genetecin-resistant cells, cell samples from 278 clones were amplified for screening of correct homologous recombination by the UC Gene Targeted Mouse

Facility. The cell lysate was supplied in a volume of 500 μl of proteinase K buffer (50 mM Tris pH=8.0, 100 mM NaCl, 5 mM EDTA, 1% SDS). To this, 10 μl of 20 mg/ml

Proteinase K solution (20 mM Tris pH=7.4, 1 mM CaCl2, 50% glycerol) was added, and this mixture was incubated at 55°C for 12 hours. To isolate DNA, one phenol followed by two chloroform extractions was performed. The DNA was then precipitated in 1 ml of ice-cold 100% ethanol. The DNA pellet was centrifuged at

12000 rpm for 5 minutes, and the supernatant was removed. The pellets were washed twice with 70% ethanol, and left to dry at room temperature for 5 minutes. Finally, the pellet was dissolved in 200 μl of 1X Tris-EDTA buffer, and the genomic DNA was stored at 4°C.

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Figure 11. PCR screening strategy of ES cells for homologous recombination.

As the first screen step for homologous recombination, a segment of the neo gene was amplified by PCR. To further screen ES cell clones for homologous recombination, a segment of the short arm contiguous with the neo gene, and a segment of the long arm following the neo gene were amplified by PCR.

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XVIII.2.2. PCR screening for the Neo gene. After DNA extraction, the genomic DNA was screened for the presence of the Neo gene to ascertain the quality of the DNA for further PCR analysis and to confirm the presence of the targeting construct in the genome. The forward primer used to amplify the Neo sequence was:

5’ GTA AAG CAC GAG GAA GCG GTC, and the reverse primer was: 5’ GAG GCT

ATT CGG CTA TGA CTG. PCR conditions are given in Table 5.

XVIII.2.3. PCR screening for the short homology arm. The clones which gave a positive Neo signal by PCR were then further screened by PCR for the presence of the short homology arm DNA sequence continuous with the Neo gene.

The forward primer was: 5’ CCA CTC CAG GCT AAC AGA ATC AG, and the reverse primer was: 5’ CGT GAT ATT GCT GAA GAG CTT GG. PCR conditions are given in Table 5. The primers were positioned to amplify the entire short homology arm only in order to narrow the field of potentially targeted cells (Figure

11).

XVIII.2.4. Southern blotting. The most complete and definitive analysis of positively targeted clones is Southern blotting. This strategy is designed to distinguish a homologous recombinant from a random integrant (Figure 12). First of all, a specific probe (500bp) was generated, which hybridized to an endogenous sequence that lies completely outside of any sequences contained in the targeting vector. To do this, one restriction site (EcoR1) was identified that lies outside of any sequence contained in the targeting vector (the other can be either inside or outside). Then, the probe was hybridized to a short sequence contained within this restriction fragment,

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but completely outside of any sequence in the targeting vector. This is the only way to distinguish a homologous recombinant from a random integrant, because upon correct homologous recombination in ES cells, the targeting construct ablates an endogenous as well as introduces an exogenous EcoR1 restriction enzyme site. After digestion with EcoR1, the probe would detect the 8.6 kb untargeted Hsp20 locus as well as the targeted 5.8 kb locus, shown in Figure 13.

XVIII.2.4.1. Digest the DNA with EcoR1. Genomic DNA (30 μl) was incubated with 4 μl restriction enzyme buffer, 4 μl of 10 mM spermidine, 1 μl of 10 mg/ml BSA, and 2 μl of XhoI at 37°C for >16 hours.

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Figure 12. Southern blot strategy. A specific probe of 500bp was designed (yellow line) to hybridize to a short sequence, upstream of 3’-short arm, which was contained within EcoR1 restriction fragment but completely outside of any sequence in the targeting vector. SA: short arm; LA: long arm.

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Figure 13. Sensitivity and specificity of Southern blot probes. To test the specificity and sensitivity of Southern blot probes, wild type tail DNA and untargeted ES cell DNA were digested with restriction enzymes (EcoR1, Xhol1, Smal1, Kpn1, BamH1), run on 0.8% agarose gels, transferred to Zeta-Probe GT Membrane (Bio-Rad), and probed with 32P-labeled Southern probes, as described in Materials and Methods. Shown are the results for Probe #3 (530bp), which yielded a single specific band at the expected molecular weight of 8.6 kb. This probe was used to screen all 278 ES cell clones.

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XVIII.2.4.2. Run the digest on an agarose gel. The digested DNA samples were loaded in a 0.8% agarose gel, and underwent electrophoresis at 14 V for ~18 hours. A

1 kb DNA ladder was used to estimate band size. A photograph of the gel was taken next to a centimeter ruler for later assessing the size of bands, which appear by autoradiography.

XVIII.2.4.3. Denature the DNA. The DNA in the gel was denatured by submerging the gel in 0.2 M HCl for 20 minutes. After rinsing with DI water, the acid was neutralized in 0.5 M NaOH for 30 minutes. Finally, the gel was equilibrated in 2X

SSC (sodiumchloride/ sodium citrate, pH=7.0).

XVIII.2.4.4. Transfer the denatured DNA to the membrane. Digested genomic

DNA was transferred from gel onto Zeta-Probe GT Membrane (Bio-Rad) under vacuum. The transfer was carried out under the pressure of 5-6 mm Hg for 2 hours.

Afterwards, the membrane was air-dried and baked at 80°C for 10 minutes. Transfer efficiency was visually assessed by UV lamp illumination.

XVIII.2.4.5. Probe the membrane with labeled ssDNA. Altogether 9 Southern probes were produced from the same BAC clones used to generate the targeting construct. Only one probe was able to specifically bind with genomic DNA. The primers and conditions used to amplify this probe are listed in Tables 6 and 7. To analyze ES cell genomic DNA for homologous recombination, Southern blot probes were 32P-labeled using the Prime-It II Random Primer Labeling Kit (Stratagene).

Briefly, 25 ng of DNA was incubated with 10 μl of random nucleotide primers in a volume of 34 μl. The reaction mixture was boiled for 5 minutes, and afterwards 10 μl

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of 5X dCTP buffer (1 mM dATP, dGTP and dTTP) 5 μl of 32P labeled dCTP and 1 μl of Exo (-) Klenow (5 U/μl) were added. This mixture was incubated at 38°C for 10 minutes. The probe was purified using NICK purification columns (Amersham), which are used to separate nick-translated DNA from unincorporated 32P -labeled nucleotides. The labeled probe was eluted in a volume of 400 μl 1X Tris-EDTA. To measure 32P incorporation, 10 μl of the probe was immediately Cerenkov counted. For probing, the membrane was prepared by incubation in 12.5 ml Rapid Hyb Buffer

(Amersham) for 30 minutes at 65°C. Simultaneously, probes were boiled for 5 minutes to denature them. The volume of probe added to the hybridization buffer was calculated according to the equation: volume (μl) = 7x106/(2 x cpm). The probes were incubated with the membrane for 16 hours. Afterwards, the hybridization buffer containing the probes was discarded, and the blot was washed in 2X SSC and 0.1%

SDS for 1 hr at 65°C. Washes in this buffer were repeated until the radioactive signal in the center of the membrane reached less than 400 cpm as measured by a pancake

Geiger counter. Finally, the blot was exposed to a PhosphoImager screen for 16 hours, and the image was developed on a Storm image scanner.

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Table 6. The sequence of primers for generation of Southern Blot probe

GC% in Component Primers Sequence PCR-

probe

pfuTurboDNA polymerase 4l 5’ 5-CTGCATAGAGAACTCTTGTC-3 Template 4 l Buffer 20l dNTP 1.6l 51.23% Primer 5’ 4l Primer 3’ 4l 3’ 5-GCACATCCAGGCATCATTCT-3 dH2O 162.4l Total 200l

Table 7. PCR cycling parameters for generation of Southern Blot probe

Segment Number of cycles Temperature Duration

1 1 95 C 1 minute

95 C 1 minute

2 30 58 C 1 minute

72 C 1.5 minute

3 1 72 C 10 minutes

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Section XIX: Generation of Hsp20S16D transgenic mice

We generated Hsp20S16D TG mice that carry the mouse cardiac Hsp20S16D cDNA under the control of the -MHC mouse promoter (Figure 14), as described.225 A 0.5kb wild-type or mutant mouse Hsp20cDNA was ligated with the murine cardiac -myosin heavy chain gene promoter (5.5-kb) (Figure 14). In all studies, male mice between 12 to 16 weeks of age for the Hsp20S16D transgene were compared with age-matched transgenics, overexpressing wild type Hsp20 and wild type controls.225 The care of all animals used in the present study was in accordance

S16D with the University of Cincinnati animal care guidelines. The Hsp20 transgenic constructs were generated by Dr. Guo-Chang Fan.

Figure 14. Diagram of transgenic Hsp20S16D mouse constructs. The mouse mutant Hsp20 cDNA, in which Serine 16 encoded by codon TCA was mutated into GAC (encoding aspartic acid), was driven by the -myosin heavy chain promoter ( -MHCp).

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Section XX: Statistical analysis

Data are expressed as mean ± SEM. Statistical analysis was performed using a 2-tailed Student T test for unpaired observations and ANOVA followed by the Bonferroni post hoc test for multiple comparisons (Systat 11). Values of P<0.05 were considered statistically significant.

Power analysis to determine groups numbers (n) is based upon retrospective analysis of data for each endpoint, using alpha=0.05, 80% power to reject the null hypothesis.

All data are expressed as meanSEM. For the studies using isolated myocytes, 5-10 cells per heart were studied, and each animal was analyzed as a single n. For quantitative immunoblotting results of the protein levels, appropriate amount of protein was separated in SDS-PAGE; three appropriate amounts of protein pooled from wild type hearts were run in the same blot as a standard linear range for each protein. A linear correlation was obtained from this range and a coefficient of above 0.97 was defined as an acceptable linear relationship. Protein levels in different hearts were quantitated using this linear relationship, and the final results were normalized to those of wild type hearts and expressed as fold of wild type. Comparison between two groups was evaluated with Student’s t test.

Comparison of the results among Hsp20WT, Hsp20S16A transgenic and non- transgenic mice was analyzed using ANOVA. Probability (P) values of <0.05 were considered to be significant.

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Chapter III

Results

Section I. Hsp20 and its phosphorylation of Ser16 in regulating cardiac function

I.1 Background and rationale

-adrenergic signaling (-AR) plays a fundamental role in cardiac function.239,

240 Inotropic responses of -AR are primarily attributed to PKA-mediated

2+ 239, phosphorylation of the L-type Ca channel (Ica-L), troponin I and phospholamban.

240 First, -AR signaling increases ICa, bringing additional Ca2+ into myocytes for larger contractions.239, 240 Second, phosphorylation of phospholamban by PKA releases its tonic inhibition of the sarcoplasmic reticulum (SR) Ca2+ pump, sequestering more Ca2+ into the SR for larger subsequent contractions.241 In addition, activation of -adrenergic signaling also increases the cardiac relaxation rate through phospholamban phosphorylation, as the increase in Ca2+ flux from cytosol to SR accelerates relaxation of the myofilaments. PKA-mediated Troponin I phosphorylation also contributes to the increase of the cardiac relaxation rate, by reducing the Ca2+ affinity of its partner protein troponin C.242 Hence, the increase in myofilament action and ATP-dependent SR Ca2+ pump rate expend additional energy in order to generate stronger contractions, during the -AR inotropic responses. To keep up with these demands, PKA also activates phosphorylase kinase, a metabolic enzyme that increases rates of glycogen breakdown, 243 thereby providing additional glucose and increasing cellular ATP.

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-adrenergic signaling (-AR) also plays a fundamental role in heart failure.244, 245 Congestive heart failure (CHF), while resulting from a number of causes, involves reduced cardiac output and contractility.246 The sympathetic nervous system is a critical component of neurohumoral responses observed in heart failure. In the early stages of the syndrome, an intrinsic decrease in myocardial function leads to an increase in sympathetic activity. Acutely, through the activation of cardiac - adrenergic receptors, heart rate and cardiac contractility are increased and compensate for the decreased cardiac output, which is returned to basal levels.246 However, as heart failure progresses, sympathetic activity is further increased in an attempt to compensate for a progressive loss of cardiac function. Unfortunately, chronic exposure of the heart to elevated levels of catecholamines, released from sympathetic nerve terminals and the adrenal gland, may lead to further pathologic changes in the heart, such as downregulation of myocardial -adrenoreceptors, uncoupling of - adrenoreceptors and adenylate cyclase, as well as modifications of downstream cAMP-mediated signaling.247 These changes will result in continued elevation of sympathetic tone but diminished contractile response to catecholamine stimulation and contribute to progressive deterioration of cardiac structure and function.244, 246, 248

One of the underlying mechanisms in the detrimental effects of prolonged -agonist stimulation involves cardiomyocyte apoptosis or cell death 249.

Recently, we found that prolonged (30 min) -agonist infusion of mouse heart induced expression and phosphorylation of Hsp20.226 Further studies in adult rat cardiomyocytes indicated that the levels of Hsp20 significantly increased by 30 min, returned to basal levels by 2 hours, and significantly decreased by 12 hour; whereas,

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there were no alterations in the levels of Hsp27 and B-crystallin after exposure to isoproterenol.155 These findings suggest that Hsp20 may play an important role in - adrenergic-mediated cardiac response. To further explore the role of Hsp20 and its phosphorylation at Ser16 in regulating cardiac contractility, adenoviral infected adult rat cardiomyocytes and transgenic mouse models were used.

I.2 Theeffects of acute overexpression of Hsp20 on cardiomyocyte contractility

Since it is known that Hsp20 regulated smooth muscle relaxation and colocalized with actin in smooth muscle and cardiac muscle,88, 108, 109, 112 we hypothesized that Hsp20 may be involved in the regulation of cardiac function and SR

Ca2+ cycling. First of all, the effects of acute overexpression of Hsp20 on rat adult cardiac myocytes were studied, using adenoviral transfection, with the assistance from

Dr. GC Fan and Dr. Q Yuan, Department of Pharmacology (Figure 15-18).

Cardiomyocytes were infected with adenoviruses carrying type (WT) Hsp20 cDNA, or GFP cDNA (ad.GFP) as control. After 24 hours of infection, almost 100% of infectivity was achieved at a MOI (multiplicity of infection, the average number of virus per cell) of 500 (Figure 15). The total Hsp20 level was upregulated by 50% in the myocytes infected with adenoviruses carrying Hsp20 cDNA, compared with controls (n=3 hearts for each group, P<0.05).

After 24 hours of incubation with adenoviruses, single cell contractile parameters were measured. We found that upregulation of Hsp20 led to increased

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myocyte contractility. Overexpression of WT-Hsp20 resulted in significant increases in fractional shortening (FS: 17.50.5% vs. 191%, GFP vs. Hsp20, P<0.05) and the maximal velocities of shortening (+dL/dt: 804 vs. 1109m/s, GFP vs. Hsp20,

P<0.05) and relengthening (-dL/dt: 705 vs. 100%10m/s, GFP vs. Hsp20, P<0.05) compared with GFP-infected cells (Figure 16). These results indicated that acute overexpression of Hsp20 increased myocyte contractility.

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Figure 15. Adenoviral infection efficiency and expression levels of Hsp20 in cardiac myocytes. (A) After 24 hours of infection, Ad.GFP and Ad.Hsp20 infected cardiomyocytes were examined under light (upper panel) and fluorescence (lower panel) microscopy. Almost 100% of infectivity was achieved at a MOI of 500 in Ad.GFP, Ad.Hsp20 groups, respectively. (B) A representative immunoblot of total Hsp20 in Ad.GFP and Ad.Hsp20 infected groups. n=3 hearts for each group, *: P<0.05 (Data generated by Dr. GC Fan)

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Figure 16. Effects of Hsp20 expression on myocyte basal contractility. (A) Representative traces of cardiomyocyte mechanics in Ad.GFP, Ad.Hsp20-infected adult rat cardiomyocytes. (B) Increased myocyte percent fractional shortening (FS) and maximal rates of contraction and relaxation (fdL/dt) were observed in Ad.Hsp20-infected myocytes. * : P<0.05, n=21 to 40 cells from 3 to 5 hearts for each group. (Data generated by Dr.GC Fan and Dr. Q Yuan)

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Ca2+ kinetics were also assessed after 24 hours incubation with the adenoviruses. The alterations of Ca2+ kinetics paralleled those of the contractile function. Figures 17 A and B showed that Ca2+ transient amplitude was increased by 16%, and time to 50% decay of Ca2+ transients was significantly shortened (by 37%) by overexpression of

Hsp20 (Fig. 17 A and B, P<0.05).

Figure 17. Effects of Hsp20 expression on myocyte basal Ca2+ transients. (A) Representative traces of Ca2+ transient in Ad.GFP, Ad.Hsp20-infected adult rat cardiomyocytes. (B) Increased myocyte Ca2+ amplitude and decreased T50 were observed in Ad.Hsp20-infected myocytes (* : P<0.05, n=21 to 40 cells from 3 to 5 hearts for each group). (Data generated by Dr. GC Fan and Dr. Q Yuan)

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I.3. Generation and characterizations of Hsp20 transgenic mice

I.3.1 Genotyping of Hsp20 transgenic mice

Since overexpression of Hsp20 increased myocyte contractility in vitro, it was of importance to investigate the in vivo role of Hsp20 in regulating cardiac function.

Thus, we generated a transgenic mouse model with cardiac overexpression of Hsp20

(Figure 7). PCR-based genotyping was performed, which specifically amplified a 600- bp fragment spanning the junction between -MHC promoter and Hsp20 cDNA from the mouse tail genomic DNA. The control PCR was set up to amplify a 350-bp fragment of TSH-. PCR products are separated on agarose gels by electrophoresis and visualized under UV light. Figure 18A shows the PCR results of Hsp20 transgenic mice.

I.3.2.Expression levels of Hsp20 and other small heat shock proteins in transgenic hearts

Western blot analysis (Figure 18B) of cardiac homogenates revealed that transgenesis resulted in a 10-fold increase of the Hsp20 protein level in the TG hearts.

In wild type mice, Hsp20 was expressed predominantly in the cardiac and in much lower abundance in skeletal and smooth muscles (Figure 18C).

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Figure 18. Generation and characterization of transgenic mice with cardiac-specific Hsp20. (A) Genotypic analysis of genomic DNA from WT (–) and TG (+) mice. Control PCR was set up to amplify a 350-bp fragment of TSH-. (B) Quantitative immunoblotting analysis showed that there was a 10-fold increase in Hsp20 protein level in TG hearts relative to WT hearts (1.0). (C) Aliquots (20 g) of different tissue homogenates from WT and TG mice were subjected to Western blotting with an anti-Hsp20 antibody (n=6, *:P<0.01). Data generated by Dr. GC Fan and Dr. Q Yuan.

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I.3.3 Morphological and histological studies of Hsp20 transgenic hearts

Standard hematoxylin-eosin staining was used for animal hearts and the sections were analyzed by light microscopy. Hsp20 TG hearts showed no apparent cardiac hypertrophy, ventricle dilation, or inflammatory cell infiltration by hematoxylin and eosin stain (Figure 19). Collagen fibers were examined by Masson’s

Trichrome staining, which revealed that there were no significant changes of “blue- stained fibers” in Hsp20 TG hearts compared with wild type (Figure 19). The heart weight/body weight and lung weight/body weight ratio were not different between

Hsp20 hearts and controls (Figure 20).

Transmission electron microscopy showed no ultrastructural alterations of the cardiomyocytes of Hsp20 transgenic mice compared with wild type (Figure 21).

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Figure 19. Histological study of Hsp20 TG hearts. Ventricular sections from 3- month-old TG hearts stained with hematoxylin and eosin (HE, magnification, x400) and Masson’s Trichrome (Masson’ T) indicated no apparent hypertrophy, inflammation or fibrosis in TG hearts compared with WT.(n=5)

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Figure 20. Heart weight/body weight ratio (HW/BW) and lung weight/body weight ratio (LW/BW) of Hsp20 TG mice. There is no difference between WT and Hsp20 TG mice in HW/BW and LW/BW ratios (n=10).

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Figure 21. Ultrastructure of cardiomyoctes from wild type and Hsp20 transgenic hearts. Electon microscopy showed that the arrangement of myofilaments and Z-bands were indistinguishable between TG and wild type hearts. A: autophagosome; M: mitochondria; Z: Z-line (Bar scale 500nm, x20000, n=3).

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I.3.4 Assessment of Ca2+ cycling protein levels in sarcolemma

Given the central role of SR Ca2+ cycling proteins in cardiac contractility, it is important to examine whether overexpression of Hsp20 poses any effects on their expression levels. By quantitative immunoblotting analysis (Figure 22), we found that there were no significant changes in SR Ca2+-ATPase (SERCA2a), phospholamban, Calsequestrin and L-type Ca2+ channel (DHPR). However, sodium/calcium exchanger (NCX) level was increased by 20%. (Figure 22)

Since -adrenergic receptor–dependent protein phosphorylation of key regulatory phosphoproteins, such as phospholamban, ryanodine receptor, troponin I, and the L-type calcium channel, constitutes a critical regulatory mechanism that governs Ca2+ cycling and cardiac contractility, we investigated the expression and phosphorylation levels of these key substrates in our transgenic model.

Phosphorylation of phospholamban at both its cAMP-dependent (Ser16) and

Ca2+/calmodulin dependent (Thr17) protein kinase sites was significantly increased compared with wild types (Figure 23). Yet, the phosphorylation level of troponin I and RyR2 was not different between WT and transgenics (Figure 23).

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Figure 22. SR Ca2+ regulatory proteins in Hsp20 TG hearts. (A) Representative blots of SR Ca-cycling protein levels in WT and Hsp20 TG hearts. (B) Quantitative results revealed that SERCA2a, phospholamban pentomer (PLNp), phospholamban monomer (PLNm), total PLN, dihydropyridine receptor (DHPR), troponin I (TnI), ryanodine receptor (RYR2) and calsequestrin (CSQ) were not altered (WT vs. TG, P>0.05, n=6 for each protein, values are mean±SEM). Sodium/calcium exchanger (NCX) level was increased in Hsp20 hearts (P<0.05).

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Figure 23. Phosphorylation of SR Ca2+ regulatory proteins in Hsp20 TG hearts. (A) Representative blots of phosphrylation of pS16-PLN, pT17-PLN, p-TnI and p- RYR2 levels in WT and Hsp20 TG hearts. (B) Quantitative results of phosphorylated SR Ca2+ regulatory proteins, TG vs. WT. Immunoblots revealed that pS16-PLN/ PLN, pT17-PLN/ PLN was significantly increased compared with wild types, but phosphorylation level of RYR2 and troponin I was not different between WT and transgenics (*:P<0.01, n=6 each, values are mean±SEM).

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I.3.5Overexpression of Hsp20 enhanced contractility and Ca2+ kinetics in cardiac myocytes

I.3.5.1 Hsp20 TG increased myocyte contractility

To evaluate the effects of Hsp20 overexpression on cardiac contractile function, mechanical parameters and Ca2+ transients were further assessed in isolated cardiomyocytes, which represent a load-independent preparation. At a stimulation frequency of 0.5Hz, overexpression of Hsp20 resulted in significant increases in FS

(11.06 ± 0.65% vs. 7.76±0.3, TG vs. WT, P<0.01), +dL/dt (122.64 ± 7.46 vs.

99.83±6.15m/s, TG vs. WT, P<0.01), as well as -dL/dt (97.85 ± 6.69 vs.

89.76±6.65m/s, TG vs. WT, P<0.01) compared to wild type (Figure 24).

To further explore the response of Hsp20 myocytes to beta-adrenergic stimulation, contractile function was measured in the presence of 0.1M isoproterenol.

After isoproterenol stimulation, +dL/dt, -dL/dt and FS were increased to 2.17, 2.16 and 1.97 fold, respectively, compared to basal levels in WTs (P<0.05). In Hsp20 cardiomyocytes, the isoproterenol-stimulated parameters were even higher than those in stimulated WTs: +dL/dt, -dL/dt and FS were increased by 1.24, 1.33 and 1.21 fold, respectively, compared to isoproterenol-stimulated wild type myocytes (Figure 25,

P<0.01). These data indicated that overexpression of Hsp20 increased cardiac myocyte contractility, and activation of cAMP-PKA pathway by isoproterenol- treatment further enhanced contractility in Hsp20 myocytes.

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Figure 24. Mechanics of isolated wild type and Hsp20 TG cardiomyocytes under basal conditions. Isolated myocytes from 12-14 weeks old hearts were suspended in 1.8mM Ca-Tyrode solution and field-stimulated at 0.5Hz. (A) Representative cell shortening traces of wild type and TG cells in the absence of isoproterenol (Iso); (B) Fractional shortening (FS); (C) Maximum rates of contraction (+dL/dt); (D) Maximum rates of relaxation (-dL/dt). n = 36-42 cells from 5 hearts for each group. Values = Mean ± SEM. *: P<0.01 vs WT.

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Figure 25. Mechanics of isolated wild type and Hsp20 TG cardiomyocytes in responses to isoproterenol. Isolated myocytes from 12-14 weeks old hearts were suspended in 1.8mM Ca-Tyrode solution and field-stimulated at 0.5Hz, in the presence of 0.1M isoproterenol (Iso). (A) Representative cell shortening traces of wild type and TG cells; (B) Fractional shortening (FS) (C) Maximum rates of contraction (+dL/dt) ; (D) Maximum rates of relaxation (-dL/dt). Isoproterenol response was performed in n=32 cells from 4 wild type hearts and 40 cells from 4 TG hearts. Values = Mean ± S.E.M. *: P<0.01 vs WT in the presence of isoproterenol.

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1.3.5.2 The Ca2+ kinetics in Hsp20 cardiac myocytes

To assess the effects of Hsp20 overexpression on SR cycling, isolated cardiac myocytes were loaded with Fura-2, and intracellular Ca transient amplitude and Ca kinetics were measured at 0.5Hz. The Ca2+ transient peak was increased by 12% in

Hsp20 cells compared with WT (0.45 ± 0.01 vs. 0.39 ± 0.02, TG vs. WT, P<0.05,

Figure 26B). Consistent with the acceleration of contraction and relaxation, the 50% time constant of Ca2+ transient decay (T50) was also significantly decreased in Hsp20 cells (0.37 ± 0.02 vs. 0.45 ± 0.02, TG vs. WT, P<0.01, Figure 26C). After isoproterenol stimulation, the peak of Ca2+ amplitude in wild type cells was significantly increased by 1.74-fold (1.07 ± 0.05 vs. 0.39 ± 0.02, +isoproterenol vs. - isoproterenol, P<0.01, Figure 26B); T50 was also significantly decreased by 63%

(0.17 ± 0.01 vs. 0.45 ± 0.02, +isoproterenol vs. -isoproterenol, P<0.01, Figure 26C) compared to its basal level. Similarly, Ca2+ peak increased by 1.47-fold and T50 decreased by 57% upon isoproterenol-stimulation in Hsp20 cells, compared to the corresponding basal parameters (Figure 26B and 26C). Interestingly, upon isoproterenol-treatment, there was no significant difference in either caffeine-induced

Ca2+ transient amplitude (1.11 ± 0.05 vs. 1.07 ± 0.05, TG vs. WT, P>0.05) or time constant of Ca2+ transient decay (0.16 ± 0.01 vs. 0.17 ± 0.01, TG vs. WT, P>0.05) in

Hsp20 TG cells compared to those of WT controls, (P>0.05, Figure 26B and 26C), suggesting that 0.1mol/L isoproterenol induced a maximal effect on both WT and

TG cells.

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Figure 26. Isolated mouse cardiac myocytes were stimulated at 0.5Hz to obtain the Ca2+ transients. (A). Representative tracings of Ca2+ transients in WT and Hsp20 TG myocytes with or without isopreterenol stimulation (100 mmol/L). (B) Ca2+ transient amplitude in WT and Hsp20 TG myocytes. (C) Time to 50% of Ca2+ decay in WT and Hsp20 TG myocytes. T50: time to 50% decay of Ca transient. n = 30-45 cells from 3 hearts. Values = mean SEM. *: P<0.05; # P<0.01, TG vs WT.

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To determine the SR Ca2+ content, Ca2+ transients were recorded upon the rapid application of 10 mM caffeine. As shown in Figure 27, the amplitude of caffeine-induced Ca2+ release was increased by 19% in Hsp20 TG myocytes (1.13 ±

0.02 vs. 0.95 ± 0.02, p<0.01, Hsp20 TG vs. WT), indicating a higher SR Ca2+ content.

However, there was no difference in T50 of caffeine-induced Ca2+ transient between

TG and WT cells (3.16±0.75 vs. 3.55±0.26, p>0.05, TG vs. WT). After isoproterenol stimulation, the caffeine-induced Ca2+ peak in wild type cells was significantly increased by 60% compared to its basal level (1.50 ± 0.06 vs. 0.95 ± 0.02, p<0.05,

+isoproterenol vs. -isoproterenol), but T50 was not significantly changed (4.05±0.45 vs. 3.55±0.26, +isoproterenol vs. -isoproterenol). Interestingly, upon isoproterenol- treatment, there were no further increases in either caffeine-induced Ca2+ transient amplitude (1.53±0.09 vs. 1.13±0.02, +isoproterenol vs. -isoproterenol) or time constant of Ca2+ transient decay in Hsp20 TG cells (3.44±0.27 vs. 3.16±0.75,

+isoproterenol vs. -isoproterenol) (Figure 27B and 27C) compared to the corresponding basal parameters, suggesting that the SR Ca2+ load had reached its maximum level under basal conditions. The further increase in the peak of Ca2+ transient after isoproterenol stimulation, without alterations in SR Ca2+ load, could be due to an increase in L-type Ca2+ current, which may trigger increased Ca2+ release from the sarcoplasmic reticulum.250

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Figure 27. Caffeine induced Ca2+ release in isolated mouse cardiac myocytes under 0.5Hz stimulation, with or without isoproterenol-treatment (100 mmol/L). (A). Representative tracings of Ca transients in WT and Hsp20 TG myocytes. (B) Caffeine induced Ca transient amplitude in WT and Hsp20 TG myocytes. (C) Caffeine-induced Ca releasing time to 50% of Ca decay in WT and Hsp20 TG myocytes. T50: time to 50% decay of Ca transient. n = 30-45 cells from 3 hearts. Values = mean SEM. *: P<0.05

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I.3.6. Enhanced global cardiac contractile function of Hsp20 transgenic mice

Overall cardiac function of Hsp20 transgenic mice at 3 months of age was compared with control littermates, which was demonstrated in the series of P-V loops obtained during transient occlusions of inferior vena cava, a procedure that reduces cardiac preload (left ventricle volume), as shown in Figure 28 (upper panel). The end- systolic pressure-volume relationship (ESPVR) describes the maximal pressure that can be developed by the ventricle at any given LV volume.251 The slope of ESPVR represents the end-systolic elastance, which provides an index of myocardial contractility.251 We observed that the slope of ESPVR of Hsp20 transgenic hearts is higher than wild type (WT: 18.350.05 mmHg/μl; Hsp20: 19.420.03 mmHg/μl; n=6,

P<0.05), implying an increase in contractile function of transgenic hearts. The end- diastolic pressure-volume relationship (EDPVR) describes the passive filling curve for the ventricle.252 The slope of the EDPVR at any point along this curve is the reciprocal of ventricular compliance (or ventricular stiffness).252 We found that the slope of EDPVR is not different between transgenic and wild type (WT: 1.693 ± 0.06 mmHg/μl; Hsp20: 2.822 ± 0.16 mmHg/μl; n=6, P>0.05), indicating that Hsp20 overexpression had no effect on ventricular compliance. Left ventricular +dP/dt and – dP/dt were significantly higher in Hsp20 hearts compared with wild types, as shown in Figure 28 (lower panel), which suggested that Hsp20 overexpression increased in vivo cardiac function.

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Figure. 28 Enhanced in vivo cardiac function of Hsp20 TG mice. Upper panel: representative left ventricular pressure-volume (P-V) loops from Hsp20 TG and wild type mice thoracic vena cava occlusion. ESPVR, end-systolic pressure volume relationship; EDPVR, end-diastolic pressure volume relationship. Lower panel: +dP/dt and –dP/dt values were higher in Hsp20 transgenic hearts than wild types. (*: P<0.01 vs. WT) Data generated with the help of Dr. Y Wang (Department of Pathology).

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I.3.7 Phosphatase activity in Hsp20 hearts

Cardiac tissue contains several subtypes of Ser/Thr protein phosphatases (PP) including PP1 and PP2A,253 which counteract protein kinase activities to balance the phosphorylation status of proteins in cells. PP1 and PP2A are actively involved in regulating cardiac contractility by dephosphorylating key phospho-proteins, after beta- adrenergic stimulation254; while PP2B, also known as calcineurin, has been shown induce hypertrophy255. To further investigate whether Hsp20 overexpression-induced hyper-contractility is related with alterations in PP1 activity, we measured the PP1 activity levels in the homogenates from wild type and Hsp20 TG hearts (Figure 29).

PP1 activity decreased significantly in the Hsp20 homogenate by ~32% compared with wild type homogenates (0.4760.0190 vs. 0.503 0.007 mmol/min/ml, n=9,

P<0.01). However, there was no difference in PP2A activity between Hsp20

(0.0210.006, mmol/min/ml) and wild type hearts (0.0440.012 mmol/min/ml).

These data suggested that in Hsp20 TG hearts, decreased PP1 activity may result in the enhanced phosphorylation of Ca2+ handling proteins, finally leading to the increase of cardiac contractility.

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Figure 29. PP1 activity in the homogenate isolated from the LV myocardium of wild type and Hsp20 transgenic hearts. PP1 and PP2A activities were determined using [32P]phosphorylase a as substrate. Values are mean SEM, n=9, *P <0.01 vs wild type.

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I.3.8 Hsp20 associated with PP1

We confirmed the direct interaction of Hsp20 and PP1 by co-

immunoprecipitation. Cardiac homogenates from wild type and Hsp20 transgenic mice

were employed for this protein interaction analysis. PP1 was detected by Western blot

analysis of anti-Hsp20 immunoprecipitates, the reverse immunoprecipitation indicated

that Hsp20 was also associated with PP1 (Figure. 30). These results showed that Hsp20

and PP1 were associated in vivo.

Figure 30. Protein interaction of Hsp20 with PP1. Co-immunoprecipitation is performed by using anti-Hsp20 (A) or anti-PP1 (B) antibody, from the cardiac homogenate of wild type and Hsp20 TG mouse. The precipitates were analysed by immunoblotting with anti-p-Hsp20 or anti-PP1 antibodies, as indicated. IP, immunoprecipitation; WB, Western blot.

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I.4. Generation of Hsp20S16A transgenic mice

I.4.1 Increased level of p-Ser16-Hsp20 in sustained isoproterenol- treated rat cardiomyocytes

We previously reported that isoproterenol stimulation for 30 minutes in mouse cardiomyocytes was associated with a distinct phosphoprotein, identified by 2-

D gel with molecular mass of 17.4 kD and pI of 5.5. This phosphoprotein was subsequently sequenced and proven to be a cardiac homologue of Hsp20.155

Here we use a non-phosphorylated Hsp20 antibody and phospho-Ser16-

Hsp20 antibody to measure the total Hsp20 and pS16-Hsp20 level in isoproterenol- treated rat cardiomyocytes. We confirmed that total Hsp20 increased after sustained iso-stimulation (Figure 31, P<0.01) which is consistent with previous results.155

Furthermore, the phosphorylated Hsp20 level was increased after isoproterenol- stimulation, while there was no obvious pS16-Hsp20 band in saline-treated controls.

(P<0.01) These results suggested that Hsp20 and its phosphorylated form play an important role in -adrenergic mediated cardiac response.

I.4.2 Increased level of phosphorylated Ser16-Hsp20 in ischemia/reperfused and failing hearts

In initial studies, we assessed the levels of phospho-Ser16-Hsp20, by quantitation of the ratio of phosphorylated Ser16 versus total Hsp20 in mouse hearts after ex vivo ischemia (45min) and reperfusion (2h). The specific anti-phospho-Ser16-Hsp20 antibody was used and the ratio of phospho-Ser16/total Hsp20 was increased by 40%,

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compared with its pre-ischemic level (Figure 32). Similarly, an increased ratio of phospho-Ser16/total Hsp20 was observed in failing human hearts compared with donor hearts (Figure 32). These results indicate that Hsp20 phosphorylation at Ser16 may be associated with cardiac ischemia/reperfusion and heart failure.

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Figure 31. Phosphorylation of Hsp20 in 30min isoproterenol -treated isolated rat adult cardiomyocytes. Western blotting analysis showed that both total and phosphorylation of Ser16-Hsp20 level increased in isoproterenol-treated (100 mmol/L) myocytes compared with saline-treated controls. CTL: control; ISO: isoproterenol- treated; CSQ: calsequestrin; pS16-Hsp20: phospho-Ser16-Hsp20. *: P<0.01, n=3.

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Figure 32. Phosphorylation of Hsp20 in ischemia/reperfusion injured wild-type mouse hearts and failing human hearts. The ratio of phospho-Ser16-Hsp20/total Hsp20 was increased in post-ischemia/reperfusion myocardium (Panel A: n=6, *: P<0.01, Post vs. Pre) and in failing human hearts (Panel B, *: P<0.01, HF vs. Donor). CSQ: calsequestrin (loading control). HF: heart failure.

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I.4.3 Transgenic mice with cardiac-overexpression of Hsp20S16A

The findings above and previous studies225 have suggested a potential role of cAMP-dependent phosphorylation of Hsp20 under stress conditions. To examine the function of Hsp20 phosphorylation at Ser16, we generated transgenic mice that carry the mouse cardiac Hsp20 cDNA, in which TCA encoding for Ser16 was substituted with GCA for alanine (Figure 33A). To verify whether the Hsp20S16A cDNA was present in the mouse genome, PCR amplification of genomic DNA, followed by DNA sequencing was performed. This confirmed that the TCA codon, encoding Ser16, was mutated into GCA, which encrypted the alanine residue (Figure 33B). Western blot analysis revealed multiple transgenic lines of Hsp20S16A with overexpression levels of

5-fold and 7-fold, relative to non-TG controls. The 5-fold and 7-fold lines shared similar basal contractile functional phenotypes with no differences from non-TG mice

(Figure 34). Subsequently, the 7-fold overexpression line of Hsp20S16A was used

(Figure 33C). The expression of non-phosphorylatable Hsp20S16A was further confirmed by proteomics.226As shown in Figure 35, only one spot of Hsp20 protein with pI 5.5 was detected by 2-D electrophoresis in non-TG hearts, and two protein spots of Hsp20 (pI 5.5 and 5.2) were observed in Hsp20S16A hearts (Figure 35A).

Mass Spectrometry revealed that the spot with a pI value of 5.2 in TG hearts contained the mutant Hsp20S16A peptide (Figure 35B).

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Figure 33. Generation of Hsp20S16A transgenic mouse models. (A) Diagram of Hsp20S16A TG constructs. The mutant mouse Hsp20 cDNA, in which Serine 16 encoded by codon TCA was mutated into GCA (encoding alanine), was driven by the -myosin heavy chain promoter (-MHCp). (B) DNA sequencing of PCR products from Hsp20S16A mouse genomic DAN confirmed the TCA to GCA mutation. (C) Quantitative immunoblotting analysis showed that in Hsp20S16A TG hearts there was a 7-fold increase in total Hsp20 levels relative to non-TG hearts (NTG).

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Figure 34. Basal functions of non-transgenic and Hsp20S16A hearts. At basal level, (A, B) the values of ± dP/dt, (C) LVDP and (D) EDP in Hsp20S16A TG (7x or 5x) hearts were not significantly different than those of non-TGs (non-TGs: n=10, Hsp20S16A: n=8). NTG: non-transgenic, TG: transgenic.

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Figure 35. 2D and Mass spectrometry results of wild type and Hsp20S16A hearts. (A) 2- D gel electrophoresis identified one non-modified Hsp20 spot (pI=5.5) in WT hearts, while there was an additional S16A-Hsp20 modified spot in Hsp20S16A hearts (pI=5.2). (B) The amino acid sequence of S16A-modified Hsp20 spot was determined by Mass spectrometry.

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Characterization of Hsp20S16A mice showed no alterations in heart weight–body weight and heart weight-tibia length ratios (Figure 36A). Histological analysis revealed that there were no signs of fibrosis, inflammation, or cardiomyocyte hypertrophy in Hsp20S16A hearts compared with non-TG controls (Figure 36B). Of importance, 7-fold overexpression of Hsp20 S16A did not alter the expression of other small heat shock proteins, such as Hsp25 or B-crystallin in the heart (Figure 36C).

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Figure 36. Characterization of Hsp20S16A hearts. (A) Heart weight/body weight (HW/BW) and heart weight/tibia length (HW/TL) ratios were not changed in Hsp20S16A TG mice compared with non-TGs at 12 weeks of age (n=10). (B) Ventricular sections from 12 week- old Hsp20S16A mice stained with HE and Masson’s trichrome (green) indicated no inflammation or cardiac fibrosis. Wheat germ agglutinin staining of Hsp20S16A hearts showed similar myocyte size with non-TGs (n=3, P>0.05). (C) Hsp25 and B-crystallin ( BC) were not altered in Hsp20S16A hearts compared with non-TGs (n=6). CSQ: calsequestrin, loading control.

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Section II. Hsp20 and its phosphorylation at Ser16 protects against cardiac ischemia/reperfusion injury

II. 1. Background and rationale

Myocardial ischemia-reperfusion (I/R) injury is one of the primary outcomes of coronary artery disease, which is the leading cause of death in the industrialized world.177. Recent studies have shown that ischemia, followed by reperfusion, impairs cellular function and signal transduction, while it triggers the cascade of necrosis, apoptosis and macroautophagy (commonly referred to as autophagy).177 It is well accepted that I/R-induced cardiomyocyte necrosis256 and apoptosis188, 257, 258 contribute to ventricular dysfunction and end-stage failure, while there is an increasing awareness that necrosis and apoptosis are not the only mechanisms for cell death. Autophagy, which involves the bulk degradation of cytoplasmic contents, may provide an alternative mechanism that determines cell fate in I/R.208, 209, 212, 213, 259

However, whether I/R-induced autophagy serves a protective or detrimental role to cell damage remains to be clarified.

Heat shock proteins (Hsps) are a family of chaperone proteins that can be constitutively expressed or induced by a variety of environmental stress conditions including ischemia/reperfusion. 260, 261Accumulating evidence has implicated Hsps as mediators of myocardial protection, particularly in experimental models of ischemia and reperfusion injury.35 260, 261The cardioprotective effects of Hsp70 have been shown in isolated adult feline cardiomyocytes and rabbit hearts after adenovirus-mediated gene transfer, and transgenic (TG) mouse hearts after global or regional ischemia.215,

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216, 262 Recently, members of the small heat shock proteins (sHsp) family, such as

Hsp27, B-crystallin and Hsp20, have also been identified as protective during myocardial ischemia.74, 220, 225, 263 The more recently identified Hsp20 is highly and constitutively expressed in muscle tissues, and shares considerable homology with

Hsp27 and B-crystallin.78 Importantly, Hsp20 is the only small heat shock protein that has the consensus motif (RRAS) for protein kinase A (PKA)/protein kinase G

(PKG)-dependent phosphorylation at Ser16.78 Recent studies indicate that Hsp20 and its phosphorylation have emerged to be functionally significant in the heart.81, 155, 225

We and others have demonstrated that the levels of cardiac Hsp20 and its phosphorylation are increased under the physiological stress of exercise training, the pharmacological treatment of doxorubicin as well as the pathological setting of chronic -adrenergic stimulation and tachycardia-induced heart failure. 221-223

Specifically, overexpression of Hsp20 protected the heart against ischemia/reperfusion

(I/R)-induced injury, -agonist-mediated cardiac apoptosis and remodeling.225, 264

More recently, we have identified a P20L substitution in human Hsp20, which was associated with diminished phosphorylation at Ser16 and complete abrogation of the

Hsp20 cardioprotective effects.224 Therefore, the phosphorylation of Hsp20 at Ser16 may be instrumental to its anti-apoptotic properties.

To further assess the functional significance of Ser16 phosphorylation of

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Hsp20 phosphorylation would compromise its role in I/R-induced cardiac injury, and whether this functional alteration is associated with cardiac autophagy. Our findings indicate that increased Hsp20S16A expression in the heart failed to protect against ex vivo and in vivo I/R injury, accompanied by impaired autophagy and increased apoptosis. Accordingly, pre-treatment of Hsp20S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery, compared with saline-treated Hsp20S16A hearts. Thus, Hsp20 and its Ser16 phosphorylation may be involved in the regulation of I/R-induced cardiac autophagy and cell death.

II. 2. Impaired functional recovery in Hsp20S16Ahearts during ex vivo ischemia/reperfusion injury

As shown in Figure 32, the levels of phosphorylated Hsp20 were increased in the heart upon ischemia/reperfusion injury. However, it is unclear whether this phosphorylation is essential to the cardioprotective effects of Hsp20 against ischemic stress. Thus, we subjected the Hsp20S16A hearts to ex vivo 45 minutes of no-flow global ischemia, followed by 2h of reperfusion. Non-TG hearts were used as controls.

There were no differences in ±dP/dt, left ventricular developed pressure (LVDP) and end diastolic pressure (EDP) between the two groups under basal conditions (Figure

37A-D). However, under ischemia/reperfusion, functional recovery of Hsp20S16A hearts was significantly depressed, as determined by the parameters of +dP/dt

(43.1±4.7% vs. non-TG control: 81.6±5.0%; Figure 37A), -dP/dt (42.8± 4.4% vs.

78.2± 3.1%; Figure 37B) and LVDP (53.7± 4.3% vs. 75.5± 2.2%; Figure 37D)

(P<0.01). In addition, EDP was significantly greater in Hsp20S16A hearts after global,

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no-flow ischemia/reperfusion, compared with non-TG controls (Figure 37C, P<0.01).

Taken together, these data suggest that blockade of Hsp20 phosphorylation in

Hsp20S16A hearts is associated with impaired functional recovery upon ischemia/reperfusion.

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Figure 37. Overexpression of Hsp20 S16A increased susceptibility to ex vivo ischemia/reperfusion injury.

During reperfusion, (A, B) recovery of ±dP/dt was significantly lower in Hsp20S16A hearts S16A compared with non-TGs,(C) LVDP recovery was lower in Hsp20 TG hearts compared S16A with non-TGs, (D) the increase of EDP in Hsp20 hearts was higher than non-TGs, (non- S16A TGs: n=10, Hsp20S16A: n=8; * : P<0.01, Hsp20 vs. non-TG).

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II.3. Increased necrosis and apoptosis in Hsp20S16A hearts upon ex vivo ischemia/reperfusion

It is recognized that maintaining an adequate numbers of myocyte is critical to the overall preservation of structural integrity and cardiac function following ischemia/reperfusion.177 Strategies to maximize post-ischemic salvage may involve preventing two forms of cell death, necrosis (an irreversible cell rupture)177, 265 and apoptosis (a reversible gene-directed, energy-dependent cell suicide). 177, 258 To delineate the detrimental effects conferred by Hsp20S16A in post-ischemic cellular damage, the extent of necrotic and apoptotic cell death was examined after ex vivo ischemia/reperfusion. Under basal conditions, LDH release, a biochemical marker of necrotic cell death, did not differ between the Hsp20S16A and control hearts. However, upon ischemia/reperfusion, LDH release was significantly increased by 2-fold in

Hsp20S16A hearts compared to non-TG hearts (Figure 38A, P<0.05). These results indicate that overexpression of Hsp20S16A promotes ischemia/reperfusion-initiated cellular disruption in the myocardium.

Furthermore, we examined whether the functional deterioration of the

Hsp20S16A TG hearts was related to increased apoptosis. Heart lysates from a subset of experimental animals were assayed for DNA fragmentation by a quantitative nucleosome assay. Hsp20S16A hearts exhibited a 1.5-fold increase over non-TGs

(61.1±7.5% versus 39.8±4.8%; n=6; P<0.05, Figure 38B). In addition, TUNEL- positive nuclei reached 3.97±0.6% in non-TGs versus 7.95±1.68% in Hsp20S16A hearts (P<0.01, Figure 38C), and the levels of active caspase-3 were significantly elevated in Hsp20S16A TG hearts relative to non-TGs (Figure 38D; P<0.01, n=6).

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Therefore, all three assays (DNA fragmentation, TUNEL assay and caspase-3 activity) demonstrate significant increases in apoptosis in Hsp20S16A hearts.

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Figure 38. Hsp20S16A overexpression increased IR-induced necrosis and apoptosis. Hsp20S16A hearts subjected to 45 minutes of no-flow ischemia followed by 2 hours of reperfusion exhibited significantly increased total LDH release (A), DNA fragmentation (B), TUNEL-positive cells (C) and caspase 3 activity (D). (non-TG: n=6, Hsp20S16A: n=6, Hsp20WT: n=8, *: P <0.01 vs. non-TG; #: P<0.01 Post vs. Pre) (E) Representative TUNEL stainings of NTG and Hsp20S16A IR hearts (x 400). Arrow: TUNEL positive nuclei. Blue- DAPI indicates nuclear counterstaining; green indicates TUNEL staining and red indicates actin staining of myocytes by Alexa 594 (Invitrogen). 157

II.4. Increased apoptosis and infarct size in Hsp20S16A hearts after in vivo ischemia/reperfusion injury.

The LAD is the major vessel that supplies blood to the left ventricle. If the

LAD is occluded, it results in infarction of the anterior wall of the left ventricle and the

anterior portion of the interventricular septum.266 Occlusion of the LAD in small

rodents has proved to be a well-established model of acute myocardial infarction.231, 267

To examine whether overexpression of Hsp20S16A may be detrimental in

ischemic/reperfused hearts in vivo, the Hsp20S16A and non-TG animals were subjected

to 30 min myocardial ischemia, via LAD occlusion, followed by 24 h reperfusion. We

found that apoptotic TUNEL-positive nuclei (with condensed chromatin) were

increased in Hsp20S16A hearts after 24h reperfusion compared with non-TGs (Figure 39).

Furthermore, infarct size was measured after 24h reperfusion. The area at risk

(AAR), determined by negative staining after reperfusion with phthalo blue dye and

expressed as percent of LV, was not significantly different between Hsp20S16A and non-

TG hearts (Hsp20S16A, 57.8±1.1%; non-TG, 59.9±2.2%; Figure 40B, n=6), indicating

that a comparable degree of ischemic jeopardy existed between these two groups after

occlusion of the LAD. The infarct size in Hsp20S16A hearts was increased by 2-fold

compared to that in non-TGs (Figure 40C, Hsp20S16A: 53.5±0.8%; non-TG: 22.0±3.0%,

n=5, *P<0.01). Consistent with the ex vivo finding, overexpression of Hsp20S16A

aggravates in vivo ischemia/reperfusion injury.

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S16A Figure 39. Evaluation of apoptosis in non-transgenic and Hsp20 transgenic myocardium subjected to in vivo 30min ischemia, followed by 24h reperfusion. (A-F)

Representative images of TUNEL-positive nuclei according to their apoptosis- and necrosis- like morphology. Arrows indicate TUNEL-positive nuclei with either condensed chromatin

(apoptosis, A-C, arrows) or diffused chromatin distribution (necrosis, D-F, arrow-head). Blue-

DAPI indicates nuclear counterstaining; green indicates TUNEL staining and red indicates actin staining of myocytes by Alexa 594 (Invitrogen). (G) The number of TUNEL-positive nuclei was evaluated in 10 fields for apoptosis-like appearance. Plotted were meanSEM for

3 animals per group. *:P<0.01.

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Figure 40. Infarct areas were increased in Hsp20S16A TG hearts after prolonged ischemia/reperfusion injury. (A) In vivo infarction was induced by 30 minutes of ischemia via occlusion of the left anterior descendant artery, followed by 24 hours of reperfusion. The entire heart was sliced transversely into 5-6 sections (~2 mm thick). The area from each region was averaged from the photographs of each side for each slice and multiplied by the weight of that tissue section. Infarct size was expressed as the percentage of the infarct area (unstained by 1%TTC) relative to the area at risk (unstained by 5% phthalocyanine blue), or to total LV mass. The images were magnified by 10-fold. (B) The area at risk (AAR) was not significantly different among groups. (C) Infarct size, which was determined as a percentage of the region at risk, in Hsp20S16A TG hearts was 2-fold larger than non-TGs (Hsp20S16A: 53.5±0.8%, n=5; non-TG: 22.0±3.0%, n=5, *P<0.01). The area at risk / LV mass was unchanged among groups (P=NS): Hsp20 S16A, 57.8±1.1%; non-TG, 59.9±2.2%). Solid dot: data from each NTG heart; open dot: data from each Hsp20S16A heart. Mean±SEM was also plotted (red for NTG, blue for Hsp20S16A TG hearts.

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II.5. Inactivation of autophagy in Hsp20S16A hearts upon ex vivo ischemia/reperfusion

Autophagy is a physiological mechanism to remove damaged organelles259

Since recent studies have shown that autophagy plays an important role in ischemia/reperfusion injury,209 we assessed the autophagy activity in Hsp20S16A hearts by measurement of the microtubuleassociated protein light chain 3 (LC3)-II/LC3-I ratio and Beclin 1 protein levels268 in comparison with non-TG hearts. Conversion of cytosolic LC3-I to membrane-conjugated LC3-II is correlated with the number of autophagosomes, indicative of autophagic activity.233, 268 Beclin 1, an autophagy related protein, is a critical player in the formation of autophagosomes.235 Western blotting analysis showed that the ratio of LC3-II/LC3-I was increased in non-TG hearts after ischemia/reperfusion (Figure 41A and B), suggesting that ischemia/reperfusion induced autophagy activity. Interestingly, the ratio of LC3-

II/LC3-I was elevated in Hsp20S16A hearts under basal conditions, but was significantly decreased following ischemia/reperfusion (Figure 41A and B).

Alterations of another autophagy-related protein, Beclin1 (Figure 41A and C) were parallel to the changes of the LC3-II/LC3-1 ratio in Hsp20S16A and non-TG hearts.

Taken together, these data indicate that suppression of ischemia/reperfusion-induced autophagy may contribute to the depressed cardiac functional recovery in Hsp20S16A hearts.

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Figure 41. Autophagy is enhanced in Hsp20WT hearts and attenuated in Hsp20S16A hearts S16A in response to ischemia/reperfusion injury. Cardiac homogenates from Hsp20 and non- TG mice were subjected to immunoblot analysis for detection of LC3-II/LC3-I and Beclin1 (A). Quantitative analysis results of LC3-II/-LC3-I ratio and LC3-II (B) and Beclin 1 level (C) are from 3 different preparations (six hearts for each group,* : P<0.05, Post- vs. Pre-ischemic group). CSQ: calsequestrin, loading control.

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II.6. Pretreatment of Hsp20S16A hearts with rapamycin improved functional recovery in response to ischemia/reperfusion

To further examine whether activation of autophagy in Hsp20S16A TG hearts would rescue its post-ischemic function, we administrated Hsp20S16A mice with rapamycin which induces autophagy by inhibiting the mammalian target of rapamycin

(mTOR).269 As shown in Figure 42, treatment with rapamycin significantly increased the LC3-II level and LC3-II/LC3-I ratio in both non-TG and Hsp20S16A hearts.

Consequently, functional recovery was greatly improved in Hsp20S16A hearts following ischemia/reperfusion, compared with saline-treated controls (Figure 43).

These findings suggest that enhancement of autophagy in post-ischemia/reperfusion

Hsp20S16A hearts may ameliorate tissue injury.

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Figure 42. Pretreatment with autophagy inducer rapamycin in Hsp20S16A hearts led to increased level of autophagy. LC3-II/LC3-1 ratio and LC3-II level in Hsp20S16A hearts following ischemia/reperfusion was significantly higher than saline-treated post I/R controls. (*: P<0.01, rapamycin vs. saline group, n=6).

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Figure 43. Pretreatment with autophagy inducer rapamycin in Hsp20S16A hearts led to increased functional recovery. LVDP and EDP recovery in S16A Hsp20 hearts following ischemia/reperfusion was significantly improved compared to saline-treated post I/R controls. (*: P<0.01, rapamycin vs. saline group, n=6).

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II.7. Effects of mutant S16A on Hsp20 oligomerization patterns following ischemia/reperfusion

The phosphorylation status of Hsp20 has been suggested to have a role in its structural organization, manifesting in different aggregation patterns of the protein, which might be associated with its function.94, 270 As a result, we examined the oligomerization pattern of Hsp20 in non-transgenic and Hsp20S16A transgenic hearts before and after global I/R by sucrose gradient ultracentrifugation. By this method, non-TG hearts displayed Hsp20 oligomers up to 150kD before I/R, and slightly right shifted after I/R. In contrast, at basal levels, Hsp20S16A hearts displayed Hsp20 aggregate patterns composed primarily of oligomers between 150kD to 250kD. After

I/R, Hsp20S16A hearts displayed Hsp20 oligomers that were shifted to a larger complex profile (>250kD, Figure 44), suggesting that phosphorylation of Ser16 may alter the ability of Hsp20 to aggregate following ischemia/reperfusion.

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Figure 44. Sucrose-gradient centrifugation analysis of Hsp20 proteins. Cardiac lysates (100g total protein) from Hsp20S16A and non-TG hearts before and after ischemia/reperfusion insults were layered on the top of 5%-40% sucrose gradients. After centrifugation, as described in Methods, fractions of the gradients (labeled 1-18 from top to bottom) were collected, and resolved by electrophoresis. A blot of the gel was probed with antiserum against Hsp20. Relative protein levels in each fraction were calculated by densitometric scans of each immunoreactive band/total Hsp20. Mutant Hsp20S16A promoted a shift of Hsp20 oligomers to larger complexes (pre-ischemia) and even larger complexes post-ischemia. Arrows indicate molecular weight standards: alcohol dehydrogenase (ADH) (150 kDa), -amylase (250 kDa), and thyroglobulin (690 kDa). Other abbreviations are as defined in the text.

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Section III: Generation of Hsp20 knock-out mice

III.1. Background and rationale

It is well-known that Hsps protect cells against deleterious effects evoked by various stressful inducers.4 The discovery and subsequent characterization of cytoprotective Hsps have been best exploited in transgenic models, with tissue specific expression of Hsps, such as inducible Hsp70,215 217, 271 B-crystallin,77, 272, 273

Hsp2730, 218, 274, and Hsp20223, 225, 264. Transgenic expression of these proteins has been used to test the hypothesis that high levels of Hsps would diminish alterations caused by cellular stress. Furthermore, these transgenic models are useful experimental tools to provide essential data in intact animals, beyond the scope provided by cultured cells.

Of interest, the Hsp70 transgenic mouse model is among the most extensively

215, 217, 271 exploited by the scientific community. The ubiquitous expression of this transgene has enabled investigators to test protection of heart, skeletal muscle, brain and embryo against ischemia, toxic exposure and genetic disease.215, 275-279 However, according to the type of transgenic lines, some discrepancy exists regarding the protective effects by Hsp70 overexpression. For example, in the same R6/2

Huntington’s model of neurodegeneration, the chicken actin-promoter driven rat

Hsp70i transgene provided some improvement280 while the human actin promoter-

281, 282 driven human Hsp70 transgene had only a modest effect on disease progression.

These conflicting results may be due to the different levels of Hsp70 expression or its co-factors in these transgenic lines. 280, 281, 282

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To better understand the in vivo role of Hsps, several knockout mouse models of

Hsps have been generated, including the Hsp70 knockout mice, 154 and the double knockout (DKO) of BC and HspB2. 274, 283, 284 Studies of the Hsp70 null model revealed that the absence of Hsp70 leads to a cardiac phenotype, characterized by impaired contractile function and altered Ca2+ handling associated with mild hypertrophy.154 In addition, deletion of Hsp70 gene leads to impaired stress response of the Hsp70 KO hearts against ischemia/reperfusion,154 which was consistent with previous observations that overexpression of Hsp70 confers cardioprotection against myocardial ischemia.216, 285 Moreover, Morrison and coworkers74 used an ex vivo model of ischemia/reperfusion and demonstrated that combined deletion of BC and

HspB2 resulted in reduced recovery of contractile performance, increased apoptosis and necrosis, compared with WT hearts. These investigators found that the double- knockout (DKO) hearts had lower levels of reduced glutathione which could contribute to enhanced I/R-induced injury. Furthermore, Kadono et al.283 reported that

BC and HspB2 deficiency increased myocyte mitochondrial permeability transition and mitochondrial Ca2+ uptake. These findings indicate an important role of BC and/or HspB2 in mitochondrial function.283

In our lab, we have successfully generated Hsp20 and Hsp20S16A transgenic mouse models to exemplify a gain-of-function study. However, a knockout strategy could provide additional valuable insights about the function of the Hsp20 gene, and this is addressed below. Furthermore, additional refinements in the experimental strategies, such as conditional knockouts and knock-ins, will undoubtedly further contribute to our precise understanding of the role of Hsp20 in stress responses.

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III.2. Generation of the Hsp20 knockout targeting Vector

Our above findings, together with the phenotype of Hsp20-overexpressing mice and data collected from adult rat cardiomyocytes infected with an Hsp20- overexpressing virus, suggest that Hsp20 may be an important regulator of cardiac contractility and protector against apoptosis induced by a beta-agonist and ischemia/reperfusion injury.81, 225 To further elucidate the functional role of Hsp20 in vivo, the Hsp20 gene locus was selected for targeted ablation to further test our hypothesis in the context of an intact animal. Importantly, the levels of Hsp20 are increased in human heart failure, as well as in some animal models of ischemia.221, 225

Our transgenic studies above indicated that increased expression of Hsp20 is associated with cardioprotection, Thus, by ablating or reducing Hsp20 expression, we would expect diminished cardioprotection in support of our hypothesis, or we would be able to address the consequences of reduced Hsp20 levels in vivo.

To ablate Hsp20 gene expression, a targeting vector was designed to replace the entire 1.3 kb Hsp20 coding region with a short length of exogenous DNA. The targeting vector was constructed to possess a total of ~5.3 kb of homologous DNA

(Figure 8); this length was selected, since other gene-targeted mice have been successfully generated in our laboratory, using constructs of this approximate homology length286. The specific lengths and locations of the homology arms were selected by examining the genomic DNA surrounding the Hsp20 locus; repetitive elements and G-C rich regions were excluded to facilitate vector construction and increase its likelihood of successfully recombining at the correct locus. To generate the targeting construct, the 2.05 kb short homology arm was first amplified by PCR

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and cloned into the TOPO-2.1 vector by Topoisomerase-catalyzed ligation as described in Materials and Methods. To confirm insertion of the short homology arm into the TOPO vector, isolated plasmids were digested with NotI and SalI to release the cloned fragment. As shown in Figure 45A, the 2.05 kb fragment was successfully released from 4 of 12 clones examined. Short homology arm fragments were gel purified from clones 2, 4, 6 and 9, and each of these were ligated separately into the

NTKV-1902 vector, as described in Materials and Methods. The full-length 2.05 kb short homology arm was successfully incorporated into the NTKV-1902 vector, and could be released by NotI and SalI digestion. The entire length of the short homology arm from 4 clones was sequenced and compared to the published C57Bl/6 genomic sequence of mouse chromosome 7. An adenine to thymidine substitution at bp position 57 was identified (Figure 46).

The 3.25 kb long homology arm was also amplified by PCR and immediately cloned into the TOPO-2.1 vector. It was released from the TOPO-2.1 vector by

HindIII and XhoI digestion, as shown in Figure 45B. The long homology arm fragments from clones 2 and 7 were gel purified and ligated separately into the NTKV vector already containing the short homology arm. The long arm could be released from clone 2 and the entire length of the long homology arm was also sequenced. And two differences from the genomic sequence were found. Both were thymine to adenine substitutions, at bp positions 258 and 686 (Figure 47). Since the rate of homologous recombination in ES cells is not significantly affected by a mismatch rate of less than 1 per kb of targeting vector length, the final ligated construct was submitted to Gene Targeting Core.

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Figure 45. Generation of Hsp20 targeting construct. (A) The 2.05 kb short homology arm was amplified from a BAC clone containing the genomic sequence of mouse Chromosome 7, flanking the Hsp20 gene, and cloned by Topoisomerase- catalyzed ligation into the TOPO 2.1 vector. This fragment was successfully released by restriction enzyme digestion in 4 of 12 clones screened. (B) The 3.25 kb long homology arm was also amplified from the same BAC clone, and cloned into the TOPO 2.1 vector. This fragment was released by HindIII and XhoI restriction enzyme digestion from 2 of 9 clones screened. The bands from clones 2 and 7 were individually cloned into the NTKV vector already containing the sequenced short homology arm. The upper band from clone 2 is TOPO plus long arm. The upper band from clone 7 is TOPO vector only.

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Figure 46. Sequencing of short arm construct. Only one a:T mutation was found in the short arm construct at bp position 57 (arrow). The mismatch rate is about 0.04%. Underlying nucleotide peptide is the primer (5’SANOT1) for DNA sequencing.

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Figure 47. Sequencing of long arm construct. Two a:T mutations were found in long arm construct at bp positions 258 and 686 (arrows). The mismatch rate is about 0.09%. The underlined nucleotide peptide is the primer sequence (5’SANOT1) for DNA sequencing

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III.3. Screening of the targeted embryonic stem (ES) cells

PCR screening of the Neo gene in each ES clone was positive. However, the correct length of PCR bands for the short arm and the long arm were not obtained. At this point, it was unclear whether the conditions of the PCR reactions were inadequate for amplifying a band of this size, or whether none of the cells were correctly targeted.

To circumvent these difficulties, Southern blotting was employed. All 278 ES cell clones were subjected to Southern blotting to ensure that previous PCR results were accurate and that no false negatives had been obtained. The strategy for differentiating targeted from untargeted cells by Southern blots was to take advantage of the fact that successful homologous recombination would ablate an endogenous and introduce an exogenous EcoR1 restriction enzyme site (Figure 8). When digested with EcoR1, the untargeted Hsp20 locus would migrate with a molecular weight of ~8.6 kb, while the targeted locus would migrate with a molecular weight of ~5.8 kb. To perform

Southern blots, 12 Southern blot probes were generated and tested for their sensitivity and specificity on tail genomic DNA from wild type mouse. Probe III proved to give the strongest specific signal, with the expected band size of 8.6 kb on test blots

(Figure 13). This probe was used for all subsequent experiments. Of the 278 clones tested, none of them gave a positive signal for possessing the correctly targeted Hsp20 gene locus at the expected molecular weight of 5.3 kb (Figure 48). It is possible that knock-out of Hsp20 gene may cause ES cell lethality, which in the future, a conditional knockout mouse model may be generated to circumvent this problem.

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Figure 48. Representative Southern blot results of ES cells for homologous recombination. After overnight-digestion, samples of genomic DNA from ES cell were probed for screening the correct homologous recombination by Southern blot. Of the 278 clones screened, none of them gave a positive 5.6kb band. Shown are Southern blots of ES cell samples K1-8.

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Section IV. Generation of Hsp20S16D transgenic mice

IV.1. Background and rationale

Several posttranslational modifications of sHsps have been detected, including phosphorylation, 287 deamidation, acylation as well as mixed intermolecular disulfide formation, oxidation and glycation.288 Phosphorylation of serine residues is a common feature of small Hsps (Table 8). For instance, Hsp27,289 B-crystallin290-293 and Hsp20

87-89 are known to be phosphorylated at serine residues in response to various stresses.

Functionally, phosphorylation of sHsps has been reported to regulate its actin-binding capacity,294 signaling to cytoskeleton294, and regulation of cell mobility.295, 296

Table 8. Phosphorylation sites of sHsp and identification of protein kinases

Phosphorylation site sHsp Kinases References in sequence 36, 297 Mouse Hsp25 LLRSPS15WEP MK 2/3, MK5/PRAK?

LNRQLSS86GV PKC- ? 37, 297 Human Hsp27 LLRGPS15WDP MK 2/3, MK5/PRAK?

YSRALS78RQL

LSRQLS82SGV

91 Bovine Hsp20 WLRRAS16APL PKG, PKA

155 Mouse Hsp20 WLRRAS16APL PKG, PKA

292 Bovine AC RYRLPS122NVD PKA?

Bovine BC 78 FFPFHSPPS19R ?

TSTLS45PFY P42/44 MAPK/ERK1/2

FLRAPS34WFD MK2/3, MK5/PRAK?

Amino acid residues which are part of the conserved kinases recognition consensus motif are underlined. AC: alpha

A-crystallin; BC: alpha B-crystallin.

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Within the sHsps family, Hsp20 is the only sHsps that contains the conserved

PKA/PKG phosphorylation site.155 In smooth muscle, phosphorylation of Hsp20 regulates muscle relaxation.106,298, 89 It has been reported that Hsp20 phosphorylation is increased during vascular smooth muscle relaxation induced by nitroglycerin,298, 89 and cAMP/cGMP-dependent inhibition of contraction of smooth muscle correlates with increased phosphorylation of Hsp20.106 In the heart, isoproterenol stimulation increases total and phosphorylated Hsp20 levels.155 Hsp20 phosphorylation is also influenced by ischemia and under conditions of ischemic preconditioning.225

Furthermore, our studies showed that blockade of Ser16 phosphorylation impaired the protective effects of Hsp20 against ischemia/reperfusion injury. However, the effects of constitutively phosphorylated Hsp20 in cardiac function and protection are not currently clear. To answer this question, we generated the Hsp20S16D transgenic mouse model, in which Ser16 is replaced with aspartic acid (D), to mimic a constitutively phosphorylated Hsp20.

IV.2. Generation of Hsp20S16D transgenic mice

Four lines of HspS16D transgenic mice were generated. The overexpression levels of total Hsp20 were revealed by Western blot analysis, as shown in Figure 50A.

There are two lines with low-level of overexpression: L1 (1.2x), L2 (1.7x), one line with medium-level overexpression: M (4.4x), and one line with high-level overexpression: H (12.7x) compared to wild type. The medium (line M: 4.4x) and high (line H: 12.7x) overexpression lines were chosen for further studies (Figure 49).

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Figure 49. Generation of Hsp20S16D TG mouse model. (A) Western blotting results of total Hsp20 level in Hsp20S16D TG (S16D) and wild type (WT) hearts. Four transgenic lines were obtained. CSQ: calsequestrin, loading control. (B) Quantitative data of Western blotting. The total Hsp20 levels in TG hearts were 1.2x (L1), 1.7x (L2). 4.4x (M) and 12.7x (H) fold compared with WT. The medium (line M) and high (line H) overexpressing lines were used for following studies (n=6).

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To confirm the expression of constitutively phosphorylatable Hsp20S16D in transgenic hearts, cardiac proteins were subjected to 2-D gel electrophoresis.226 Three wild type and three Hsp20S16D hearts (12x overexpression) were used in this study, and all samples were analyzed in triplicate. Ventricular proteins from wild type and

Hsp20 S16D mice were solubilized, processed in parallel, and loaded onto pH 4–7 immobilized pH gradient (IPG) strips. Vertical second-dimension SDS-PAGE (12% gels) was performed immediately following isoelectric focusing (IEF). As shown in

Figure 50A, two Hsp20 spots appeared in 2-D gels. The non-modified wild type

Hsp20 appeared in spot 1 and the mutant Hsp20S16D in spot 2. The single mutation at

Ser16 into aspartic acid (D) was confirmed by mass spectroscopy (Figure 50B). The non-modified AS16AP sequence was replaced by AD16AP.

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Figure 50. Confirmation of Hsp20S16D expression in transgenic hearts. (A) 150 μg of cardiac homogenates were isoelectrically focused (IEF) on 18-cm narrow range strips (pH 4-

7) and immediately subjected to vertical SDS-PAGE, as described under "Methods." Analysis of the two-dimensional fluorescent-stained gel images of Hsp20 showed one additional spot

(2) compared with the non-modified form of Hsp20 (spot 1). Each heart was subjected to two-dimensional gel electrophoresis in triplicate. (B) Mass spectrum analysis of protein peptide from spot 2. The modified AD16AP peptide sequence replaced the non-modified

AS16AP sequence.

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To study whether overexpression of mutant S16D in the heart may lead to hypertrophy by 10-12 weeks of age, we assessed heart weight, body weight, and lung weight of the mice with medium (S16D-M: 4.4x) or high-level (S16D-H: 12.7x) overexpression of Hsp20S16D. We did not observe any changes in these indices. As a consequence, the heart weight–body weight and lung weight-body weight ratios

(Figure 51A-B) were not different compared with those of wild type sibling littermates, suggesting that overexpression of Hsp20S16D did not result in hypertrophy or lung congestion. To explore whether overexpression of mutant Hsp20 triggered compensatory increases of other small heat shock proteins, Western Blotting of -BC and Hsp25, which share the crystallin-domain with Hsp20, was performed. The protein expression level of these two proteins was not changed compared with WT

(P>0.05, n=6, Figure 51C).

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Figure 51. Characterization of Hsp20S16D TG mouse model. Comparing line M (4.4x) and line H (12.7x) to WT, there is no difference in heat weight-body weight (HW/BW) ratio (A) and lung weight-body weight (LW/BW) ratio (B) of Hsp20S16DTG.(n=6) (C) Expression level of two other sHsps detected by Western Blotting, -BC and Hsp25, was not changed.(n=6)

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IV.3. Overexpression of Hsp20S16D reduced myocyte contractility

To evaluate the effects of Hsp20S16D overexpression on cardiac contractile function, mechanical parameters were assessed in isolated cardiomyocytes, which represent a load-independent preparation. At a stimulation frequency of 0.5Hz, overexpression of 4.4x (S16D-M) and 12.7x (S16D-H) Hsp20S16D resulted in significant decreases in +dL/dt (S16D-M: 71.76.0 m/s; S16D-H: 53.05.3m/s vs.

WT: 89.13.7m/s; P<0.01), -dL/dt (S16D-M: 54.74.6m/s; S16D-H:

37.53.5m/s vs. WT: 67.64.0m/s; P<0.01), and FS (S16D-M: 6.0 0.3%;

S16D-H: 5.0 0.3% vs. WT:7.3 0.3%; P<0.01 ) compared to wild types (Figure 52).

To further explore the response of Hsp20S16D myocytes to beta-adrenergic stimulation, contractile function was measured in the presence of 0.1M isoproterenol.

After isoproterenol stimulation of wild type cardiomyocytes, +dL/dt, -dL/dt and FS were increased to 187.87.8m/s, 153.96.8m/s and 12.40.4%, respectively.

However, in cardiomyocytes with 12-fold (S16D-H) overexpression of Hsp20S16D,

+dL/dt, -dL/dt and FS were significantly decreased relative to wild type (+dL/dt:

1258.0m/s, -dL/dt: 81.27.7m/s, FS: 9.68 0.6%, P<0.01). In cardiomyocytes with 4x (S16D-M) overexpression of Hsp20S16D, only +dL/dt (136.516.5m/s) was significantly decreased (P<0.01), although -dL/dt and FS of transgenic myocytes also showed a tendency of decrease, (no statistical difference) compared with wild types

(Figure 53).

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These data indicated that overexpression of Hsp20S16D decreases basal cardiac

contractility at the myocyte level, and Hsp20S16D myocytes had blunted isoproterenol

responses.

Figure 52. Basal contractility of isolated myocytes from WT and Hsp20S16D (line #M and #H) TG hearts. (A) Representative traces of basal cardiomyocyte mechanics. (B) Decreased myocyte percent fractional shortening (FS) and maximal rates of contraction and relaxation (f dL/dt) were observed in myocytes from 2 lines of Hsp20S16D myocytes (S16D-M: medium level, 4.4X Overexpression; S16D-H: high level, 12x overexpression) compared with WT cells under baseline. Values = Mean ± S.E.M. * P<0.01, TG v.s. WT. n=45 to 60 cells from 4 hearts for each group.

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S16D Figure 53. Mechanics of isolated wild type and Hsp20 TG cardiomyocytes in responses to isoproterenol. Isolated myocytes from 12-14 weeks old hearts were suspended in 1.8mM Ca-Tyrode solution and field-stimulated at 0.5Hz. (A) Representative cell shortening traces of wild type and TG cells; (B) myocytes from two TG lines (S16D-M: medium level, 4.4X Overexpression; S16D-H: high level, 12x overexpression) showed blunted fractional shortening (FS), maximum rates of contraction (+dL/dt) and maximum rates of relaxation (-dL/dt) in the presence of 0.1M isoproterenol (Iso). Isoproterenol response was determined in n=20-30 cells from total 4 wild type hearts and 4 TG hearts. Values = Mean ± S.E.M. *:P<0.01 TG vs WT in the presence of isoproterenol.

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IV.4. Overexpression of Hsp20S16D depressed ex vivo and in vivo cardiac function

Further studies were carried out with the high overexpression (12x) line of

Hsp20S16D TG hearts. Ex vivo cardiac function of these hearts was assessed by

Langendorff perfusion. As shown in Figure 54, there is no significant difference in

EDP between wild type and Hsp20S16D TG hearts. LVDP, +dP/dt and –dP/dt were significantly lower in the hearts with 12x overexpression of Hsp20S16D hearts, compared with wild type. These data suggested that 12x overexpression of constitutively phosphorylated Hsp20 decreased ex vivo cardiac function.

LV function was also assessed noninvasively by using M-mode echocardiography on anesthetized transgenic (S16D-H: 12x) and age-matched wild type mice. Among all parameters, LV fractional shortening (FS) and ejection fraction

(EF) are the most commonly used ejection phase indices to measure LV systolic performance 299, 300. Consistent with ex vivo data, both EF% and FS% of Hsp20S16D hearts were decreased by about 15% and 26%, respectively, compared with wild types

(Figure 55), suggesting that the in vivo function of Hsp20S16D hearts (S16D-H: 12x) was also significantly depressed. In addition, end-systolic LV internal diameter

(LVIDs) of Hsp20S16D hearts increased by 38% compared with wild types, suggesting that LV chamber size, of TG hearts was increased at the end of contraction (Figure

55).

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Figure 54. Ex vivo cardiac function of WT and Hsp20 S16D TG mice by Langendorff perfusion. Isovolumic contraction and relaxation of WT and S16D-H (12-fold overexpression of Hsp20S16D) TG hearts was measured under basal conditions. S16D-H hearts showed decreased contractile function (LVDP, +dP/dt) and rate of relaxation (-dP/dt), compared to WT. * P< 0.05, n=6.

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* * *

* Figure 55. In vivo function of Hsp20S16D hearts assessed by echocardiography. (A) Representative M-mode echocardiographic images of the LV of 12-week-old male Hsp20S16D (12x) mice and wild type littermates (n=6 for each group). (B) Left ventricular parameters as assessed by echocardiographic analysis. LVPWs, systolic LV posterior wall thickness; LVIDs, end-systolic LV internal diameter; IVSs, systolic interventricular septal thickness; LVPWd, diastolic LV posterior wall thickness; LVIDd, end-diastolic LV internal diameter; IVSd, diastolic interventricular septal thickness; EF: ejection fraction, FS, fractional shortening. *: P < 0.01, TG vs. WT, n=6.

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IV.5 Overexpression of Hsp20S16D induced interstitial fibrosis in the heart

Since results from echocardiography showed that LVIDs increased, which implied cardiac remodeling in 12x overexpressing Hsp20S16D mice, we further assessed the existence of fibrosis in these transgenic hearts by using Masson’s

Trichrome staining of histological sections. Interstitial cardiac fibrosis is an important feature of cardiac remodeling. 301 It decreases ventricle compliance, impedes oxygen diffusion to cardiomyocytes, and may alter the transmission of force from individual cells to global chamber contraction, all of which hinder the cardiac function.301

Masson’s Trichrome stains the nuclei by blue color, plasma by red, and the febrile collagen by blue. We found that massive green-staining was observed in 12x fold

(Figure 56B) overexpressing Hsp20S16D hearts, and fibrosis occurred as early as 6 weeks of age compared with same age WT hearts (Figure 56A).

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Figure 56. Representative sections of Masson’s Trichrome stained cardiac sections from 12-weeks old transgenic and wild type mice. Interstitial fibrosis stained in blue (arrows) was observed in myocardium from 12x fold (B) overexpressing Hsp20 S16D hearts, but not in WT (A) hearts.

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IV.6. Overexpression of Hsp20S16D maintains myofilament arrangement

Myofibrillar assembly is an important factor in regulating cardiac function. 302

Since we observed depressed function of Hsp20S16D myocytes, it is of our great interest to investigate whether the S16D mutation causes any ultrastructure alterations, such as myofibrillar disarray. We examined the ultrastructure of both wild type and

Hsp20S16D myocytes by transmission electron microscopy (Figure 57). The structure of sarcomere, the fundamental unit of cardiac myocyte, which is made of thin filaments (actin) and thick filaments (myosin), can be clearly visualized by electron microscopy. Surprisingly, we found that sarcomeres, which extend from Z-line to Z- line, were not altered in Hsp20S16D myocytes. In addition, there was no myofilament disarray in wild type or Hsp20S16D cardiac sections, suggesting that overexpression of constitutively phosphorylated Hsp20 do not alter myofibrillar assembly.

Ventricular myocytes were rich in mitochondria, which constituted about 35% of the cell volume and were situated between myofibrils.303 the morphology of mitochondria was observed by electron microscopy in longitudinal sections among specimens of the WT and Hsp20S16D heart (Figure 57). In the WT control group, spherical-formed mitochondria were located abundantly between the myofibrils, with a light matrix and a well-developed pattern of cristae membrane. In Hsp20S16D hearts, although the mitochondria appeared roundish, their membrane integrity was conserved and there was no alteration in matrix.

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Figure 57. Electron microscopy showing the normal arrangement of myofilaments in the wild type and Hsp20S16D cardiomyocytes. The sarcomere is the "fundamental unit" of the cardiac muscle cell, extended from Z line (A) to Z line (A). The thin actin filaments extend from both Z line in towards the center of the cell. The thick myosin filaments are found towards the center of the sarcomere. There is no myofilament dislodgment or disarray in either wild type or Hsp20S16D transgenic cell. Z line (A) corresponds to the Z disk, the dark M band (B) is made up of thick myosin filaments in the middle of a sarcomere. C: mitochondria; Bar scale 500nm, 15000x)

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IV.7. Overexpression of Hsp20S16D stimulates autophagosome accumulation

Macroautophagy (which refers to as `autophagy'), is an intracellular bulk degradation process whereby cytosolic, long-lived proteins and organelles are degraded and recycled.208, 209, 304 It involves the sequestration of cytosol or cytoplasmic organelles within double membranes, thus creating autophagosomes (also called autophagic vacuoles).208, 209, 304 Autophagosomes, the key players in autophagy, subsequently fuse with endosomes and eventually with lysosomes, thereby creating autophagolysosomes or autolysosomes. In the lumen of these latter structures, lysosomal enzymes operating at low pH then catabolize the autophagic material.305, 306

The principal role of autophagy is to supply nutrients for survival.307, 308 In addition, a low level of constitutive autophagy is also important for controlling the quality of proteins and organelles, in order to maintain cell function.309 It has been reported that constitutive autophagy in the heart under baseline conditions is a homeostatic mechanism for maintaining cardiomyocyte size and global cardiac structure and function.310

First, the basal level of autophagy activity Hsp20S16D hearts was examined.

Using transmition electron microscopy, we investigated whether the number of autophagsomes is altered in Hsp20S16D hearts compared to littermate controls (n=3). In cardiomyocytes, the majority of double-membraned autophagsomes is localized close to mitochondria, and some of them contain pieces of degraded mitochondria. We counted total autophagosome number in 10 randomly selected fields (20000x

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magnification), from 3 different tissue sections. We observed that the number of autophagosomes in Hsp20S16D myocytes was significantly larger than wild types

(Figure 58). Interestingly, in Hsp20S16D myocytes, the number of mitochondria seemed also to be increased (Figure 58).

To determine whether this accumulation of autophagosomes was due to an increase in autophagosome formation, or was a secondary consequence of lysosome dysfunction preventing autophagosome clearance,311, 312 we performed immunohisto- stainging for the lysosomal marker, lysosomal-associated membrane protein-1

(LAMP-1) in Hsp20S16D hearts. LAMP1, homologous C-type transmembrane protein

(37% identity in humans) specific for lysosomes, is involved in lysosomal biogenesis, and/or the fusion between autophagosomes and lysosomes required for the final catabolism of autophagic material. 311, 312 We found that the lysosome abundance was increased in Hsp20S16D hearts relative to control at basal level (Figure 59), suggesting the diminished lysosomal function did not contribute to autophagosome accumulation in Hsp20S16D cardiomyocytes.

Our above data suggest that chronic overexpression of Hsp20S16D increases the accumulation of autophagosome, which may lead to an increased autophagic degradation of organelles, such as mitochondria. However, whether this disregulated autophagy contributes to cardiac dysfunction and remodeling in Hsp20S16D hearts is still under investigation. Besides, the role of necrosis or apoptosis in this process is required further studies.

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Figure 58. Electron microscopy of autophagosomes in wild type and Hsp20S16D myocytes. (A) Accumulation of autophagic vacuoles with their characteristic double membranes is visible in Hsp20S16D myocyte. Arrow: autophagsome; M: mitochondria. Bar scale is 500nm, 20000x. (B) Quantitative results of autophagosome from transgenic and wild type myocytes. The number of total autophagosome in Hsp20S16D was significantly higher than wild type (n=10 fields for each group, *: P<0.01). Myocytes were from 3 different hearts for each group.

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Figure 59. Increased abundance of lysosomal markers in Hsp20S16D ventricles. Colocalization of the lysosomal marker LAMP-1 (red) and Hsp20 (green) is detected by immunohistochemistry. Distribution of LAMP-1 was changed from a WT pattern of discrete cytoplasmic punctae to a more diffuse and more intense staining in Hsp20S16D myocardium, indicating an increased lysosomal activity in these hearts.

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IV.8. Survival rate of Hsp20S16D transgenic mice

Both in vivo and in vitro measurements showed that cardiac contractile function was depressed in Hsp20S16D TG mice, and severe interstitial fibrosis was observed in the TG hearts. During the 11-month life span study, the 12x overexpressing Hsp20S16D transgenic mice died sporadically and suddenly, compared with their wild type littermates. About 60% of the TG mice died at 5 months of age, and more than 80% of the mice died at 9 months (Figure 60), which is around the midpoint of the life-span in normal mice. The premature death may be due to cardiac dysfunction, as suggested by echocardiography (Figure 55).

Figure 60. Premature death in Hsp20S16D transgenic mice. Percentage of survival rate was plotted against age (months) for Hsp20S16D transgenic (12x) and wild type (n=20 for both wild type and Hsp20S16D TG mice). None of the wild type mice died during the 11-month observation. However, about 60% Hsp20S16D transgenic mice died as early as 5-month old, and only 20% Hsp20S16D transgenic mice were still alive by 9 months of age. Data were analyzed using logistic regression with exact method, *: P<0.05, n=20.

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Chapter IV

Discussion

Dissertation Summary

The objective of this study was to determine the role of Hsp20 in the regulation of SR Ca cycling and cardiac function. This study revealed that acute upregulation of Hsp20 resulted in enhanced Ca2+ kinetics and contractile parameters.

Consistent with in vitro data, overexpression of Hsp20 in vivo led to significant increases in SR Ca2+ load, SR Ca2+ cycling, and the corresponding enhanced contractile function, assessed in single cardiomyocytes and intact animals.

Furthermore, the enhanced contractile function of Hsp20 transgenic hearts was associated with increased phosphorylation of phospholamban at both Ser16 and Thr17 sites, which was inversely correlated with the protein phosphatase (PP1) activity.

Interestingly, Hsp20 appeared to associate with PP1 as revealed by pull-down assays, indicating a regulatory role of Hsp20 on PP1 activity. Taken together, these results suggest that Hsp20 is a prominent regulator of SR Ca2+ cycling and cardiac function.

Since increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, small heat-shock protein Hsp20 may be a promising therapeutic target for human heart failure treatment.

Another objective of our study is to determine the role of Hsp20 and its phosphorylation in protecting against cardiac ischemia/reperfusion injury. We generated a cardiac-specific overexpression mouse model carrying non-

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phosphorylatable Hsp20, where serine 16 was substituted with alanine (Hsp20S16A).

By subjecting this model to ischemia/reperfusion, we addressed whether: 1) the cardioprotective effects of Hsp20 are associated with serine 16 phosphorylation; 2) blockade of Hsp20 phosphorylation influences the balance between autophagy and cell death; and 3) the aggregation pattern of Hsp20 is altered by its phosphorylation.

Our results demonstrated that Hsp20S16A hearts were more sensitive to ischemia/reperfusion injury, evidenced by lower recovery of contractile function and increased necrosis and apoptosis compared with non-transgenic hearts. Interestingly, autophagy was activated in non-TG hearts, but significantly inhibited in Hsp20S16A hearts following ischemia/reperfusion. Accordingly, pre-treatment of Hsp20S16A hearts with rapamycin, an activator of autophagy, resulted in improvement of functional recovery compared with saline-treated Hsp20S16A hearts. Furthermore, upon ischemia/reperfusion, the oligomerization pattern of Hsp20 appeared to shift to higher aggregates in Hsp20S16A hearts. Collectively, these data indicate that blockade of Ser16-Hsp20 phosphorylation attenuates the cardioprotective effects of Hsp20 against ischemia/reperfusion injury, which may be due to suppressed autophagy and increased cell death. Therefore, phosphorylation of Hsp20 at serine 16 may represent a potential therapeutic target in ischemic heart disease.

Furthermore, two mouse models with Hsp20 ablation and Hsp20S16D cardiac overexpression, were designed. The knockout model was important to investigate the in vivo role of Hsp20 via loss-of-function studies, while the Hsp20S16D transgenic model provided a useful tool to study the effects of chronically phosphorylated Hsp20 in the heart. Initially, we generated the Hsp20 gene targeting construct, and it was

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subsequently electroporated into mouse ES cells. Unfortunately, we did not obtain any positively targeted ES cells. It is possible that ablation of Hsp20 gene might be lethal to ES cells. Therefore, a conditional KO mouse model is under generation. In the future, the availability of the Hsp20 knockout mouse in combination with site-specific mutagenesis will provide us with an excellent opportunity to examine structure- function relations of Hsp20 in a genetic background free of the endogenous protein.

Furthermore, we successfully generated the Hsp20S16D transgenic mouse model. Two transgenic lines with protein overexpression level of 4-fold and 12-fold were used for characterization studies. Chronic overexpression of Hsp20S16D significantly decreased contractility at the myocyte and whole heart levels. As early as

6 weeks of age, transgenic mice exhibited severe interstitial fibrosis. However, the ultrastructure of transgenic myocytes revealed normal myofibrillar arrangement, but there was increased number of autophagosomes, a direct marker of autophagy activity.

Accordingly, the lysosomal marker LAMP-1 was increased in transgenic hearts, also indicating increased autophagy levels in Hsp20S16D hearts. Thus, chronic overexpression of Hsp20S16D impairs cardiac function and triggers cardiac remodeling.

It will be of importance to explore whether autophagy plays a protective or detrimental role in this model. Future studies should be focused on the underlying mechanisms of cardiac dysfunction and remodeling of Hsp20S16D hearts.

In summary, Hsp20 and its phosphorylation play an important role in regulating cardiac contractility and protecting against ischemia/reperfusion injury.

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Section I. Hsp20 regulates cardiac function

I.1 The balance of protein kinase and phosphatase fine-tunes cardiac function

The primary function of the heart is to pump oxygenated blood to all parts of the body, and sustain an arterial blood pressure necessary to provide adequate perfusion of organs.313 The inability of the heart to supply adequate blood flow and therefore oxygen delivery to peripheral tissues and organs is termed heart failure.313

Heart failure is a major and growing public health problem in the United States. It affects an estimated 5 million people in the United States and has an annual mortality rate approaching 20%.314 The disorder is the primary reason for 12 to 15 million office visits and 6.5 million hospital days each year. More than half of the patients have depressed cardiac function.315 316 Despite advances in treatment, the number of deaths has increased steadily.314

At the cellular level, abnormal Ca2+-homeostasis317 and depressed Ca2+ cycling318, 319 are the hallmark characteristics of the failing heart. In the normal myocardium, the sarcoplasmic reticulum (SR) is a principal organelle,that controls intracellular Ca2+ concentration during the cardiac cycle.144 In general, it regulates

Ca2+ dynamics on a beat-to-beat basis through Ca2+ release during contraction and

Ca2+ sequestration during relaxation.143, 320 Importantly, Ca2+ homeostasis is also subject to regulation by the phosphorylation status of key proteins, which is very tightly regulated by the balance of kinases and phosphatases in the cardiomyocyte.321,

322 This becomes of particular importance in the failing heart, where abnormalities in

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the activity of enzymes in the kinase and phosphatase families disturb this fine equilibrium of phosphorylation.236

Protein kinase A (PKA) and type 1/2 protein phosphatase (PP1 and PP2) are the major counter-players in balancing phosphorylation and dephoshorylation processes in cardiomyocytes.321, 322 In response to catecholamine stimulation

(epinephrine or norepinephrine), beta-adrenergic receptors become activated and stimulate adenylate cyclase to catalyze the conversion of ATP to 3’-5’-cyclic-AMP

(cAMP), which subsequently binds to the regulatory subunit of PKA and allows activation of its catalytic subunit.323 Consequently, PKA phosphorylates several target proteins, including the L-type calcium channel (LTCC), phospholamban (PLN), ryanodine receptor (RyR), Troponin I (TnI), and Myosin Binding Protein C (MyBPC), which lead to enhanced contractile function.323

The process of phosphorylation is counter-acted by dephosphorylation, carried out by phosphatases, which facilitate restoration of contractility to basal levels. 321, 322

The type 1 protein phosphatase PP1, and the type 2 phosphatases, PP2A and PP2B, constitute the majority of phosphatase activity in the heart.321, 322 PP1 is regulated by two heat- and acid-stable proteins, inhibitor 1 (I-1) and inhibitor 2 (I-2).322 I-1 becomes active upon phosphorylation on threonine-35 by PKA. This results in inhibition of PP1 and therefore enhanced PKA-mediated protein phosphorylation, leading to amplification of the -agonist responses in the heart.322

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In the context of the failing hearts, the delicate balance of protein phosphorylation is disrupted. On one side, the initial benefits of increased circulating catecholamines become maladaptive over the long-term.324, 325 As a result, the total number of beta-adrenergic receptors is reduced245, 326 and their response to catecholamine stimulation is also diminished.327 One the other side, altered phosphatase activation may also play an important role in heart disease pathogenesis.

Indeed, accumulating evidence has indicated that PP1 activity is increased in human and experimental failing hearts.328-330 Additionally, it has been postulated that the increased PP1 activity in failing hearts is associated with diminished cAMP-dependent phosphorylation and activation of I-1328 Increased PP1 activity in the SR can account for reduced SR Ca2+ uptake and therefore reduced LV function, which is a characteristic of heart failure. 329

I.2 Hsp20 is a prominent regulator of SR Ca2+ cycling and contractile function in cardiomyocytes

It has been reported that the total and phosphorylated levels of small Hsps, including Hsp20, may play a role in animal heart failure.221 Consistently, we found that the expression levels of total and phosphorylated Hsp20 were elevated in heart failure patients, probably as a compensatory mechanism in this disease. It is well- recognized that Hsp20 is the only member of small heat-shock proteins that contains a conserved phosphorylation site of PKA/PKG.155 Hsp20 and its phosphorylation is known to modulate smooth muscle relaxation109 and increase cardiac muscle shortening.72 More interestingly, microcystin-affinity chromatography experiments

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suggested that Hsp20 may interact with the catalytic subunit of Type 1 Protein

331 Phosphatase (PP1C) in rabbit skeletal muscle. Whether Hsp20 could also interact with PP1 C and affect contractile function in the heart is not clear.

We proposed that overexpression of Hsp20 regulates cardiac contractility by affecting PP1 activity. Indeed, our findings demonstrated that acute expression of

Hsp20 enhanced myocyte contractility and SR Ca2+-cycling (Figure 16, 17). These studies were performed in cultured adult rat cardiomyocytes, a well-controlled experimental setting, to avoid adaptive alterations occurring during cardiac development in vivo, and are consistent with a previous report.155 Furthermore, chronic overexpression of Hsp20 resulted in increases in contractility and Ca2+ transients in mouse ventricular myocytes at basal level and under isoproterenol stimulation (Figure 24 - 26).These increases were associated with enhanced caffeine- induced Ca2+-amplitude in Hsp20 transgenic myocytes (Figure 27B), suggesting a

2+ 332 higher SR Ca content. Importantly, using 2D gel electrophoresis, we observed that 161% of total Hsp20 was phosphorylated in Hsp20 TG hearts.225 However, phosphorylated Hsp20 was undetectable in wild type hearts.225 These findings imply that Hsp20 phosphorylation may play an important role in cardiac contractility.

Previously, the role of phosphorylated Hsp20 in regulating cardiac contractility was reported in rat cardiac myocytes.80 Consistent with our results, Pipkin et al. 80 showed that incubation of transiently permeabilized myocytes with phosphopeptide analogues of Hsp20 led to an increase in the rate of shortening. The increased shortening rate was associated with an increase in the rate of relengthening 80. However, these investigators claimed that there was no change in the magnitude of the Ca2+ transient

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after treatment with the phosphopeptide analogues of Hsp20, although the decay of the Ca2+ transient was accelerated, suggesting that the phosphopeptide analogue of

Hsp20 stimulate a more rapid uptake of Ca2+ by the sarcoplasmic reticulum. This apparent discrepancy between our findings and those by Pipkin et al. 80 may be due to transient vs. chronic overexpression of Hsp20, the Hsp20 peptide length, and experimental conditions, such as temperature for measuring Ca2+ transient.

Based on our Ca2+ transient data, a question is raised: what mechanisms are involved in the increased SR Ca2+ load in Hsp20 overexpressing cardiomyocytes? It is known that the calsequestrin (CSQ)-binding capacity, SR Ca2+ uptake rate, and sodium-calcium exchanger (NCX) activity are key determinants of SR Ca2+ load.333-

336 CSQ is the major Ca2+ storage protein in the SR with a binding capacity of 40-50

Ca2+ ions per molecule.337 SR luminal Ca2+ binds to CSQ during diastole to prevent

Ca2+ precipitation and lower the free Ca2+ concentration, to facilitate efficient storage.337 In our studies, we found that the CSQ protein levels were not altered in

Hsp20 TG hearts (Figure 22), suggesting that the CSQ-binding capacity may not be a contributory factor to increase SR Ca2+ load. By contrast, the expression levels of the

NCX were enhanced by 1.2 fold in Hsp20 overexpressing cardiomyocytes (Figure 22).

In mouse cardiomyocytes, more than 90% of the SR-released Ca2+ is reuptaken by the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA); whereas the remaining Ca2+ is pumped out of the cell via Na+/Ca2+ exchanger (NCX).338 Upon treatment of caffeine, which keeps ryanodine receptor (RyRs open) and depletes SR Ca storage, the decline of Ca2+ transient depends on Ca2+ extrusion through NCX.332 As shown in Figure 26, there was no difference in T50 between Hsp20 TG myocytes and WT controls,

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suggesting that the NCX activity was the same between these two groups. However, it should be noted here that the NCX current is the direct evidence for alterations in

NCX activity, which will be measured by whole-cell patch clamp in the future.

Of importance, we observed no alterations in the expression levels of Ca2+ cycling proteins, including triadin, RyR2, L-type Ca2+ channel, sarcoplasmic reticulum (SR) Ca2+ pump (SERCA2a) and phospholamban (PLN) in Hsp20 overexpressing mouse hearts (Figure 22). Interestingly, the phosphorylation of phospholamban at Ser16 and Thr17 appeared to specifically increase, without alterations in the phosphorylation levels of the ryanodine receptor and troponin I in the transgenic hearts (Figure 23). It has been well documented that phosphorylation of PLN would relieve its inhibitory effects on SERCA2a, with subsequent acceleration of Ca2+ uptake by SERCA2a into the SR lumen, resulting in increased SR

Ca2+-load.333, 339, 340

Phospholamban, a low molecular weight phosphoprotein in cardiac sarcoplasmic reticulum (SR), is the regulator of the affinity of SERCA2a for Ca2+.333,

339, 340 Dephosphorylated phospholamban is an inhibitor of the affinity of SERCA2a for Ca2+.339, 340 Phosphorylation of PLN relieves its inhibition on SERCA2a, with subsequent acceleration of Ca2+ transport into the SR lumen.339, 340 In vitro studies have shown that PLN can be phosphorylated on Ser10 by protein kinase C (PKC),

Ser16 by cAMP-dependent protein kinase (PKA) and Thr17 by Ca2+-calmodulin- dependent protein kinase (CaMKII) 341-343. However, in vivo studies have shown that only Ser16 and Thr17 are phosphorylated in cardiac myocytes or perfused hearts, 344,

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345 while phosphorylation of PLN by PKC has not been detected in the intact heart.

Phosphorylation of PLN on Ser16 or Thr17 is associated with stimulation of the initial rates of sarcoplasmic reticulum Ca2+ uptake, which is mainly pronounced at low

[Ca2+], resulting in an overall increase in the affinity of the Ca2+ pump for Ca2+. 346, 347

In the present studies, phosphorylation of phospholamban at Ser16 and Thr17 was increased in Hsp20 TG hearts. It is plausible that Ser16 phosphorylation may precede phosphorylation of Thr17 and the increased SR Ca2+-cycling may lead to activation of CaM Kinase and subsequent phosphorylation of Thr17. Phosphorylation of either Ser16 or Thr17 may occur independently of each other347-350; however, several in vivo studies have indicated that Ser16 phosphorylation or dephosphorylation precedes Thr17 phosphorylation or dephosphorylation during exposure or removal of beta-agonist stimulation, respectively351-353,354. These results suggested that Thr17 phosphorylation requires phosphorylation of Ser16 as a prerequisite in vivo. However, the mechanisms underlying the interdependence between dual site PLN phosphorylation in vivo are not clear. It has been proposed that initially Ser16 phosphorylation may be required to enhance the SR Ca2+ uptake rates and SR Ca2+ load. This in turn, would lead to increased Ca2+ levels released by the SR,

340 activation of the CaMKII, and subsequent phosphorylation of Thr17 in PLN Thus, in current study, the Ser16-PLN may be phosphorylated prior to the Thr17-PLN.

Interestingly, expression of Hsp20 in the transgenic mouse heart appeared to specifically increase phosphorylation of phospholamban, without altering the phosphorylation levels of the ryanodine receptor (PKA-Ser2809 site) and troponin I,

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similar to our previous findings in the hyperdynamic inhibitor-1 transgenic hearts355.

This finding was unexpected because both the ryanodine receptor and troponin I are substrates for protein kinase A, similar to phospholamban.321, 322 Increases in troponin

I phosphorylation would be expected to decrease myofilament sensitivity to Ca2+; whereas increases in ryanodine receptor phosphorylation would cause SR Ca2+ leakage and arrhythmias,356 both contrary to the beneficial effects of overexpressing

Hsp20 observed in this study.

The apparent specificity for increased phosphorylation of PLN by Hsp20 overexpression appears to be due to association of Hsp20 with PP1 and inhibition of the PP1-PLN activity (Figure 29 and 30). This is the first evidence demonstrating that

Hsp20 associates with PP1 in cardiomyocytes and decreases its activity in vivo. Other heat shock proteins such as Hsp90, Hsp59, and Hsp70 are known to be involved in stabilizing the steroid/thyroid receptors in a dephosphorylated form in the cytoplasm.357 Hsp90 and Hsp50 are also known to regulate the activity of kinases such as Ras, Raf, and pp60v-src kinase.358, 359 Overexpressed Hsp70 significantly inhibits the enzymatic activities of protein kinase A and protein kinase C, but it stimulates the activity of protein serine/threonine phosphatases, protein phosphatase-1 and protein phosphatase-2A.360 There is also direct interaction of Hsp70 with calcineurin

(phosphatase-2B).361 Thus, the interaction of Hsps and phosphatases plays an important role in altering the balance between protein kinase and protein phosphatase activities.

Protein dephosphorylation by protein phosphatases, acting in concert with

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protein kinases, is a pivotal regulatory mechanism of protein phosphorylation, participating in SR Ca2+-cycling.321, 322 Previously, it has been shown that endogenous protein phosphatase activity in cardiac SR could dephosphorylate the cAMP- dependent sites362 and the Ca2+-calmodulin-dependent sites on phospholamban,363 which was associated with a decrease in the initial rate(s) of Ca2+ transport by cardiac

SR. It has also been reported that PP1, a serine/threonine protein phosphatase, effectively dephosphorylated the PKA sites of TnI in the thin filament.364 Furthermore, it is known that there is a multimeric complex associated with RyR2, controlling its phosphorylation status.356 PKA is bound to RyR2 via the anchoring protein mAKAP356 and Ser2809 on RyR2 is the target site of PKA.356, 365 The protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) that participate in RyR2 dephosphorylation are bound to RyR2 via spinophilin and PR130, respectively.365

Importantly, the catalytic activity and substrate specificity of PP1 is determined by its regulatory proteins, which associate with the catalytic subunit of PP1 to form a holoenzyme.366 Therefore, the observed “specificity” for PLN phosphorylation in

Hsp20 TG hearts could be the result of PP1 regulatory proteins, subcellular localization or the delicate balance between protein kinases and phosphatases.366

In 1998, Damer et al.331 used microcystin-affinity chromatography to purify type 1 phosphatase (PP1)-binding proteins from the myofibrillar fraction of rabbit skeletal muscle and surprisingly found that human Hsp20 complexed with the catalytic subunit of PP-1 (PP-1C), suggesting that Hsp20 may regulate the activity of

PP1. Interestingly, we found that the PP1 activity was decreased in Hsp20 TG hearts

(Figure 29) compared with WTs. It has been suggested that besides protein

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phosphatase 1, other phosphatases, such as protein phosphatase 2A, also regulate cardiac function.367 However, the activity of PP2A was unaffected in the Hsp20 TG hearts (Figure 29), suggesting a specificity of Hsp20 for PP1.

Furthermore, co-immunoprecipitation studies of PP1 and Hsp20 showed that

Hsp20 associates with PP1, suggesting that these proteins may directly interact

(Figure 30). It has been reported that the catalytic subunits of PP1 (PP1c), exhibiting phosphatase activity, do not freely exist in the cell, but they associate with a host of different regulatory proteins to form a variety of distinct multimeric holoenzymes.322,

366 These proteins target PP1 to appropriate subcellular compartments, supposedly dictating substrate specificity, and modulating its catalytic activity.366 The regulatory subunits of PP1 holoenzyme typically bind to PP1 catalytic subunit via short peptide regions (4-6 residues) and most regulatory subunits have multiple points of interaction with the catalytic moiety. 307 In addition, it has been suggested that the regulatory subunits have multiple contacts with PP1c and that they can share binding sites, and specificity is achieved by interaction with specific subsets of binding pockets on PP1c

(combinatorial-control model).366 Even with a limited number of interaction sites for the regulatory subunits, the catalytic subunit can "combine" with the regulatory subunit in many different ways and form a large variety of holoenzymes with distinct specific activities and substrate specificities.368

An intriguing question is how a relatively small protein like Hsp20 can have distinct effects on the activity and substrate specificity of PP1. Hsp20, like other Hsps, has long been considered as a chaperone protein, which recognizes and renaturates

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misfolded proteins (aggregate).94 It is reported that most small heat shock proteins assemble into high molecular weight complexes, ranging in size from 200 kDa to more than 1 MDa. 369 Consistently, we observed that in wild type hearts, Hsp20 forms small oligomers with molecular weight around 150kD (Figure 43). Thus, it is possible that overexpression of Hsp20 results in larger oligomers in transgenic hearts, which may bind with the catalytic subunit of PP1 and compete with other PP1 regulators, resulting in a unique holoenzyme with substrate specificity for PLN.

Another possibility is that Hsp20 oligomers may act as activity modulators, facilitating the role of other regulatory proteins on PP1 catalytic activity, such as inhibitor-1. Inhibitor-1 (I-1) was the first recognized endogenous inhibitor of PP1.370

It is phosphorylated by PKA or -adrenergic stimulation on threonine 35 and potently inhibits PP1 activity, allowing PKA phosphorylation to propagate and increase cardiac contractility in an unopposed manner. 329, 330 Indeed, expression of a truncated (amino acids 1–65) and constitutively phosphorylated form (T35D) of inhibitor-1 enhanced cardiac contractility by favoring Ser16 and Thr 17 phosphorylation of PLN, without alterations in phospho-RyR and phospho-TnI levels.355 These transgenic hearts exhibited a significant decrease (15%) in cardiac protein phosphatase 1 activity.

Furthermore, the -adrenergic response is enhanced in transgenic hearts expressing the active inhibitor-1 compared with wild types.355 Whether Hsp20 is associated with

I-1 and modulates its activity in Hsp20 transgenic hearts requires further experiments.

I.3 Future Studies

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The findings in our study beg further investigation into following issues. First, since SR Ca2+ uptake is mediated by SERCA2a and its regulatory protein PLN,333, 339,

340 analysis that accurately quantifies Ca2+ uptake is important in determining the underlying mechanisms of cardiac contractility. In our current study, we assess the rate of SR Ca2+ uptake by measuring the time constant (´, T50) of the Ca2+ transient decline. The problem of this method is that the time constant of decline in the Ca2+ is influenced by the function of Na+/Ca2+ exchanger (NCX), which is extruding Ca2+ from the myocyte in parallel to the reuptake of Ca2+ by SERCA2a.336 In addition, the time constant of Ca2+ transient is related to the peak of the Ca2+ achieved during the transient.371 Therefore, an in vitro measurement of oxalate-stimulated Ca2+ uptake will be used to directly quantify the SERCA2a activity.

Second, we estimated the NCX function by measuring the rate of decline of the

Ca2+ during a caffeine-induced Ca2+ transient. The theory here is that reuptake of Ca2+ released by caffeine is prevented by the continued presence of caffeine, and the decline of the Ca2+ and associated relaxation are due to extrusion of Ca2+ from the cell by NCX. Thus the rate of this extrusion can be taken as an index of the function of

NCX. However, this NCX function is dependent on the transsarcolemmal Na+ concentration gradient, and membrane potential.336 Unless the cell is voltage clamped to control the membrane potential, and the cell dialyzed with a specific Na+ concentration solution, these factors cannot be adequately controlled to allow precise assessment of NCX function.372 Therefore, in the future it is important to measure the

NCX function in Hsp20 TG hearts by whole-cell patch clamp.

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Third, L-type Ca2+ channel is also a substrate of PKA. Phosphorylation of L- type Ca 2+ channels by PKA increases the probability and duration of the open state of the channels.150, 373-377 Functionally, the L-type Ca2+ current links the membrane depolarization to contraction of the heart. During the depolarization, Ca2+ enters the cell through L-type Ca2+ channel, and induces a subsequent massive Ca2+ release from the SR, which finally triggers contraction.374, 375 Furthermore, the L-type Ca2+ current contributes to maintain the long duration of cardiac action potential, which leads to a refractoriness, preventing the cell from activation by another action potential.373 This limitation can be avoided by measuring the L-type Ca2+ channel activity by patch clamp.

Furthermore, we did not determine the localization of Hsp20 in the myocyte. It has been reported that under basal conditions, Hsp20 is mainly localized in cytosol.225

Whether it is also localized on SR membrane, where it would associate with PP1 and phosphorylate PLN, remains unknown. In the future, assessment of Hsp20 levels in subcellular fraction of nuclei, cytosol and SR membrane from myocytes of transgenic and wild type mice by Western blot would allow avoiding this limitation.

1.4 Summary

In conclusion, the present study significantly advanced our understanding on the effects of Hsp20 in cardiac function. Collectively, acute or chronic overexpression of Hsp20 plays an important role in SR Ca2+ cycling and cardiac contractility. Our data suggest that Hsp20 interacts with PP1 and regulated its activity in the mouse heart. Since increases in type 1 phosphatase (PP1) activity have been observed in end

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stage human heart failure, the small heat-shock protein Hsp20 could represent a potential therapeutic target for heart failure.

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Section II: Hsp20 and its phosphorylation at Ser16 protects against cardiac ischemia/reperfusion injury

II.1 Apoptosis, necrosis and autophagy in cardiac ischemia/reperfusion injury

Out of the approximately one million people suffering myocardial infarctions in the United States, nearly a third will succumb in the first year, while many survivors will eventually develop significant systolic dysfunction resulting in congestive heart failure and mortality.378 Reperfusion strategies, which aim to restore blood flow in damaged tissue, is the primary therapeutic strategy to blunt the progression of coronary artery disease to congestive heart failure.177, 379 However, coronary reperfusion may at first exacerbate cellular damage sustained during the ischemic period, which leads to so-called ischemia/reperfusion injury.177, 379 Thus, the maintenance of cardiomyocyte numbers is critical to the overall preservation of both structural integrity and function of the heart. 177, 379

Studies indicate that, following ischemia-reperfusion, cardiac myocytes may undergo three forms of cell death, i.e., apoptosis, necrosis and autophagic cell death.177, 208, 209 It is well defined that ischemia/reperfusion-induced cardiomyocyte apoptosis258 and necrosis177, 265 contribute to ventricular dysfunction and end-stage failure. Pharmacologic and transgenic studies in animal models indicate that inhibition of both apoptosis and necrosis reduces the impact of myocardial infarction and thus, arrest the progression to heart failure.177 However, the role of macroautophagy

(commonly referred to as autophagy) is still controversial for a potential source of

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programmed cell death.177, 208, 209

Apoptosis is a gene-directed, energy-dependent cell suicide process.

Morphologically, apoptosis is characterized by chromatin condensation and fragmentation, cell shrinkage, and plasma membrane budding with release of apoptotic bodies that are phagocytized by either macrophages or neighboring cells.177,

200, 202, 380-382 Since intracellular components are not released during apoptotic process, there is little or no inflammatory response.177, 381, 382 Initiation of apoptosis occurs at cell surface death receptors (e.g., Fas, TNF-alpha etc.) endoplasmic/sarcoplasmic reticulum (e.g., procaspase-12), mitochondria (e.g., Bax, Bak and cyclophilin D), and via nuclear proteins in response to genotoxic stress (e.g., p53).177, 381, 382 Integration of these stimuli occurs via the activation and release of cysteinyl-aspartate proteases, known as, caspases into the cytosol.383 These proteins exist in the cell as inactive proenzymes and can be broadly classified into the upstream initiator caspases (e.g., procaspases-2, 8, 9, 10, and 12) and downstream effector caspases (e.g., procaspases-

3, 6, and 7). Activated caspase-3, 6 and 7 bind to and cleave a variety of structural proteins in the cell leading to the morphological changes of apoptosis.177, 381-383

Necrotic cell death is characterized by the irreversible rupture of the plasma membrane and release of cytosolic components, usually exciting a strong inflammatory response.198, 265 For a long time, necrosis has been considered as an unregulated process. However, recent studies suggest that necrosis can be regulated and that interventions can reduce necrotic cell death.177, 384 Unlike apoptosis, though, molecular pathways that regulate the necrotic programmed cell death in

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ischemia/reperfusion injury are not fully elucidated. It has been reported necrosis and apoptosis share several regulators. Of those, Ca2+ signaling, as well as the role of calpains appear to be central.177 The initiation phase of necrosis can occur either via intrinsic mechanisms, or by external stimuli through death receptors on cell surface.

These events result in provoking catastrophic energy depletion and Ca2+ accumulation, as well as an increase in availability of reactive oxygen species (ROS).177 It appears that energy depletion results in inhibition of cell surface Na+ and Ca2+ channels resulting in cytosolic Ca2+ accumulation.385 ER proteins calnexin and calreticulin, as well as the IP3 and ryanodine receptors, appear to provoke this Ca2+ accumulation.385

The execution phase of the necrotic program is conducted by calpain activation, and subsequently activation of lysosomal cathepsins that result in widespread proteolysis and cellular destruction.386 The mitochondria appear to play an important role in necrosis beyond just regulating energy levels. Inhibition of mitochondrial permeability transition with cyclosporine and deletion of cyclophilin D appears to have significant impact in models of necrosis.386 In studies using mice lacking cyclophilin D, reductions in infarct size commensurate to decreases in apoptosis and necrosis have been reported.386 Furthermore, Ca2+ overload, increased ROS production, and severe energy depletion are all associated with mitochondrial dysfunction, highlighting the central role of this vital organelle in both apoptosis and necrosis.177

Autophagy is another process that has been suggested to play a role in ischemia-reperfusion injury.208, 209, 304, 208, 387 Autophagy is a physiological mechanism that is used to remove damaged organelles, such as mitochondria or endoplasmic

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reticulum.304, 306, 388, 389 Autophagy was first found to be initiated by starvation to provide nutrients.304, 306, 388, 389 The conduction of autophagy is strictly regulated by the ATG (autophagy related gene) family of proteins, such as Atg5390, Atg6/Beclin235,

391, 392, Atg8/LC3233, Atg10393 and Atg12.394,395 However, extensive autophagy can cause cell death.306, 388, 389 There are conflicting data as to whether the increased autophagy that occurs during ischemia and reperfusion is beneficial or detrimental.208,

209, 304, 387 There are studies showing that inhibition of autophagy during ischemia or anoxia is detrimental, suggesting a beneficial role for stimulation of autophagy during ischemia.208, 209, 304 However, in contrast to these studies, a decrease in Beclin1 (a protein that stimulates autophagy) reduces ischemia-reperfusion-mediated autophagy and myocyte death.392 Consistent with a beneficial role for autophagy in ischemia- reperfusion, in HL-1 cells an increase in beclin1, which increased autophagy, was also shown to decrease activation of BAX, and knockdown of beclin1 increased activation of BAX392. Thus, the role of autophagy in cell death during ischemia-reperfusion is still unclear.

Recent studies indicate that several overlapping pathways intersect between apoptosis, autophagy, and necrosis during ischemia/reperfusion.178,396 For instance, an increase in Ca2+, as occurs during ischemia, has been shown to increase autophagy, and at the same time convert some of the autophagic machinery to apoptosis.396

Activation of calpain, a Ca2+-activated protease in necrosis, has been reported to cleave Atg5, a protein involved in autophagy; cleaved Atg5 translocates to the mitochondria where it is reported to bind Bcl-2, thereby stimulating apoptosis.178

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II.2 Hsp20 and its phosphorylation at Ser16 protect against ischemia/reperfusion injury

Upon ischemia/reperfusion injury, adaptive stress responses occur immediately, some of which are involved with upregulation of heat shock proteins

(Hsps).35 In fact, Hsp levels increase transiently under a wide spectrum of stressful stimuli as a protective mechanism.260 Numerous studies over the past decade have demonstrated that increased expression of Hsps may protect the heart from stressful environments such as ischemia and reperfusion injury.260, 397 Hearts isolated from transgenic mice that express human Hsp70 in the myocardium have shown greatly improved functional recovery, with decreased infarct size after experimental induction of ischemia and reperfusion.271,215 Within the superfamily of Hsps, the small Hsps with molecular weights ranging from 12 ~ 43 kD have received particular attention.74,

225, 263 Recently, several members of the small Hsp family have been identified as protective mediators during myocardial ischemia, including B-crystallin, Hsp27 and

Hsp20. 74, 225, 263

Hsp20, sharing a conserved domain with B-crystallin and Hsp27,91 is the only member within the sHsps family that contains a consensus peptide motif (RRAS) for protein kinase A (PKA)/protein kinase G (PKG)-dependent phosphorylation at

Ser16.155 We and others have demonstrated that the levels of cardiac Hsp20 and its phosphorylation were significantly increased, compared with Hsp27 and B-crystallin, in animal hearts upon ischemic conditions, exercise training, rapid right ventricular pacing, and pharmacological treatment by doxorubicin and chronic -adrenergic

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stimulation.221, 223, 225, 398 More recently, we have identified a P20L substitution in human Hsp20, which was associated with diminished phosphorylation at Ser16 and complete abrogation of the protective effects of Hsp20, suggesting an instrumental role of phosphorylated Hsp20 in cardioprotection.224 Indeed, the Hsp20S16D, mimic the constitutive phosphorylation of Hsp20, conferred protection against -agonist-induced apoptosis in cultured myocytes.81 Conversely, the mutant Hsp20S16A, mimicking constitutively dephosphorylation, displayed no anti-apoptotic properties,81 implying a mechanistic link between phosphorylated Hsp20 and its protection.

It is noteworthy that in vivo or ex vivo myocardial ischemia involves a large and progressive release of catecholamines from adrenergic nerve terminals, and excessive stimulation of myocardial -adrenergic receptors by catecholamines may further accelerate the ischemia-induced cell damage.399, 400 Indeed, we observed an increased ratio of phospho-Ser16/total Hsp20 in wild type mouse hearts upon ischemia/reperfusion injury, as well as in failing human hearts. (Figure 32) This could be interpreted as a compensatory protective response to the accumulated catecholamines in the ischemic myocardial tissue. In fact, our previous study has shown that the protective effect of Hsp20 against ischemia/reperfusion was associated with increased phosphorylation of Hsp20.224, 225

According to our hypothesis, phosphorylation of Hsp20 at its Ser16 site is essential for protection against ischemia/reperfusion injury.

Our results revealed the following novel findings: (1) blockade of the Ser16 phosphorylation site in a model with cardiac overexpression of Hsp20S16A resulted in

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loss of cardioprotective effects exhibited by Hsp20,225 and fostered worse functional recovery following ischemia/reperfusion; (2) More interestingly, this detrimental effect of Hsp20S16A was associated with increased necrosis and apoptosis, but decreased autophagy (Figure 61). To our knowledge, this is the first study showing that blockade of Hsp20 phosphorylation reduces ischemia/reperfusion-induced autophagy and compromises its cardioprotection.

Figure 61. Proposed scheme for phosphorylation of Ser16-Hsp20 protecting against cardiac ischemia/reperfusion injury. Upon phosphorylation, Hsp20 tends to form small oligomers which increase autophagy activity and decrease apoptosis, therefore preventing cardiac injury during ischemia/reperfusion. When phosphorylation of the Ser16 site is blocked (Hsp20S16A hearts), under basal conditions and further during ischemia/reperfusion injury, Hsp20 forms large oligomers, suppresses autophagy activity and increases apoptosis, which leads to cardiac injury.

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It has been well-documented that Hsps protect the heart against ischemia/reperfusion-induced apoptosis.73, 74, 218, 283, 401 However, the role of Hsps in ischemia/reperfusion-induced autophagy is not clear. Hsp20 and Hsp70 are the only two heat shock protein members, which are currently known to play a role in autophagy. Previously, Hsp70402, 403 has been reported to be involved in BAG-1 (Bcl-

2-associated athanogene-1) induced autophagy for cardiac survival. BAG-1 acts as a co-chaperone and exerts many of its actions via binding with the chaperone molecules, such as heat shock protein 70 (Hsp70) and heat shock cognate protein (Hsc70, the constitutive isoform of Hsp70).404 BAG-1 modulates the chaperone activity of

Hsc70/Hsp70, and it has been proposed to link these heat shock proteins to the autophagosome to facilitate protein degradation.405 In fact, it has been demonstrated that the autophagosomal membrane contains significantly higher amount of Hsc70 proteins.406 Therefore, Hsp70 could conceivably serve a recognition function in the autophagic scavenging of denatured or aggregated proteins.406 However, the role of

Hsp70 in ischemia/reperfusion-induced autophagy remains unknown.

The mechanisms underlying the detrimental effects of Hsp20S16A on ischemia/reperfusion may involve several pathways. Firstly, the increase in apoptosis is likely an important factor responsible for severe injuries in post-ischemic Hsp20S16A hearts. It is well accepted that cardiomyocyte apoptosis plays a fundamental role in the myocardial processes that initiate or aggravate cardiac injury.177, 257, 380, 407 For example, conditional overexpression of active caspase-8 demonstrated that very low levels of myocyte apoptosis were sufficient to cause lethal, dilated cardiomyopathy.408

Furthermore, anti-apoptotic sHsps play an important role in cardiac injury. For instance, transgenic overexpression of B-crystallin73 confers simultaneous protection

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against cardiomyocyte apoptosis and necrosis during myocardial ischemia and reperfusion. In contrast, B-crystallin and HspB2 double-knockout mouse hearts exhibited a twofold reduction in contractile recovery, as well as increased necrosis and apoptosis.74 Our laboratory has also observed that reduced cardiac apoptosis significantly contributed to better functional recovery in Hsp20-overexpressing hearts upon ischemia/reperfusion.225 Hence, blockade of Hsp20 phosphorylation at Ser16 is associated with increased cardiomyocyte apoptosis triggered by ischemia/reperfusion, leading to lower functional recovery.

Secondly, there is an increasing awareness that autophagy plays a critical role in ischemia/reperfusion injury.208, 209, 259 Under basal conditions, low level of autophagy is necessary for the turnover of long-lived proteins and cytoplasmic organelles in the heart.305, 389 In response to ischemia, the extent of autophagy depends on the severity and duration of ischemic insults. For example, modest levels of autophagy, induced by mild to moderate hypoxia/ischemia, appear to be protective by degrading and removing damaged mitochondria, therefore preventing activation of apoptosis.178, 213, 259 On the other hand, high levels of autophagy triggered by severe hypoxia or ischemia/reperfusion may cause self-digestion and eventual cell death.208,

209 Accordingly, Decker and Wildenthal259 reported that as early as 40min of ischemia led to upregulation of autophagy, and that subsequent reperfusion of 1h induced a drastic enhancement of autophagy in Langendorff perfused rabbit hearts. In their model, induction of modest levels of autophagy was correlated with functional recovery of rabbit hearts after ischemia/reperfusion.259 Therefore, the level of autophagy may determine whether it is protective or detrimental to the heart in

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response to ischemia/reperfusion. The beneficial increase of autophagy may be responsible for elimination of damaged, presumably nonfunctional organelles, including mitochondria, along with the restoration of normal cardiac structure and function. 208, 209

It is known that deficiency in autophagy can promote necrotic cell death.409

Specifically, a deficit of LAMP-2, a lysosomal protein required for the proper fusion of autophagosomes with lysosomes, caused the accumulation of autophagic vacuole.

Thus, leading to the inhibited autophagy.409,410 Indeed, autophagy is required for the recycling of small molecules generated by the catabolism of cytoplasmic macromolecules, which produces ATP.177, 200, 202, 380-382 Additionally, autophagy can lead to the removal of damaged organelles including uncoupled and permeabilized mitochondria.177, 200, 202, 380-382 Hence, the inhibition of autophagy may accelerate the bioenergetic failure of damaged cells and lead to necrosis.

Consistently, we found that a brief period (20 minutes) of ischemia followed by 2 hours reperfusion induced reversible cardiac injury (complete contractile recovery) in wild type murine hearts. By contrast, prolonged ischemia (60 minutes) and 2 hours of reperfusion resulted in a large infarct along with minimal recovery of contractility (~10%) (Figure 35). The degree of injury inflicted during 45 minutes of ischemia followed by 2 hours of reperfusion was intermediate, and correlated with a modest activation of autophagy (1.3-fold increase of the LC3-II/LC3-I ratio). Thus, we selected a 45min/2h of ischemia-reperfusion protocol for our present study. As expected, autophagy was increased in our non-TG hearts upon ischemia/reperfusion, which corresponded to ~75% of functional recovery. However, Hsp20S16A hearts

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displayed inhibition of ischemia/reperfusion-activated autophagy, and this may contribute to deterioration of energy homeostasis, leading to impaired functional recovery (~54%). Accordingly, activation of the autophagy process in Hsp20S16A hearts by pretreatment with rapamycin restored their functional recovery upon ischemia/reperfusion. Furthermore, we have noticed a modest (1.27 fold) increase of autophagy in Hsp20S16A hearts under basal conditions, which did not affect its basal myocardial function, compared with non-TG controls. We also found that pretreatment of Hsp20S16A hearts with rapamycin did not alter basal contractile function (Figure 36). These observations suggest that a modest increase of autophagy has no effects on basal cardiac function. However, it should be admitted that rapamycin might have some other protective effects beyond activating autophagy, such as inducing potent preconditioning-like effects against myocardial infarction

411 through opening of mitochondrial KATP channels.

Non-phosphorylatable Hsp20 may be abnormal in its oligomerization, which offsets the protective effects of Hsp20. It is well-known that the phosphorylative capacity and the resulting aggregation patterns of small heat shock proteins may influence their cytoprotective ability during cellular stress.412-414 For example, the phosphorylated form of Hsp27 was concentrated in small and medium-sized oligomers, whereas its non-phosphorylated form was present in larger oligomers.412, 413

Unexpectedly, non-phosphorylatable mutants of Hsp25, the rodent form of Hsp27, conferred better protection than wild type Hsp25 in L929 cells subjected to tumor

414 necrosis factor (TNF)-alpha and H2O2-induced cytotoxicity. Furthermore, overexpression of wild type Hsp27 or a non-phosphorylatable Hsp27 mutant was

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equally capable of protecting from an ischemic insult in adult rat myocytes412 and transgenic mouse models.218 These conflicting results may be partially due to the use of different models and variability in Hsp27 protein levels between studies. Previous in vitro studies have shown that phosphorylation at Ser16 regulates the aggregation pattern of Hsp20, 270 which is consistent with the observation that the N-terminal residues of sHsps are necessary for complex formation.415 Interestingly, our pull- down assay indicated that phospho-Ser16-Hsp20 is associated with autophagy-related

Beclin-1 (Figure 37), suggesting that Beclin-1 may be a potential target of phosphorylated Hsp20 in regulating autophagy. The blockade of Hsp20 phosphorylation at Ser16 promoted formation of large aggregates and possibly disassociated Beclin-1, which may promote the detrimental effects under stress conditions.

II.3 Study limitations

The major limitation of this study is that in our Hsp20S16A transgenic mouse model, the mutant Hsp20S16A transgenes were expressed on wild-type genetic background in which endogenous Hsp20 was also expressed. It would be better to express this mutant protein in a Hsp20-null background, to eliminate the effects of endogenous Hsp20. Furthermore, using a knock-in approach,416 the targeting vector which contains mouse Hsp20S16A cDNA (or a mouse gene with S16A) can be inserted in-frame in the position of Hsp20 gene. The Hsp20S16A cDNA would thus be expressed in place of the endogenous gene product. The main advantage of this approach is that it allows tight control of the expression of Hsp20S16A cDNA, because it is placed in the context of the complete set of cis-acting regulatory elements that

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normally control the expression of the endogenous Hsp20 gene; furthermore, this approach avoids the position effects encountered in gene-addition transgenesis, and it allows the expression of mutant Hsp20S16A to a level comparable to that of endogenous Hsp20.416 Moreover, an "inducible knock-in" strategy417 is capable of conferring tissue- or developmental stage-specific control the expression of mutant

Hsp20S16A on the Hsp20 null-background. The principal advantage of this "inducible knock-in" is that their genome contains a single copy per cell of a mutation causing a typical serine substitution, under the control of the endogenous promoter and with dominant genetic transmission. Meanwhile, by removing endogenous Hsp20, the phenotypes of the transgenic mice will be determined by the mutant protein, instead of the combination of the endogenous Hsp20 and mutant, which will avoid mis- interpretations in experimental data.

Limitations also exit in apoptosis assay by using TUNEL staining. Introduced in 1992 ,418 TUNEL is the most widely used histochemical technique for in situ labeling and localization of DNA breaks in individual nuclei on tissue sections. It is based on the fact that apoptosis is associated with cleavage of genomic DNA within internucleosomal DNA segments by selectively activated Ca2+/Mg2+-dependent endonucleases.419, 420 An apoptotic index based on TUNEL can be calculated by expressing the number of positive nuclei as a ratio of the number of cells counted, thereby enabling quantification. TUNEL is highly sensitive. However, TUNEL has its pitfalls and lacks specificity421. It may not distinguish apoptosis from necrosis with absolute certainty and the specificity of a TUNELpositive nucleus may not be definite.

In the other hand, TUNEL may be oversensitive and can detect minimal amounts of

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DNA strand breaks due to necrosis 422, 423 and 3’ DNA ends due to DNA repair. 424, 425

For this reason, TUNEL is heavily criticized and multiple biochemical methods (such as DNA laddering426) or other molecular markers (such as caspase-3427) are recommended to complement apoptosis. Consequently, in the present study, we have used altogether three methods to detect apoptosis, including TUNEL, DNA fragmentation and caspase 3 activity.

Another limitation is lack of assessing autophagic flux. Since autophagy is a dynamic, multi-step process,177, 257, 380, 407 an accumulation of autophagosomes

(measured by electron microscopy (EM) image analysis, as fluorescent GFP-LC3 dots, or as LC3 lipidation on a Western blot), could, reflect either increased autophagosome formation due to increases in autophagic activity or reduced turnover of autophagosomes.409,410 A defect in autophagosome turnover due, for example, to a block in fusion with lysosomes or disruption of lysosomal function will result in an increased number of autophagosomes (Figure 60) In this scenario, autophagy is induced, but there is no or limited autophagic flux.409,410 In our current studies, we measured the autophagy activity mainly by LC3 and Beclin-1 activity. Therefore, the lysosomal marker LAMP-1 levels in myocardium by Western blot or immunohistostaining should be determined in future studies.

There is also limitation in studying the role of phospo-Ser16-Hsp20 in regulating Beclin-1. The co-immunoprecipitation assay indicated that phospho-Ser16-

Hsp20 is associated with autophagy-related Beclin-1Alternatively, the yeast two- hybrid can be used to confirm the protein-protein interaction.428 This method bears the

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advantage of detecting transient and weak interactions, which are often important in signaling cascades, since the genetic reporter gene strategy results in significant signal amplification.428, 429 However, howphospho-Ser16-Hsp20 regulates Beclin-1 activity is not clear. Therefore, isolated rat cardiomyocyte can be used and upon infection with

Ad.Hsp20WT, Ad. Hsp20S16D and Ad. GFP (control), then subjected to stimulated ischemia/reperfusion,224 to determine the underlying mechanisms.

II.4 Future Studies

The data presented in this study significantly contributed to our understanding of the critical role of phospho-Ser16-Hsp20 in cardiac injuries. In deed, our lab has started screening the Hsp20 polymorphisms in dilated cardiac myopathy patients.

Until now we have identified a P20L substitution in human Hsp20, which was associated with diminished phosphorylation at Ser16 and complete abrogation of the protective effects of Hsp20, suggesting an instrumental role of phosphorylated Hsp20 in cardioprotection.224 In the future, we will continue identifying the Hsp20 polymorphisms and determine if any of them would associate with pathophysiology of the failing heart. Furthermore, to explore the potential of applying phospho-Ser16-

Hsp20 in treating myocardial ischemia, gene therapy based on Adeno-associated virus

(AAV) will be carried out in mouse model. Since AAV has not been shown to contribute to any human pathology, it becomes the safest of the candidate gene therapy vectors aside from naked DNA.430

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Figure 62. Schematic model demonstrating normal autophagic flux (A) and the induction of autophagosome formation when turnover is blocked (B). (A) Autophagy involves five steps: (1) vesicle nucleation: formation of an isolated membrane or phagophore; (2) vesicle elongation: expansion of the phagophore; (3) vesicle completion: closing of the edges of the phagophore to form an double-membraned autophagosome, enclosing selected cytoplasmic materials including organelles; (4) docking and fusion: the fusion of the autophagosome with a lysosome to form an autolysosome; and (5) degradation: digestion of the luminal content along with the inner layer of membrane by the lysosome hydrolases. (B) A defect in autophagosome turnover due, for example, to a block in fusion with lysosomes or disruption of lysosomal function will result in an increased number of autophagosomes. In this scenario, autophagy is induced, but there is no or limited autophagic flux. This is a different outcome than the situation shown in (A), where autophagosome formation is followed by fusion with lysosomes and degradation of the contents, allowing complete flux, or flow, through the entire pathway.

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II.4 Summary

In conclusion, the present findings indicate that blockade of Hsp20 phosphorylation at Ser16 is associated with increased cardiac ischemia/reperfusion injury, partially through reduced activation of autophagy and increased apoptosis as well as necrosis. Thus, Hsp20 and its phosphorylation may constitute an important modality for cardioprotection against myocardial infarction.

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Section III: Generation of Hsp20 KO mice

III.1 Design of Hsp20 gene targeting construct

Gene targeting is a genetic method that uses homologous recombination to change the endogenous gene.431-433 This technique can be used to delete a gene, remove exons, or introduce point mutations.434 By successful transmission through the mouse germline, a remarkable number of novel and informative mouse strains can be generated, which have been invaluable not only for the study of gene function in vivo, but also serving as model systems for human disease.435, 436

Gene-targeting strategies can employ two different types of vectors, sequence replacement or sequence insertion vectors. Replacement vectors431 are used to disrupt target gene function by deleting specific sequences, and replacing them with heterologous DNA, usually a drug selection gene or a marker gene to analyze target gene expression. Insertion vectors,431, 432 which may offer increased gene-targeting frequencies at a given genetic locus compared to a replacement vector,437 can also be used to disrupt gene function by inserting heterologous DNA, but also allow for the introduction of more subtle genetic alterations, such as point mutations.438, 439

Homologous recombination between the targeting vector and the endogenous locus in ES cells is a rare event, but marker genes incorporated in targeting vectors for drug selection help to enrich the targeted ES cells.431-433 In addition, there is considerable collective experience on the optimal design features for the targeting construct, such as the nature of the targeting site, size, and composition of flanking genomic DNA.434-438, 439 Despite the improved technology, there still exist important

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limitations of gene-targeting techniques. For example, about 15 percent of gene knockouts are developmentally lethal,440 which means that the genetically altered embryos cannot grow into adult mice. Thus, studies are limited to embryonic development and this may make it difficult to determine a gene's function in relation to human disease.

Gene-targeting strategies have been applied in studying the in vivo function of heat shock protein genes. Early studies have found that heat shock proteins (Hsps) expressed in mouse embryonic development,441, 442 which led to the proposal that

Hsps serve pivotal roles during early embryogenesis, and Hsps knockouts are developmentally lethalHowever, this assumption was not supported by data from gene-targeted mouse models. There have been several gene-targeted mouse models generated, including Hsp70 knockout,154 the double knockout (DKO) of BC and

HspB2, 74, 246, 247 and a Hsp25 knockin model,442 where the coding sequence of Hsp25 was replaced by a lacZ reporter gene. Surprisingly, the offspring of both knockout models,74,154, 246, 247 as well as the Hsp25 knockin442 were viable. Even the cross model of Hsp25-null mice (derived from Hsp25 knockin) with DKO (HspB2 and BC-null) mice, which generated a model deficient in three major sHsps, had no apparent effects on embryonic development, although these mice died early in adulthood with skeletal muscle degeneration.442 These data suggested that, the above heat shock proteins are dispensable for mouse development.

One plausible explanation for such apparent functional redundancy could be the unusual complexity in the sHsp system, in which individual members of the sHsp

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family may act in concert and display distinct functions in tissues expressing these chaperone molecules.260 For example, several sHsps (BC, Hsp25, Hsp20, HspB3,

HspB7, and HspB8) are abundant in cardiac or skeletal muscle 23, 443. In such tissues, sHsps may form heterodimers and hetero-oligomers (e.g., Hsp25, Hsp20 and BC form one type of complex, while HspB2 and HspB3 form a different type)18 and fulfill specific and perhaps overlapping functions. Thus, the functional consequences of

BC, HspB2, and/or Hsp25 ablation could easily be compensated by formation of structural units of other interacting sHsps.

As mentioned earlier, we were not able to generate any positively targeted ES cells by the conventional Hsp20 knockout construct. Given its unique phosphorylation characteristics within the sHsps family,155 and its unique expression pattern during heart development,23 it is plausible that Hsp20 may perform a special, nonredundant function in embryonic development. Therefore, it is possible that ablation of the

Hsp20 gene may be developmentally lethal. To circumvent this problem, a conditional knockout mouse model may be generated.

Presently, two site-specific recombinases, Cre and Flp, are predominantly used as the basis of the conditional knockout approach.444, 445, 446, 447 Cre recombinase is the 38-kDa product of the Cre gene of bacteriophage P1 and recognizes a 34-bp site within the P1 genome, called loxP. Cre efficiently catalyzes reciprocal conservative

DNA recombination, that is planned to be excised, between pairs of loxP sites.444, 445

Mice are generated with this (silent) modification in their genome. These mice can be mated with transgenic animals, expressing the CRE recombinase driven by a

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particular promoter.444, 445 An inducible promoter may be used in the case where a gene deletion results in fetal lethality, 444, 445 or a tissue/cell specific the promoter may used in the case where it is required to study the consequences of loss of gene function in a specific cell type in isolation.448-450

Introduction of LoxP sites by homologous recombination in embryonic stem cells utilizes the neomycin phosphotransferase (neo) gene, for example, as a positive selection marker.444, 445 However, such marker genes can interfere the transcription and splicing of the target and neighboring gene, thereby resulting in ambiguous genotype–phenotype relationships.451 One way to avoid such effects is the use of Flp recombinase to eliminate marker genes flanked by Flp recognition target (FRT) sites.446 Similar to Cre/LoxP system, Flp/FRT involves the recombination of sequences between short Flippase Recognition Target (FRT) sites by the Flippase recombination enzyme (Flp).446, 447

III. 2 Future studies

An inducible cardiac-specific Hsp20 deficient mouse model may be generated by crossing two genetically-modified mouse lines452: one expressing a tamoxifen- inducible Cre recombinase protein fused to two mutant estrogen-receptor ligand- binding domains (MerCreMer), under the control of the cardiac specific -MHC promoter453 (Figure 63A); and one where the loxP sites will be introduced outside exon1 and exon 3 of the Hsp20 gene (Figure 63B). Transgene expression driven by the -MHC promoter is mainly constitutive throughout development and in the adult heart. The Cre recombinase fusion protein is inactivated by two mutant estrogen

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receptor ligand-binding domains. In the cross model, Cre protein will be expressed in the cardiac tissue, where it will excise the whole Hsp20 gene flanked by loxP sites. In the end, we will establish a mouse model with a cardiac-specific, inducible inactivation of the Hsp20 gene.

III.3 Summary

A cardiac inducible Hsp20 knockout model will be generated in the future.

This model will circumvent embryonic or fetal lethality associated with complete somatic disruption of the Hsp20 gene, thus permitting temporally regulated activation or inactivation of Hsp20 gene in the heart. Such applications will undoubtedly expand our ability to genetically dissect the function of the Hsp20 gene within either the developing or adult heart.

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Figure 63. Proposed Tamoxifen-inducible disruption of the Hsp20 gene.

(A) cDNA encoding the mutant estrogen receptor ligand-binding domain (Mer) flanking Cre recombinase (Cre) will be subcloned downstream of the -MHC cardiac-specific promoter, which was used to generate transgenic mice. (B)

Modification of the Hsp20 by insertion of loxP sites (triangles). Exons 1, 2 and 3 will be deleted (Hsp20 allele) by Cre recombination of the LoxP sites in the presence of tamoxifen.

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Section IV: Generation of Hsp20S16D transgenic mice

IV. 1 Chronic expression of Hsp20S16D resulted in cardiac dysfunction and remodeling

Activation of the sympathetic nervous system is a common compensatory feature in heart failure,244, 245 but sustained -adrenergic activation induces cardiomyocyte death, leading to cardiac remodeling and dysfunction.454 Our lab has reported that prolonged exposure to-agonists was associated with transient increases in expression and phosphorylation of Hsp20 in mouse cardiomyocytes.155 To determine the functional significance of Hsp20, this protein and its constitutively phosphorylated mimetic (S16D) or nonphosphorylated mimetic (S16A) were overexpressed in adult rat cardiomyocytes by adenoviral infection.81 We observed that

Hsp20 protected cardiomyocytes from apoptosis triggered by activation of the cAMP-

PKA pathway, as indicated by decreases in the number of positive-stained nuclei by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL), and DNA laddering, which were associated with inhibition of caspase-3 activity.81

These protective effects were further increased by the constitutively phosphorylated mimetic Hsp20 mutant (S16D), which conferred full protection from apoptosis. In contrast, the nonphosphorylatable mutant (S16A) exhibited no antiapoptotic properties.81 These findings suggest that Hsp20 and its phosphorylation at Ser16 may provide cardioprotection against -agonist-induced apoptosis.

To further explore the in vivo role of phospho-Hsp20, we generated the

Hsp20S16D transgenic mouse model, in which Ser16 is replaced with aspartic acid (D).

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Surprisingly, we observed that contractile parameters were significantly depressed in transgenic (TG) cardiomyocytes compared with non-TGs. In vivo contractile function was also significantly impaired in TGs compared with their non-transgenic littermates.

In addition, TG mice developed left ventricular (LV) fibrosis around 8 weeks of age, without evidence of LV hypertrophy or dilation, and their life span was markedly shortened (mean age at death: 9 months). Further electron microscopy examination of the hearts revealed that double-membrane autophagosomes were more prominent in

TGs. This was associated with increased lysosomal activity, as demonstrated by immunostaining for lysosomal-associated membrane protein-1 (LAMP-1), suggesting that autophagosome accumulation was not due to diminished activity of distal lysosomal pathways. Our data indicate that long-term augmentation of cardiac Hsp20 phosphorylation impairs cardiac function, accentuates pathological remodeling, and increases autophagic activity, leading to premature death. Therefore, it would be very interesting to investigate the mechanisms underlying the cardiac dysfunction and remodeling in Hsp20S16D transgenic mice.

Many mechanisms contribute to cardiac dysfunction and remodeling. Recent studies found that activation of the innate immune system could play an important role in various cardiac diseases without the direct involvement of infectious pathogens.455 In cardiac immune responses, circulating heat shock proteins are thought to act as endogenous danger signals augmenting inflammatory cytokines by triggering monocyte Toll-like receptors (TLRs).456-458 For example, in acute myocardial infarction patients, elevated circulating levels of Hsp70 were involved in

TLR4 signal-mediated immune response and the progression of heart failure,459 and

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extracellular Hsp60 (exHSP60) induced cardiomyocyte apoptosis via TLR-4.460 The extracellular release of Hsps may through different pathways, such as through necrotic cell lysis,461 secretary vesicles,462 or secretary lysosomal endosomes.463, 464

We propose that Hsp20S16D may be secreted from the cardiomyocytes into extracellular fluid in similar manners.

TLRs serve as pattern recognition receptors within the innate immune system.465 To date, 11 proteins have been identified that belong to the mammalian

TLR family.465 All TLRs are transmembrane proteins that consist of a leucine-rich repeat extracellular domain (for recognizing specific pathogens), a transmembrane region, and a Toll-IL-1R domain (for initiating intracellular signaling events).465

Specifically, activation of TLR4 results in an inflammatory response and is involved in extracellular matrix degradation, both key processes of LV remodeling.466 During inflammation and oxidative stress, TLR4 is also activated in response to endogenous ligands, such as heat shock protein Hsp60, resulting in the release of proinflammatory factors.467 Besides its role in inflammation, TLR4 stimulation in monocytes induces the production of matrix metalloproteinase (MMP9), which has been suggested to be a marker for extracellular matrix degradation.468 This points to a regulatory role for

TLR4 in inflammation and matrix turnover. In animal models, TLR4 has been shown to be involved in outward vascular remodeling, probably via activation by endogenous ligands and affecting collagen accumulation in the artery.469 Hence, we propose that cardiac overexpression of Hsp20S16D may serve as a circulating ligand for TLR4, and the subsequent activation of TLR4 may lead to inflammation responses in the heart, triggering cardiac dysfunction and remodeling.

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Several recent studies revealed that basal activation of autophagy was required for normal cellular function; whereas stress-related increases in autophagy can be maladaptive470 and contributed to cardiac remodeling.470 Consistently, in

Hsp20S16D overexpressing hearts, we observed an increase of autophagy activity at basal level. Furthermore, apoptosis and necrosis may also participate in cardiac remodeling,471 by triggering the interstitial fibrosis as a reaction to the loss of cardiomyocyte.472 Moreover, apoptosis, autophagy, and necrosis share several overlapping pathways.473 The activated autophagy in Hsp20S16D overexpressing hearts may increase apoptosis and necrosis, through their common regulators.396,178 Hence, we propose that overexpressing of Hsp20S16D may promote cell death through activated autophagy, apoptosis, or necrosis, resulting in cardiac dysfunction and remodeling.

IV.2 Future Studies

The mechanisms underlying cardiac dysfunction and remodeling in Hsp20S16D transgenic hearts may be explored, by measuring the expression level of TLRs

(especially TLR4), and autophagy related proteins (Beclin-1). Apoptosis will be compared in TG and wild type hearts by measuring Caspase 3 activity and DNA fragmentation. Levels of high-mobility group box 1 (HMGB1, also known as amphoterin), a proinflammatory mediator,474 released by necrotic cardiomyocytes will be measured by Western blot from TG and wild type cardiac homogenates.

IV.3 Summary

Chronic overexpression of constitutively phosphorylated Hsp20S16D resulted in

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depressed cardiac contractility and severe interstitial fibrosis. These transgenic mice died in their early adulthood. Furthermore, electron microscopy studies found that autophagosomes were accumulated in transgenic hearts, which were associated with increased lysosomal activity, suggesting that autophagosome accumulation was not due to diminished activity of distal lysosomal pathways. Our data indicate that long- term augmentation of cardiac Hsp20 phosphorylation impairs cardiac function, promotes pathological remodeling, and upregulates autophagic activity, leading to premature death. Future studies will be focused on exploring the underlying mechanisms.

Conclusion of the dissertation

Our current findings show that overexpression of Hsp20 increased cardiomyocyte SR Ca2+ content and contractile function through its direct association and regulation of PP1 activity, establishing that Hsp20 is an important modulator of

SR Ca2+ cycling. Furthermore, blockade of Hsp20 phosphorylation at Ser16 exacerbates cardiac ischemia/reperfusion injury by suppressing autophagy and increased cell death. Moreover, chronic overexpression of Hsp20S16D resulted in cardiac dysfunction and remodeling. Thus, maintenance of appropriate Hsp20 and its phosphorylation levels appears necessary for proper SR Ca2+-handling, cardiac function, and resistance to ischemia/reperfusion injury.

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Appendix A. Peer-Reviewed Publications

1. Journal of Molecular and Cellular Cardiology 2004 Aug; 37: 607-612 Threonine-17 phosphorylation of phospholamban: a key determinant of frequency- dependent increase of cardiac contractility. Wen zhao, Yoshiki Uehara, Guoxiang Chu, Qiujing Song, Jiang Qian, Karen Young, Evagelia G.Kranias

2. Circulation Research. 2005;96:756 Enhancement of Cardiac Function and Suppression of Heart Failure Progression by Inhibition of Protein Phosphatase 1 Anand Pathak, Federica del Monte, Wen Zhao, Bryan Mitton, Andrew Carr, Jo- ElShultz, Harvey Hahn, Faisal Syed , Ilona Bodi, Nirmala Mavila, Leena Jha, Emmett O’Neal, Jiang Qian, Guo-Chang Fan, Yehia Marreez, Dennis W. McGraw, E.KevinHeist, J. Luis Guerrero, Anna A. DePaoli-Roach,Roger J. Hajjar and Evangelia G. Kranias

3. Circulation. 2005;111:1792-1799 Novel Cardioprotective Role of a Small Heat-Shock Protein, Hsp20, Against Ischemia/Reperfusion Injury Guo-chang Fan, Xiaoping Ren, Jiang Qian, Qunying Yuan, Persoulla Nicolaou, Yang Wang, W.Keith Jones, Guoxiang Chu, Evagelia G. Kranias

4. Circulation. 2006 Feb 21;113(7):995-1004 The presence of Lys27 instead of Asn27 in human phospholamban promotes sarcoplasmic reticulum Ca2+-ATPase superinhibition and cardiac remodeling. Zhao W, Yuan Q, Qian J, Waggoner JR, Pathak A, Chu G, Mitton B, Sun X, Jin J, Braz JC, Hahn HS, Marreez Y, Syed F, Pollesello P, Annila A, Wang HS, Schultz Jel J, Molkentin JD, Liggett SB, Dorn GW 2nd, Kranias EG.

5. Circulation Research 2006;99:1233-1242

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Small Heat Shock Protein Hsp20 Attenuates -Agonist-Mediated Cardiac Remodeling Through Apoptosis Signal-Regulating Kinase 1 Guo-Chang Fan, Qunying Yuan, Guojie Song, Yigang Wang, Guoli Chen, Jiang Qian, Xiaoyang Zhou, Yong J. Lee, Muhammad Ashraf, Evangelia Kranias

6. Circ Res. 2008;21;103(11):1270-9 Heat shock protein 20 interacting with phosphorylated Akt reduces doxorubicin-triggered oxidative stress and cardiotoxicity Guo-Chang Fan, Xiaoyang Zhou, Xiaohong Wang, Guojie Song, Jiang Qian, Persoulla Nicolaou, Guoli Chen, Xiaoping Ren, Evangelia G. Kranias.

7. Circ Res. 2009;104(8):1012-20 Inducible expression of active protein phosphatase-1 inhibitor-1 enhances basal cardiac function and protects against ischemia/reperfusion injury. Persoulla Nicolaou, Patricia Rodriguez, Xiaoping Ren, Xiaoyang Zhou, Jiang Qian, Sakthivel Sadayappan, Bryan Mitton, Anand Pathak, Jeffrey Robbins, Roger J. Hajjar, Keith Jones, Evangelia G. Kranias

8. Circulation. 2009;119(17):2357-66 MicroRNA-320 is involved in the regulation of cardiac ischemia/reperfusion injury by targeting heat-shock protein 20. Xiao-Ping Ren, Jinghai Wu, Xiaohong Wang,Maureen A. Sartor, Jiang Qian, Keith Jones,Persoulla Nicolaou,Tracy J. Pritchard, Guo-Chang Fan.

9. Circ Res. 2009;105(12):1223-31 Blockade of Hsp20 Phosphorylation Exacerbates Cardiac Ischemia/Reperfusion Injury by Suppressed Autophagy and Increased Cell Death Jiang Qian, Xiaoping Ren, Xiaohong Wang, W. Keith Jones, Guo-Chang Fan, Evangelia G. Kranias

10. Stem Cells 2009;27(12):3021-31

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Hsp20-Engineered Mesenchymal Stem Cells Are Resistant to Oxidative Stress via Enhanced Activation of Akt and Secretion of Growth Factors Xiaohong Wang, Tiemin Zhao, Wei Huang, Tao Wang, Jiang Qian, Meifeng Xu, Evangelia G. Kranias, Yigang Wang, Guo-Chang Fan

11. PNAS 2009 Nov 17 The Anti-apoptotic Protein HAX-1 is a Novel Regulator of Cardiac Function Wen Zhao, Jason R. Waggoner, Zhi-Guo Zhang, Chi Keung Lam, Peidong Han, Jiang Qian, Paul M. Schroder, Bryan Mitton, Aikaterini Kontrogianni-Konstantopoulos, Seth L. Robia, Evangelia G. Kranias

B. Presentations, abstracts and meetings

1. 2004 AHA (American Heart Association) Scientific Session Cardiac-specific overexpression of small heat-shock protein HSP20 alleviates ischemia/reperfusion injury and myocardial dysfunction Guo-chang Fan, Xiaoping Ren, Jiang Qian, Qunying yuan, Persoulla Nicolaou,k Yang Wang, W.Keith Jones, Guoxiang Chu, Evangelia G. Kranias

2. 2004, 25th Graduate Student Research Forum, UC Collage of Medicine Cardiac-Specific Overexpression of Small Heat-Shock Protein Hsp20 Alleviates Ischemia/Reperfusion Injury and Myocardial Dysfunction Jiang Qian#, Guo-chang Fan#, Qunying Yuan, Xiaoping Ren, W.Keith Jones, Guoxiang Chu, Evangelia G. Kranias (# First author)

3. 2005 AHA Scientific Session (Session No: APS.31.4) Suppression of p38MAPK activation by a small heat shock protein HSP20 attenuates isoproterenol-mediated myocardial injury Guo-chang Fan, Qunying Yuan, Jiang Qian, Persoulla Nicolaou, GuoJie Song, Guoli Chen, Guoxiang Chu, Evangelia G. Kranias

4. 2005 BCVS (AHA Council on Basic Cardiovacular Sciences Session No:I)

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Novel Cardioprotective Effects by the Small Heat Shock Protein Hsp20 Guo-Chang Fan, Qunying Yuan, Jiang Qian, Persoulla Nicolaou, Guojie Song, Guoli Chen, Guoxiang Chu, Evangelia G Kranias

5. ISP 2006 (5th International Congress of Pathology), Beijing, China Small heat shock protein 20 (HSP20) protects against ischemia/reperfusion injury in Hearts Jiang Qian, Guo-chang Fan, Qunying Yuan, Xiaoping Ren, W.Keith Jones, Guoxiang Chu, Evangelia G. Kranias

6. 2006 AHA Scientific Session Phosphorylation of Small Heat Shock Protein 20 (Hsp20) at Serine 16 Modulates Cardiac Contractility Circulation Research 2006,114(18):II-315 Jiang Qian, Guo-Chang Fan, Qunying Yuan, Evangelia Kranias

7. 2006 AHA Scientific Session Temporally Induced Expression of Active Protein Phosphatase Inhibitor-1 in the Adult Mouse Heart Enhances Cardiac Function Circulation Research 2006,114(18):II-314 Patricia Rodriguez, Persoulla Nicolaou, Jiang Qian, Bryan Mitton, Anand Pathak, Jeffrey Robbins, Evangelia Kranias

8. 2007, 28th Graduate Student Research Forum, UC Collage of Medicine Phosphorylation of Small Heat Shock Protein 20 at Serine 16 Protect against Cardiac Ischemia/Reperfusion Injury Jiang Qian, Guo-Chang Fan, Evangelia Kranias

9. 2007 AHA Scientific Session Phosphorylation of Small Heat Shock Protein 20 at Serine 16 Is Essential in Cardiac Protection against Ischemia/Reperfusion Injury Circulation. 2007;116:II-68

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Jiang Qian, Guo-Chang Fan, Evangelia Kranias

10. 2007 AHA Scientific Session Hsp20 Inhibits Doxorubicin-triggered Cardiac Injury Via PI3 Kinase-Akt Dependent Pathway Circulation. 2007;116:II-9 Guo-Chang Fan; Xiaoyang Zhou; Guojie Song; Jiang Qian; Sarah Figueria; Guoli Chen; Evangelia G Kranias

11. 2007 AHA Scientific Session Enhancement of Myocardial Function by Hsp20 is Associated with Decreased Type-1 Phosphatase Activity Circulation. 2007;116:II-189 Guo-Chang Fan; Qunying Yuan; Jiang Qian; Xiaoyang Zhou; Jason R Waggoner; Guoli Chen; Evangelia G Kranias

12. 2008 18th World Congress of the International Society of Heart Research Cincinnati Phosphorylation of Small Heat Shock Protein 20 at Serine 16 Is Essential in Cardiac Protection against Ischemia/Reperfusion Injury Jiang Qian, Guo-Chang Fan, Evangelia Kranias

C. Honors and Awards

1. 3rd Place in 25th Graduate Student Research Forum UC Collage of Medicine, Nov 2004 2. Travel Awards for Graduate Student, University of Cincinnati, 2006 3. 2nd Place in 28th Graduate Student Research Forum UC Collage of Medicine, Nov 2007 4. Travel Awards for Graduate Student, University of Cincinnati, 2007

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