Effect of Post-Translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle

Effect of Post-translational Modification Crosstalk on Thin Filament Regulatory Function in Cardiac Muscle

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Benjamin R. Nixon

Graduate Program in Biomedical Sciences

The Ohio State University

2014

Dissertation Committee:

Brandon J. Biesiadecki, Ph. D. (Advisor)

Jonathan P. Davis, Ph. D.

Michael A. Freitas, Ph. D.

Mark T. Ziolo, Ph. D.

Benjamin R. Nixon

2014

Abstract

Heart disease, with an anticipated $316 billion in economic expenses, affects one in three adults and is the leading cause of death in the United States. In the diseased heart a multitude of cellular changes occur, either as a compensatory mechanism to deter the modifications brought on as a result of cardiac disease or a direct result of the pathophysiology. One such avenue for uncovering the molecular mechanisms underlying cardiac disease and their functional changes is to study post-translational modification

(PTM) of proteins. While extensive work has been done characterizing phosphorylation of cardiac contractile regulatory proteins, this work has been conducted investigating the modifications in isolation. Despite the fact that a single phosphorylation site may be sufficient to alter function, the additive functional effect of multiple phosphorylation sites to crosstalk inter-molecularly and change function differently than that evoked by a single phosphorylation must be taken into consideration.

Initially, we sought to determine if the metabolic regulatory kinase AMPK phosphorylates cardiac troponin I (cTnI) at Ser-150 in vivo to alter cardiac contractile function directly at the level of the myofilament. Rabbit cardiac myofibrils separated by two-dimensional isoelectric focusing subjected to a Western blot with a cTnI phosphorylation-specific antibody demonstrate that cTnI is endogenously phosphorylated at Ser-150 in the heart. Treatment of myofibrils with the AMPK holoenzyme increased cTnI Ser-150 phosphorylation within the constraints of the muscle lattice. Compared with ii controls, cardiac fiber bundles exchanged with troponin containing cTnI pseudo- phosphorylated at Ser-150 demonstrate increased sensitivity of calcium-dependent force development, blunting of both PKA-dependent calcium desensitization, and PKA- dependent increases in length dependent activation.

We next wanted to investigate the effect of ischemic pH on Ser-150 and Ser-

23/24 phosphorylation. We demonstrate phosphorylation of cTnI is simultaneously increased at Ser-150 and Ser-23/24 during in vivo myocardial ischemia. Myocardial ischemia is known to decrease intracellular pH directly resulting in depressed Ca2+ binding to Tn and impaired contraction. To determine the pathological relevance of these simultaneous TnI phosphorylations in ischemia we measured the individual effects of TnI

Ser-150 (S150D), Ser-23/24 (S23/24D) or their combined (S23/24/150D) pseudo- phosphorylation on thin filament regulation at acidic pH similar to that in myocardial ischemia. Results demonstrate that while acidic pH decreased thin filament Ca2+ binding of all TnIs, TnI S150D attenuated this decrease such that it was similar to non- phosphorylated TnI at normal pH. The dissociation of Ca2+ from troponin C (TnC) was unaltered by pH, such that TnI S150D remained slow, S23/24D remained accelerated and the combination of Ser-150 and Ser-23/24 pseudo-phosphorylation on the same TnI molecule retained accelerated dissociation.

Lastly, to investigate the remaining component of thin filament regulation, tropomyosin nitration or phosphorylation was investigated to determine the effect of

PTM on structure and function. To investigate the kinetic regulatory role of αTm phosphorylation we expressed and purified native N-terminal acetylated Ser-283 wild-

iii type, S283A phosphorylation null and S283D pseudo-phosphorylation Tm mutants in insect cells. Purified Tm's regulate thin filaments similar to that reported for muscle purified Tm. Steady-state Ca2+ binding to TnC in reconstituted thin filaments did not differ between the three Tm's, however dissociation of Ca2+ from filaments containing pseudo-phosphorylated Tm was slowed compared to wild-type Tm. Replacement of pseudo-phosphorylated Tm into myofibrils similarly prolonged the slow phase of relaxation and decreased the rate of the fast phase without altering activation kinetics.

Additionally, we sought to investigate the effect of reactive nitrogen species to nitrate Tm

Tyr residues, its structure-function impact and develop a mass spectrometry approach to identify Tm 3-nitotyrosin (3-NT) PTM. Our data demonstrates the pathologically relevant reactive nitrogen species peroxynitrite modifies Tm Tyr residues to 3-NT with structural impact significant to modulate Tm function. We further developed and validated a novel and highly versatile target-driven MS/MS strategy to facilitate identification and quantification of Tm 3-NT without a priori knowledge of target residue modification.

In conclusion, the structural and functional modification of thin filament regulatory proteins described above provide an intricate glimpse into the mechanisms in which the heart can undergo to alter function both in normal physiology and in disease states. Gaining a better understanding of protein PTMs associated with cardiac disease will play a major role in the continued development of therapeutics to treat cardiovascular disease.

Dedication

IN LOVING MEMORY OF JOSEPH AND DORIS COTTER, THEODORE NIXON, &

KATHERINE WELLS

Acknowledgments

Much of the work completed during my dissertation could not have been done without insight and help from others and I would be remiss not to mention and thank those who continuously directed and assisted me over the years. First and foremost I would like to humbly thank my graduate advisor Dr. Brandon Biesiadecki. Brandon’s experience and incessant enthusiasm for his research facilitated my own interest in muscle physiology giving me a fantastic research experience. Furthermore, his invaluable guidance has always taught me to evaluate work critically and maintain my own scientific integrity by not cutting corners when carrying out and conveying my research. Likewise, I would sincerely like to thank Dr. Jonathan Davis, Dr. Mark Ziolo, and Dr. Michael Freitas as their lively conversations and insight as my committee proved to be an aspect instrumental in my research. I would like to thank Dr. Gregory Caputo for not only being likely the best teacher during my time at Rowan University but for being an outstanding lab mentor and fostering my passion for scientific research (as well as frequent Tecmo sessions). Thanks to my advanced chemistry teacher in high school Gerald Biggs for undoubtedly showing me that chemistry, and science in general, is a significantly better career path for me than my initially planned accounting. I cannot thank him enough for kick starting the foundation of interest in science I have today. I would also like to sincerely thank all of the labs and members associated with the Molecular and Cellular

Cardiophysiology group at The Ohio State University. This group has made presenting scientific work a beneficial and spirited event which I can only hope exists in a similar fashion in my future endeavors. To Elizabeth Brundage and Nathan Hassel in the

Biesiadecki lab, it has been a great experience working together and I thank them for all their help both technically and intellectually during my studies in the lab. Many thanks to my fellow Biomedical Sciences graduate program student in the Biesiadecki lab Hussam

Salhi who has provided me with numerous conversations discussing our work and the work of the field. Such dialogue has helped guide my thoughts and open my mind to new possibilities. You are going to do good things in the future. A special thanks to my friend that I met while at Rowan University Joe Garofalo for consistently being a great friend and providing me with a way to temporarily forget the stresses associated with completing my doctoral work by means of audiovisual competitions. Lastly, I would like to thank my family and loved ones for their undying support during my time at The Ohio

State University. I want to give a special thanks to my loving girlfriend Mellissa Hicks for always offering an ear to talk to and being incredibly supporting and understanding when times were bumpy, albeit because of school or health. I also want to thank my family for continuously supporting me as well as recurrently making the trek out to

Columbus from New Jersey to visit. I cannot stress how important it was to be able to see a piece of home frequently during my five years away. All of my successes would not be possible without those mentioned above, and for that I am forever grateful.

vii

Vita

2005...... Palmyra High School

2009...... B.S. Biochemistry, Rowan University

2009 to present ...... Graduate Research Associate, Department

of Physiology and Cell Biology, The Ohio

State University

Publications

1. Nixon, BR, A Thawornkaiwong, J Jin, EA Brundage, SC Little, JP Davis, RJ Solaro, and BJ Biesiadecki. "AMP-activated Protein Kinase Phosphorylates Cardiac Troponin I at Ser-150 to Increase Myofilament Calcium Sensitivity and Blunt PKA-dependent

Function." J. Biol. Chem. 287 (2012): 19136-9147.

2. Nixon, BR, B Liu, B Scellini, C Tesi, N Pirrodi, O Ogut, RJ Solaro, MT Ziolo, PML

Janssen, JP Davis, C Pogessi, and BJ Biesiadecki. "Tropomyosin Ser-283 Pseudo- phosphorylation Slows Myofibril Relaxation." Arch. Biochem. Biophys. (2013): 30-381.

viii

3. Nixon, BR, SD Walton, B Zhang, EA Brundage, SC Little, MT Ziolo, JP Davis, and

BJ Biesiadecki. "Combined Troponin I Ser-150 and Ser-23/24 Phosphorylation Sustains

Thin Filament Ca2+ Sensitivity and Accelerates Deactivation in an Acidic Environment."

J. Mol. Cell. Cardiol. Forthcoming 2014.

Fields of Study

Major Field: Biomedical Sciences Graduate Program

1. Emphasis: Cellular and Molecular Physiology

2. Cardiac and Muscle Physiology

Table of Contents

Abstract ...... ii

Dedication ...... v

Acknowledgments...... vi

Vita ...... viii

Table of Contents ...... x

List of Tables ...... xix

List of Figures ...... xx

Chapter 1: Introduction ...... 1

1.1 General Introduction ...... 1

1.2 Ca2+ Cycling in the Cardiomyocyte ...... 2

1.2.1 Excitation-contraction Coupling to Produce Force ...... 2

1.3 Sarcomeric Structure and Functional Components ...... 4

1.3.1 Sarcomere Structure ...... 4

1.3.2 The Cardiac Thick Filament ...... 5

1.3.3 The Cardiac Thin Filament ...... 8

1.4 Cardiac Troponin I Structure and Function...... 11

1.4.1 Troponin I Structure Overview...... 11

1.4.2 Unique N-terminal Extension ...... 12

1.4.3 The Inhibitory Peptide ...... 13

1.4.4 The Switch Peptide ...... 15

1.4.5 C-terminal Region ...... 16

1.4.6 pH-sensitive Histidine Button ...... 17

1.5 Tropomyosin Structure and Function ...... 18

1.5.1 Tropomyosin Structure Overview ...... 18

1.5.2 Flexibility and Actin Binding ...... 19

1.5.3 N and C-terminal Domains ...... 21

1.5.4 TnT Binding ...... 22

1.5.5 Role in Cardiac Contraction and Cooperativity ...... 23

1.6 Modulation of Cardiac Output ...... 24

1.6.1 β-adrenergic Signaling Pathway ...... 25

1.6.2 Phosphorylation of Troponin I ...... 25

1.6.3 Phosphorylation of Tropomyosin ...... 28

1.7 Cardiac Ischemia ...... 30

1.7.1 Metabolic Alterations and Decreased pH in Ischemia ...... 30

1.7.2 Role of AMPK ...... 31

1.7.3 Generation of Free Radicals During Reperfusion ...... 33

1.8 Overall Goal of Dissertation ...... 35

Chapter 2: AMP Activated Protein Kinase Phosphorylates Cardiac Troponin I at Ser-150 to Increase Myofilament Calcium Sensitivity and Blunt PKA Dependent Function ...... 45

2.1 Introduction ...... 45

2.2 Materials and Methods ...... 47

2.2.1 cDNA constructs...... 47

2.2.2 Proteins ...... 47

2.2.3 Kinase treatments ...... 48

2.2.4 Protein electrophoresis, staining and Western blot ...... 48

2.2.5 2-D isoelectric focusing ...... 50

2.2.6 Exchange of recombinant cardiac troponin into skinned mouse cardiac fiber

bundles ...... 50

2.2.7 Measurement of isometric tension ...... 51

2.2.8 Measurement of thin filament steady-state Ca2+ binding to TnC ...... 51

2.2.9 Data processing and statistical analysis ...... 52

2.3 Results ...... 52

2.3.1 The AMPK holoenzyme complex phosphorylates cTnI at Ser-150...... 52

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2.3.2 cTnI Ser-150 phosphorylation increases myofilament Ca2+ sensitive force

development and blunts PKA dependent Ca2+ desensitization...... 55

2.3.3 cTnI Ser-150 phosphorylation increases Ca2+ sensitivity by altering Ca2+

binding to TnC...... 56

2.3.4 cTnI Ser-150 phosphorylation alone does not alter length dependent activation

but blunts cTnI PKA dependent induced length dependent activation...... 58

2.3.5 cTnI Ser-150 phosphorylation does not alter cTnI PKA phosphorylation...... 59

2.4 Discussion ...... 60

2.4.1 AMPK cTnI Ser-150 phosphorylation...... 60

2.4.2 The effects of cTnI Ser-150 phosphorylation on Ca2+ regulation of the thin

filament...... 62

2.4.3 AMPK signaling and the modulation of myofilament cardiac contractility. ... 64

2.5 Conclusions ...... 66

Chapter 3: Combined Troponin I Ser-150 and Ser-23/24 Phosphorylation Sustains Thin

Filament Ca2+ Sensitivity and Accelerates Deactivation in an Acidic Environment ...... 77

3.1 Introduction ...... 77

3.2 Materials and Methods ...... 80

3.2.1 In vivo myocardial ischemia ...... 80

3.2.2 Protein electrophoresis and Western blot ...... 80

3.2.3 cDNA constructs...... 81 xiii

3.2.4 Proteins ...... 82

3.2.5 Measurement of Tn steady-state Ca2+ binding to TnC ...... 82

3.2.6 Measurement of thin filament and Tn Ca2+ dissociation from TnC ...... 82

3.2.7 Measurement of myosin S1 dissociation from the thin filament ...... 83

3.2.8 Data processing and statistical analysis ...... 83

3.3 Results ...... 84

3.3.1 In vivo myocardial ischemia increases both TnI Ser-150 and Ser-23/24

phosphorylation...... 84

3.3.2 TnI Ser-150 and Ser-23/24 phosphorylation alters the acidic effects of thin

filament regulation...... 84

3.3.3 The phosphorylation of TnI Ser-150 and Ser-23/24 affect function through

different thin filament interactions...... 85

3.3.4 The combination of TnI Ser-150 with Ser-23/24 phosphorylation retains

accelerated thin filament deactivation...... 86

3.4 Discussion ...... 88

3.4.1 Myocardial ischemia simultaneously increases phosphorylation of both TnI

Ser-150 and Ser-23/24...... 89

3.4.2 TnI Ser-150 phosphorylation differentially modulates thin filament Ca2+

regulation...... 90

3.4.3 TnI Ser-150 and Ser-23/24 structure-function...... 93 xiv

3.4.4 Functional consequences of ischemia-induced TnI phosphorylation...... 94

3.5 Conclusions ...... 95

Chapter 4: Tropomyosin Ser-283 Phosphorylation Slows Myofibril Relaxation ...... 105

4.1 Introduction ...... 105

4.2 Materials and Methods ...... 107

4.2.1 Baculoviral Spodoptera frugiperda (Sf9) insect cell expression and purification

of recombinant Tm...... 107

4.2.2 Protein purification...... 109

4.2.3 Gel electrophoresis and Western Blot...... 110

4.2.4 Myosin S1 ATPase in reconstituted thin filaments...... 110

4.2.5 Ca2+ binding to TnC in reconstituted thin filaments...... 111

4.2.6 Myosin S1 binding to Tm decorated actin...... 111

4.2.7 Ca2+ dissociation from TnC in reconstituted thin filaments...... 112

4.2.8 Myofibril experiments: Preparation, Tm-Tn replacement, force and kinetic

measurements...... 112

4.2.9 Statistical analysis...... 115

4.3 Results ...... 115

4.3.1 Expression and purification of N-terminal acetylated Tm...... 115

4.3.2 Pseudo-phosphorylated Tm increases maximal ATPase Activity...... 116

4.3.3 Tm pseudo-phosphorylation does not alter steady-state Ca2+ sensitivity of the

thin filament...... 117

4.3.4 Tm pseudo-phosphorylation decreases the rate Ca2+ of dissociation from

regulated thin filaments...... 119

4.3.5 Tm pseudo-phosphorylation slows muscle force relaxation...... 119

4.4 Discussion ...... 121

4.4.1 Pseudo-phosphorylated Tm functions as a model of Ser-283 phosphorylated

muscle Tm...... 121

4.4.2 Tm Ser-283 pseudo-phosphorylation does not alter steady-state myofilament

regulatory function...... 123

4.4.3 Tm Ser-283 pseudo-phosphorylation slows dynamic muscle relaxation...... 123

4.5 Conclusions ...... 126

Chapter 5: Identification and Characterization of Tropomyosin Nitration ...... 137

5.1 Introduction ...... 137

5.2 Materials and Methods ...... 139

5.2.1 Expression of Tm...... 139

5.2.2 Nitration of Tm Tyrosine ...... 140

5.2.3 Protein Electrophoresis And Western Blot ...... 140

5.2.4 Circular Dichroism and Thermal Denaturation ...... 141

5.2.5 Measurement of nitrated Tm binding to isolated TnT or whole Tn ...... 141 xvi

5.2.6 Steady-state Ca2+ binding in the reconstituted thin filament...... 142

5.2.7 Protein Digestion ...... 142

5.2.8 LC-MS ...... 143

5.2.9 LC-MS/MS ...... 144

5.2.10 Selected Ion Monitoring (SIM) ...... 144

5.2.11 Data analysis ...... 145

5.3 Results ...... 145

5.3.1 Western blot and structure-function analysis...... 145

5.3.2 Mass spectrometric identification of Tm tyr nitration...... 147

5.3.4 Quantification of nitrotyrosine and nitrosyltyrosine...... 148

5.4 Discussion ...... 149

5.4.1 Characteristics of Tm nitration ...... 149

5.4.2 Tm nitration occurs in a specific, reproducible manner...... 150

5.4.3 Effect of Tm nitration on flexibility...... 151

5.4.4 In vivo implications of Tm nitration...... 152

5.5 Conclusions ...... 153

Chapter 6: Discussion ...... 164

6.1 Principle Findings ...... 164

6.1.1 Chapter 2...... 164

xvii

6.1.2 Chapter 3...... 164

6.1.3 Chapter 4...... 165

6.1.4 Chapter 5...... 165

6.2 Overall Implications and Therapeutic Potential ...... 166

6.2.1 TnI Ser-150 and Ser-23/24 Phosphorylation ...... 166

6.2.2 Tm Phosphorylation ...... 170

6.2.3 Tm Nitration ...... 172

6.3 Future Directions ...... 174

6.4 Final Remarks ...... 180

References ...... 181

xviii

List of Tables

Table 1. Mechanical characteristics of exchanged fiber bundles...... 67

Table 2. Thin filament TnC Ca2+ binding characteristics...... 68

Table 3. Steady-state Ca2+ binding to TnC in reconstituted thin filaments containing WT,

S150D, S23/24D, or S23/24/150D Tn at pH 6.5 or pH 7.0...... 96

Table 4. Steady-state Ca2+ binding to TnC in isolated Tn containing WT, S150D,

S23/24D, or S23/24/150D TnI at pH 6.5 or pH 7.0...... 97

Table 5. Ca2+ dissociation from TnC in reconstituted thin filaments containing WT,

S150D, S23/24D, or S23/24/150D Tn at pH 6.5 or pH 7.0...... 98

Table 6. Ca2+ dissociation from TnC in reconstituted thin filaments containing WT,

S150D, S23/24D, or S23/24/150D Tn and myosin S1 at pH 6.5 or pH 7.0...... 99

Table 7. Effects of Tm S283 WT, Tm S283A phosphorylation null or Tm S283D pseudo- phosphorylation reconstitution on thin filament calcium binding characteristics...... 127

Table 8. Effects of Tn–Tm extraction and reconstitution with skeletal Tn and WT, phosphorylation null, or pseudo-phosphorylation Tm on rabbit psoas myofibrils tension kinetics...... 128

Table 9. Percent nitration and nitrosylation at specific Tm Tyr residues ...... 155

xix

List of Figures

Figure 1. Schematic representation of excitation-contraction in the cardiomyocyte...... 37

Figure 2. Schematic representation of distinct regions across two sarcomeres...... 38

Figure 3. Five-step schematic representation of crossbridge cycling...... 39

Figure 4. Schematic representation of the TnI in the thin filament ...... 40

Figure 5. Schematic representation of Tm dimer structure and interactions...... 41

Figure 6. Parameters of cardiac output...... 42

Figure 7. Schematic representation of β-adrenderic stimulation pathway...... 43

Figure 8. Schematic representation of possible phosphorylation sites in TnI...... 44

Figure 9. The AMPK holoenzyme complex phosphorylates cTnI Ser-150 in Tn...... 69

Figure 10. Native rabbit cardiac muscle contains cTnI phosphorylated at Ser-150...... 70

Figure 11. The AMPK holoenzyme phosphorylates cTnI Ser-150 in the cardiac muscle lattice...... 71

Figure 12. Cardiac TnI Ser-150 phosphorylation increases myofilament Ca2+ sensitive force development and blunts cTnI PKA-dependent desensitization...... 72

Figure 13. Cardiac TnI Ser-150 phosphorylation induced increase in sub-maximal force development results from increased Ca2+ binding to TnC...... 73

Figure 14. The incorporation of native phosphate at cTnI Ser-150 blunts Ser-23/24 decreased Ca2+ binding to TnC...... 74

Figure 15. Cardiac TnI Ser-150 phosphorylation blunts the cTnI PKA dependent increase in length-dependent activation...... 75

Figure 16. Cardiac TnI Ser-150 phosphorylation does not alter cTnI PKA-dependent phosphorylation...... 76

Figure 17. Western blot of TnI Ser-150 and Ser-23/24 phosphorylation in ischemia cardiac tissue...... 100

Figure 18. TnI phosphorylation steady-state Ca2+ binding in the reconstituted thin filament at ischemic and normal pH...... 101

Figure 19. TnI phosphorylation steady-state Ca2+ binding in isolated Tn at ischemic and normal pH...... 102

Figure 20. Ca2+ dissociation kinetics of phosphorylated TnI in the reconstituted thin filament at ischemic and normal pH...... 103

Figure 21. Myosin dissociation kinetics of phosphorylated TnI in the reconstituted thin filament with myosin S1 at ischemic and normal pH...... 104

Figure 22. Baculoviral Tm expression and purification in Sf9 cells...... 129

Figure 23. Sf9 expressed Tm is N-terminal acetylated...... 130

Figure 24. Myosin S1 ATPase of Tm variant reconstituted thin filaments...... 131

Figure 25. Ca2+ binding to TnC in reconstituted thin filaments...... 132

Figure 26. Myosin S1 binding to Tm decorated actin...... 133

Figure 27. Dynamic Ca2+ dissociation from TnC...... 134

Figure 28. Tm exchanged myofibril tension kinetics...... 135

Figure 29. Reaction of peroxynitrite (ONOO-) with Tyr...... 156

xxi

Figure 30. Nitration of αTm by ONOO- occurs in a dose-dependent manner...... 157

Figure 31. αTm nitration results in altered structure and decrease in stability...... 158

Figure 32. Nitrated Tm alters binding affinity of both isolated TnT and whole Tn...... 159

Figure 33. Steady-state Ca2+ sensitivity is reduced in thin filaments containing nitrated

Tm...... 160

Figure 34. LC-MS of nitrated or non-nitrated αTm...... 161

Figure 35. Sequence coverage of αTm exposed to ONOO- and identification of peptides with nitrated Tyr by LC-MS/MS...... 162

Figure 36. Selected ion monitoring experiment data of nitration and nitrosylation on each

Tyr site...... 163

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Chapter 1: Introduction

1.1 General Introduction

The heart, a four-chambered organ located in the center of the chest between the lobes of the lungs, has widely been viewed as a symbolic representation of emotion and love. Yet, at its core the heart functions as a workhorse to drive the circulation of nutrient rich blood to and from various organs in the body, a process necessary for survival. To do so, the heart muscle contracts to expel blood from its chambers and subsequently relaxes to allow for the refilling of blood. When this physiological process becomes interrupted by direct insult to the heart, the heart is no longer capable of keeping up with the body’s demand for oxygenated blood leading to heart disease. Heart disease, resulting in

~600,000 deaths per year and totaling over 100 billion dollars in health care costs, is the leading cause of death among men and women in the United States [1]. Great strides have been made in the field of medicine to diagnose and treat the damaged heart such as the development and use of β-adrenergic receptor blockers. However, heart disease remains prevalent even with such therapeutic developments underscoring the need for novel therapeutic approaches. The work discussed below investigates modifications made to the contractile proteins of the heart and how they may play a role in physiology and disease to potentially provide a new target for therapeutic development to improve quality of life.

1.2 Ca2+ Cycling in the Cardiomyocyte

In order for the heart to function properly it must undergo two events – electrical stimulation and mechanical contraction of the heart muscle. To produce one beat of the heart, electrical stimulation of the muscle occurs to induce contraction and subsequent expulsion of blood from the ventricles. Following ejection of blood, the heart relaxes to complete one round in the cardiac cycle and begins filling with blood from the superior and inferior vena cava to initiate the next cycle. A detailed view of how the electrical signal is transmitted to mechanical force will be discussed below.

1.2.1 Excitation-contraction Coupling to Produce Force

Generation of an electrical impulse that spreads from the base of the heart to the apex occurs as a means to induce contraction of the heart. Depicted in Figure 1, cardiomyocytes contain a specific process to transmit the electrical signal to a mechanical signal to produce force termed excitation-contraction coupling (ECC). In a resting state, cardiomyocytes maintain a negative membrane potential; however, upon stimulation that crosses a threshold for action potential firing, cardiomyocyte permeability to sodium increases greatly resulting in an increase in membrane potential depolarizing the myocyte. Subsequently there is a rapid increase in intracellular Ca2+ accounting for 30% of total Ca2+ primarily because of the voltage-sensitive L-type Ca2+ channels (LTCC ) opening to allowing Ca2+ to enter the cell [2]; however, this contribution is much higher in smaller mammals. Once Ca2+ has entered the cell it binds to the ryanodine receptor

(RyR) located in the membrane of the sarcoplasmic reticulum (SR), the major Ca2+

2 storage compartment in the cardiomyocyte capable of storing millimolar amounts of

Ca2+. Upon binding of Ca2+ to the RyR, Ca2+ is further released from the SR, a processed known as calcium-induced calcium release (CICR). During CICR intracellular Ca2+ becomes elevated increasing diastolic, or resting, Ca2+ levels from 100nM to 600nM, a level suitable to activate the myofilaments and allow for contraction [3]. However, it should be noted that the amount of Ca2+ released from the SR, known as the SR load, can vary depending on events occurring from beat to beat in the heart and that only ~20% of

2+ Tn are occupied by Ca providing a reservoir to modulate function [4].

Following contraction of the cardiomyocyte, Ca2+ levels will begin to fall to allow for the cell to exit the contracted state and return to the relaxed state, a process facilitated by the sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA). Through the hydrolysis of one ATP molecule, two Ca2+ ions are transported across the SR membrane however the small protein phospholamban (PLB) plays a role in regulating the amount of

Ca2+ being transported. In its unphosphorylated state, PLB acts to inhibit the SERCA pump and this inhibition is removed upon phosphorylation during β-adrenergic stimulation [5]. Additionally, while playing a lesser role but a role nonetheless, NCX aids in the removal of Ca2+ initially brought in by the LTCC by extruding one Ca2+ ion from the cell and bringing in three Na+ ions. The process of ECC is a tightly coupled mechanism in which two goals are accomplished: 1) to increase Ca2+ to a level for myofilament activation and contraction and 2) decrease Ca2+ to a level for myofilament deactivation and relaxation. These two goals are critical to allow for proper contraction and relaxation of the myofilament, as detailed in the next section.

1.3 Sarcomeric Structure and Functional Components

The sarcomere is the most basic functional unit in the cardiomyocyte at 1.6-

2.2µm and is responsible for force generation in each cardiomyocyte. It is composed of a variety of proteins that help maintain structure and contribute to the overall function, however the thin and thick filament are the primary contributors to contraction of the sarcomere. In this section, the ultrastructure of the sarcomere will be discussed as well as a detailing of the thin and thick filaments and how they contribute to the function of the sarcomere.

1.3.1 Sarcomere Structure

As previously mentioned, the sarcomere is composed of myosin thick filament and actin thin filament. The organization of these filaments in the muscle lattice results in an appearance of striation and can be categorized into five different areas (see Fig. 2). A single sarcomere is defined between two dark bands on an electron micrograph known as the Z-line or Z-disc. The Z-disc is primarily responsible for the physical connection of thin filaments, and as a whole the myofibrils to the extracellular matrix and the sarcolemma. Additionally, it serves a function to allow for the uniform transmission of force across the myofibril [6]. The M-line, located at the center of the sarcomere, is responsible for organizing and stabilizing the myosin filament that extends in opposite directions of the M-line. The I-band is the region of thin filament actin that is not overlaid with thick filament myosin. The A-band is comprised of the entire thick filament myosin length; however there is some overlap with the thin filament actin. Lastly, the H-zone

4 represents the region of thick filament that is not overlaid with thin filament. All of these regions together form the contractile quantum of the heart with the ultimate goal being to generate shortening and force. Identified in 1953 by Huxley, the process of contraction was observed as a result of thin filament actin sliding from repetitive association and dissociation of thick filament myosin giving rise to the sliding filament theory [7, 8].

1.3.2 The Cardiac Thick Filament

The thick filament is comprised of helical bipolar polymers containing approximately 300 hexameric myosin motor proteins as well as additional regulatory proteins myosin light chain-2 (MLC-2), essential light chain (ECL), and myosin binding protein C (MyBP-C) [9]. Myosin acts as the mechanical lever arm in the thick filament and consists of a head domain, a neck domain, and a tail domain. The N-terminal head domain is where two subfragment-1 (S1) are located to form the globular head region of myosin that binds ATP and divalent cations to interact with actin to contract the sarcomere. While it was shown that the S1 fragment alone is sufficient to move actin filaments in vitro, function in vivo requires multiple domains [10]. The neck domain, located just below the head domain, is responsible for transducing the force generated from the catalytic domain of myosin and contains two MLC-2 and two ECL wrapped around it for stabilization. Lastly, the tail region of myosin forms a coiled-coil α-helical region at the C-terminus and functions to form a scaffold for the thick filament as well as a binding site for the myosin regulatory protein MyBP-C. Myosin can exist in either the slower high force generating β or the faster low force generating α isoform but it is β

5 myosin that exists predominantly in the ventricle (90:10); however, this ratio differs in rodents as higher frequency contractions are required [11].

During contraction it is believed that no more than four myosin S1 heads attach to one 7-actin component with only a total of 20-40% of total myosin attaching indicating one to two myosin per seven actin [12] through an ATP-dependent mechanism called the crossbridge cycle (for in depth description see [13]). As shown in Figure 3, the filaments are initially found in the non-activated state where Tm-covered actin and ATP-bound myosin remain detached. Next, the hydrolysis of the bound ATP to form ADP and inorganic phosphate (Pi) places the myosin molecule in an activated conformation to weakly interact with actin if present. Following the Ca2+ transient, Tm undergoes a 30° azimuthal shift on the thin filament to allow for the strong attachment of myosin to actin

[14]. Release of Pi initiates a power stroke in which myosin utilizes its lever arm conformation to induce a sliding of the thin filament in the opposite direction. Myosin then undergoes isomerization and subsequent release of the remaining ADP molecule.

Following the power stroke and sliding of the thin filament, myosin rapidly dissociates from actin upon binding of ATP and the cycle can repeat. The cyclic nature of this process is ultimately what allows the heart to circulate blood efficiently throughout the body.

Acting as a regulator of myosin function is MyBP-C (for in depth review see

[15]). Located within the A-band and accounting for 1-2% of total myofibrillar proteins,

MyBP-C interacts with an array of myofilament proteins including myosin, actin and titin, as well as within itself to also regulate the rate and magnitude of force generated by

6 myosin [16]. MyBP-C contains four phosphorylation sites, two targeted by

Ca2+/calmodulin kinase (CAMK) and two by protein kinase A (PKA), that have the potential to act as a regulator to alter myosin crossbridge formation with actin by means of reducing lag time in the crossbridge cycle and thus accelerating crossbridge cycling rates [17]. Additionally, the myosin neck stabilizer MLC-2 can undergo PTM to alter the crossbridge cycle. Initially discovered in 1973, Perrie and colleagues demonstrated that

MLC-2 phosphorylation could potentiate force development in intact fibers [18, 19] later shown to occur by inducing an extension of the myosin backbone to place the crossbridge in a more favorable position to interact with the actin thin filament [20]. The regulatory capacity of MyBP-C and MLC-2 is a crucial component in modulating the interaction between the thick filament and the components of the thin filament during contraction.

As part of the first proposed two-state mechanism of muscle contraction [21], tropomyosin (Tm) was believed to block myosin binding sites on actin in the absence of

Ca2+ and upon an increase in Ca2+ Tm shifts to expose myosin binding sites on actin with subsequent contraction. However, this simplistic mechanism lacked the explanation behind cooperative activation of the thin filament [22-24]. A 3-state model was proposed initially proposed by McKillop and Geeves [25] and was later confirmed through atomic modeling [26]. In this model, at a period of diastole, Tm lies in the outer domain of actin and is in the “blocked” state in which myosin binding sites on actin are sterically blocked by Tm to inhibit contraction. Upon an increase in intracellular Ca2+, Tm is repositioned to the inner domain of actin in a manner termed the “closed” state such that weak myosin binding sites are exposed to allow for weakly bound crossbridges. As higher amounts of

Ca2+ occurs, more myosin are able to bind to actin and further shift Tm into the “open” state ultimately resulting in the interaction of actin and myosin to induce contraction [27].

1.3.3 The Cardiac Thin Filament

It is easy to understand why the thick filament is considered the motor of the heart. However, as with a vehicle motor, a regulatory component must be in place so that the motor doesn’t continue to run out of control and burn fuel unnecessarily. As a result, the thin filament protein complex provides an external regulatory component to dictate whether or not contraction occurs. The proteins involved in this mechanism are actin, Tm and the troponin (Tn) complex and as a whole form the thin filament at a ratio of 7:1:1, respectively. The Tn complex can further be broken down into subunits troponin T

(TnT), troponin I (TnI) and troponin C (TnC). The thin filament functions two-fold in regulating contraction: 1) in a state of low Ca2+ it prevents the interaction of actin and myosin to inhibit contraction and 2) in the presence of high Ca2+ this inhibition is removed and the interaction of actin and myosin is permitted. In this section the proteins of the thin filament will be generally discussed with in depth chapter discussion of specific components later.

While actin serves as a cytoskeletal scaffold to maintain cellular structural integrity across all cells it serves an additional specialized function in cardiac cells by acting as the attachment site for thick filament myosin to produce force. Existing in three isoforms, either α, β, or γ, the predominate isoform found in human muscle is the α isoform [6]. Actin can exist in a globular monomer form (G-actin) or a filamentous form

(F-actin) in which G-actin polymerizes by means of ATP hydrolysis to form a two- stranded double helix filament. Additionally, there are numerous accessory proteins (for complete review see [28]) that actin utilizes to preserve structural integrity with α-actinin playing a major role at the Z-disc in maintaining a static distance between thin filaments

[29]. The role of actin is simple; remain situated in the sarcomere and wait to be seized by myosin to induce contraction. It is the remaining components of the thin filament, Tm and the Tn complex, that act as the regulatory mechanism in the sarcomere in a dynamic manner.

Tm, often considered the gatekeeper of the actin thin filament, forms a coiled-coil helical dimer that extends the length of seven actin. Tm dimers form in a head-to-tail fashion with Tm’s 284 amino acid sequence producing heptad repeats integral to the structure and function of Tm [30]. Multiple species of Tm exist (α/β/γ/δ) but only α, β, and γ are incorporated into striated muscle sarcomeres with α-Tm being the dominate human heart isoform in addition to low levels of β-Tm. A more in depth discussion will be presented in a later chapter dedicated to Tm structure and function.

In 1965, the Tn complex was isolated from native Tm by Ebashi and Kodama establishing the third component of the thin filament [31]. TnC, part of the E-F hand family of proteins, is the Ca2+-sensing subunit of Tn. At a molecular weight of 18 kDa,

TnC consists of two globular domains, one regulatory and one structural, connected by a linker region to take on the appearance of a dumbbell. The structural C-lobe of TnC consist of two E-F hand motifs and has two high affinity divalent ion binding sites for

Ca2+ or Mg2+ regardless of changing intracellular Ca2+ levels; therefore C-lobe binding of

Ca2+ does not contribute to muscle contraction but plays a critical role in upholding the structural integrity of not only TnC but they entire Tn complex [32, 33]. Differing from the skeletal isoform in which two Ca2+ binding sites are present, the N-lobe of cardiac

TnC has one Ca2+ binding site that plays an important role in activation and deactivation of the thin filament. Upon binding of Ca2+ to the N-lobe, structural rearrangements ensue resulting in the exposure of a hydrophobic portion, called the hydrophobic sticky patch, of TnC where the TnI C-terminal region interacts to activate the thin filament.

TnT is a 37 kDa containing numerous charged residues throughout the structure; however, interestingly the N-terminal portion of TnT is highly acidic while conversely the C-terminal portion is highly basic. Two distinct regions are found with TnT, T1 and

T2 as identified by mild digestion by chymotrypsin [34]. It is believed that the T1 region of TnT is responsible for interacting with the Tm overlap between each dimer pair and aids in cooperative myosin binding to the thin filament while T2 primarily interacts with

TnI and TnC [35]. These interactions serve to tether the Tn complex to Tm, and in turn, the thin filament to regulate the interaction of actin and myosin.

The inhibitory component of the Tn complex derives its name from the ability to inhibit the interaction of actin-activated myosin ATPase activity by pinning Tm to actin in a low Ca2+ environment [35]. Acting as a central hub for modulation of contraction,

TnI has a large number of sites along all of the listed regions available for PTM to alter function. How these sites function to alter TnI will be discussed in a later chapter. A complete understanding of the structural components of TnI and how they structurally

10 contribute to the overall function of TnI are required to appreciate how modification by

PTM contributes to altered function as detailed in the next section.

1.4 Cardiac Troponin I Structure and Function

The inhibitory component of the Tn complex TnI is the 24 kDa, 210 amino acid subunit discovered in 1969 by Schaub and Perry [36]. While TnI has long been appreciated by clinicians as a suitable biomarker for myocardial infarction taking the place of the lesser sensitive creatine kinase, the function of TnI as it relates to the heart is much more than a byproduct of muscle damage. In this section of the chapter the inhibitory component of the Tn complex will be discussed in depth, with focus on the important structural regions and how they contribute to function of not only TnI, but the

Tn complex as a whole.

1.4.1 Troponin I Structure Overview

The structure of the TnI molecule can be broken into six distinct regions: 1) the cardiac-specific N-terminal extension; 2) a region that interacts with the C lobe of TnC;

3) a region which binds to a portion of TnT to form the IT arm; 4) the inhibitory peptide;

5) the switch peptide; and 6) a C-terminal actin binding region (see Fig. 4). Each region of TnI serves a unique function to contribute to the overall regulatory mechanisms associated with the Tn complex (for complete review see [37]) and is ultimately integral to the function of the thin filament regulation in the sarcomere.

1.4.2 Unique N-terminal Extension

Differing from fast and slow skeletal isoforms of TnI, the cardiac isoform contains a unique 32 amino acid N-terminal sequence that can be utilized to modulate sarcomere contraction. Within this N-terminal extension resides a terminal acidic region, a rigid polyproline helix, and a helix containing PKA bisphosphorylation sites at Ser-23 and 24. Through the use of nuclear magnetic resonance (NMR), it was determined that in the non-phosphorylated state, the N-terminal extension of TnI comes in contact with N- lobe of TnC to alter Ca2+ sensitivity of the myofilament. The mechanism of bisphosphorylation at Ser-23/24 was demonstrated to weaken the aforementioned interactions resulting in TnI N-terminal extension repositioning to interact with basic inhibitory region to decrease Ca2+ sensitivity [38]. Interestingly, removal of this N- terminal extension by proteolysis has been shown to produce similar structural and functional effects as bisphosphorylation potentially by putting TnI in a closed state similar to that which is present during bisphosphorylation [39, 40]. The phosphorylation of the N-terminal extension is arguably the most important purpose of the unique extension. However, as previously mentioned the N-terminal extension is thought to interact with basic residues in the inhibitory peptide of TnI and this occurs by means of an acidic region in the N-terminal extension [41]. Little was known with regards to the functional impact of the acidic region of the N-terminal extension on function until relatively recently. Sadayappan and colleagues examined the role of the acidic N-terminal region on TnI structure and function under β-adrenergic stimulation. Deletion of the acidic N-terminal region resulted in decreased cardiac contractility and it was concluded

12 that the bisphosphorylation of Ser-23/24 situated the acidic N-terminal region in a position to interact with the inhibitory peptide highlighting the mechanisms underlying decreased Ca2+ sensitivity [41].

1.4.3 The Inhibitory Peptide

The inhibitory peptide (IP) of TnI, defined by Takeda and colleagues as residues 137-148, is the highly basic minimum portion of TnI that is required to inhibit myosin ATPase activity [33, 42] and plays a key role in inhibiting contraction at low

Ca2+. Skeletal TnI residues 96-115 were initially shown to interact with actin, believed to be the inhibitory mechanism resulting in the pinning of Tm to actin, and this work was recently followed up in the cardiac isoform identifying the IP as interacting with actin as shown by förster resonance energy transfer (FRET) [43, 44]. Conversely, upon Ca2+ activation of the thin filament and binding of the switch peptide to TnC, it was initially believed that the IP interacted with both domains of TnC as shown by Pearlstone and

Smillie [45]; however, several years later it was discovered through NMR spectroscopy that TnI IP likely interacted with the linker region between the N and C lobes of TnC rather than the previously believed C-lobe [46]. Highlighting the importance of this region of TnI, mice expressing slow skeletal TnI, whose IP sequence differs from the cardiac isoform in one proline residue, exhibited a marked decrease in sarcomere length- dependent activation [47].

When examining TnI IP in complex with TnC, it was observed that residues 135-

140 took on an α-helical structure [48]. Additionally, it was identified that when in the Tn

13 complex, the structure of the IP in residues 129-137 also took on a α-helical arrangement with no distinct structure in the remaining molecule [49]. Taking a different approach than NMR, Dong and colleagues utilized FRET to measure intramolecular distance changes in TnI. They demonstrated that in the Ca2+ bound state, the IP changes from a less extended conformation to a more extended conformation to potentially act as a switch to slingshot the switch peptide in the direction of the exposed hydrophobic patch of the N-lobe of TnC; however, it was later confirmed that Ca2+-induced movement of the switch peptide towards TnC N-lobe sticky patch resulted in a dragging of the inhibitory peptide to an extended conformation and subsequent release from actin through a mechanism termed “drag and release” [50-52]. While these studies investigated the structure of the IP in the Ca2+ state, it was unknown as to how the transition from Ca2+ free TnC to Ca2+ bound TnC altered the structure of the IP. To address this, Dong and colleagues furthered their studies to measure intramolecular distance changes in TnI, either the binary (TnI/TnC) or ternary (TnI/TnC/TnT) complex, in the absence and presence of Ca2+. It was concluded that upon binding of Ca2+ to TnC, the TnI IP altered its structure from a β-turn to a less-structured α-helix upon release from actin as compared to the switch peptide which remains helical regardless of Ca2+ state [53]. The function of the release of the IP from actin is critical component in allowing for the switch peptide segment of TnI to interact with TnC for activation of the thin filament.

1.4.4 The Switch Peptide

The C-terminal switch segment of TnI, referred to as the switch peptide, serves to bind the N-lobe of TnC to initiate myofilament activation. The switch peptide, as reported by Takeda and colleagues as residues 150-159, has been shown to bind to the exposed hydrophobic region of TnC N-lobe upon Ca2+ binding through numerous van der

Waals contacts [33]. While numerous studies have been carried out to investigate the interaction of TnC and TnI, McKay and colleagues provided the first high-resolution three dimensional structure of this interaction between skeletal TnI and TnC. It was determined that residues 115-131, analogous to the aforementioned cardiac residues, bound to the hydrophobic pocket of the skeletal TnC N-lobe [54]. This work was then further translated into the cardiac isoform of TnI and similar results demonstrate amphiphilic TnI α helical switch peptide binding to the N-lobe hydrophobic pocket of

TnC [55]. Interestingly, only residues 150-158 adopt a structured α-helical content while the remaining portion of the switch peptide appears disordered. Further aiding in this interaction is the presence of the TnI N-terminal extension that helps induce an open confirmation in TnC N-lobe which exposes the hydrophobic cleft and helps to further stabilize the open conformation of TnC [56]. As a result of this interaction, the C- terminal actin-tropomyosin binding region of TnI is released in turn exposing myosin binding sites on actin and subsequent contraction of the sarcomere. The switch peptide represents the segment in the C-terminal region responsible for inducing activation of the thin filament upon interaction with TnC; however, it is known that the remaining region

15 of TnI plays an integral role in modulating the function of not only TnI but the Tn complex as a whole.

1.4.5 C-terminal Region

The C-terminal region of TnI as defined by Takeda and colleagues as residues

192-210 [33] is largely associated with the binding of actin and the “fly-casting” mechanism. It was proposed by Hoffman et al. in 2006 that in the absence of Ca2+ the C- terminal region, referred to as the mobile domain, is largely disordered with no particular structure; however, in the presence of Ca2+ and subsequent release from actin, the mobile domain takes on a structure favorable to promote switch peptide binding to TnC [57].

This mobile domain of TnI functions to participate in “long-range” sampling to determine favorable interactions, either TnC in the presence of Ca2+ or actin in the absence of Ca2+, by means of ionic residue interactions [58]. While proposed to play a role in environment sensing as a means of regulating the activated or deactivated state of the thin filament, this region of TnI can become disrupted as a result of C-terminal truncation brought on by cardiac ischemia.

As a result of ischemia, it was observed that the C-terminal portion of TnI is selectively truncated forming the C-terminal cleave product TnI1-192 [59, 60]. With regards to the fly casting model, it is easy to see that TnI1-192 would alter the interactions of the mobile domain and interfere with the normal regulation. Of significance to this altered interaction is the 17 reside shorter C-terminus which has a shorter “line” to detect actin as well as a decrease in the overall positive charge further altering the sensing

16 ability [58]. It is highly likely that the loss in ability to sense and bind actin results in an increase in Ca2+ sensitivity and a shift toward an activated state of the thin filament. The functional implications of TnI1-192 have been established in transgenic mice expressing

TnI1-192 exhibiting depressed contractility and dilatation of the heart [61]. Conversely, the molecular mechanisms underlying this dysfunction were further examined in exchanged skinned rat trabeculae demonstrating enhanced crossbridge cycling kinetics, decreased maximal force and cooperativity, and increased Ca2+ sensitivity [62]. The C-terminal region of TnI is necessary to allow for the inhibition of contraction with truncation of the representing a mechanism to modulate function within the heart, albeit under pathological conditions of ischemia.

1.4.6 pH-sensitive Histidine Button

One of the unique features of the slow skeletal isoform of Tn (ssTn) C-terminus is that it contains a single TnI His residue that has been shown to confer resistance to functional decline associated with acidic pH. Slow skeletal Tn was also shown to diminish metabolic decline following cardiac ischemic insult and enhances contractile function when expressed in mice or cardiomyocytes, respectively [63, 64]. In the cardiac isoform of TnI, an alanine is located in the corresponding residue position of the pH- resistant His residue. Dargis and colleagues used a reconstituted thin filament-myosin S1 system to investigate how the presence of a histidine residue would alter pH-sensitivity in the cardiac isoform of Tn (TnI A162H). They demonstrate that the presence of this His residue, later termed histidine button, resulted in a diminished pH effect on myosin

ATPase activity to preserve function at low pH [65]. Translating this protective capability, a report demonstrated that transgenic mice hearts expressing A162H exhibited preserved function and energetics during acidosis or ischemia. Remarkably, expression of

TnI A162H in human failing cardiomyocytes obtained from transplant significantly improved contraction and relaxation compared to non-transduced controls [66]. While this modification of TnI was induced through mutation, endogenous mechanisms in the cardiomyocyte exist to alter function by means of phosphorylation as discussed in the next section.

1.5 Tropomyosin Structure and Function

Tm is a ubiquitously expressed regulator of actin filaments that serves a unique purpose in cardiomyocytes. Using a similar purification process used to obtain actin,

Bailey isolated Tm in 1946 and crystalized Tm to gain a better understanding of its structure a few years later [67, 68]. In this section, structurally important regions of Tm will be discussed as well the role these structural components play in function. For a complete review of Tm structure and function see [69].

1.5.1 Tropomyosin Structure Overview

As previously stated, Tm forms a ~40nm coiled-coil helical dimer that lies along actin to cover and block the myosin binding sites. Tm can exist in the heart as either an α or β isoform with the α isoform associated with fast muscle and β with slow muscle. The

γ isoform is expressed in slow-twitch muscle but is absent in the heart [70]. Sequence 18 variations are minimal as little difference in function and 86% homology with the notable presence of an additional cysteine residue in the β isoform [69]. Isoforms in the adult heart are coexistent (α:β 90:10) and typically found in heterodimers as they provide more structural flexibility than that of homodimers [71, 72]. Additionally, Tm has a patterned amino acid sequence to further aid in the proper structure formation for adequate flexibility for actin binding. Residues are arranged in a special formation commonly denoted a-b-c-d-e-f-g and the arrangement of specific residues helps to give Tm its shape

(see Fig. 5). Commonly found in the a and d positions are hydrophobic residues clustering at the dimer interface, while charged residues commonly found in the e and g positions providing stability through opposite charge interactions between Tm monomers

[30]. Of particular interest is the region of Tm containing Y214-E218-Y221 where the inter-dimer distance is at its farthest apart representing a potential PTM site. These interactions play a crucial role in proper formation of Tm structure to allow for sufficient actin binding capabilities. Deviations from these patterns pose significant complications for normal contraction and relaxation of the heart.

1.5.2 Flexibility and Actin Binding

One of the most important structural features of Tm is the bending and flexibility to provide the most efficient binding of Tm to actin. Aiding in this bending ability are clusters of non-polar residues, typically alanine, commonly referred to as Ala clusters.

While lower resolution crystal structures of native Tm had been generated, Brown and colleagues attempted to crystalize smaller portions of Tm to obtain high resolution

19 structural information. By crystalizing residues 1-81 (Tm1-81) they were able to provide a

2 Å high resolution depiction demonstrating these Ala clusters for the first time as well as the remaining portion of the Tm fragment comprised of predominantly Leu residues [73].

Furthermore, it was suggested that these clusters are required for proper bending of Tm on actin. It appears that these residue clusters have opposing effects in that Ala clusters provide instability and thus bending of regions of the Tm molecule while Leu clusters provide stability within the Tm structure and elicit rigidity.

Other residues were also shown to play a role in stabilizing or destabilizing the

Tm dimer. In an intricate study examining the a position of the Tm sequence in which all residues other than Ala were inserted into the hydrophobic core domain, it was determined that Leu, Ile, Val, Met, Phe and Tyr residues were considered stabilizing while the remaining residues were destabilizing [74]. In skeletal Tm, positions a and d were demonstrated to harbor 42% destabilizing residues and 59% stabilizing residues highlighting the heterogeneity within the Tm sequence to obtain the proper flexibility.

While other residues could participate in destabilization, Ala was again confirmed to be the predominant destabilizing residue accounting for 78% of destabilizing clusters and concluded to destabilize the hydrophobic core while retaining the ability to maintain the

α-helical structure of Tm dimers [75].

Singh and Hitchcock-DeGregori furthered the notion of Tm destabilization necessity for actin binding by performing mutational studies in which destabilizing Ala was replaced with stabilizing Leu to see how this altered association with actin [30].

They report that incorporation of Leu significantly stabilized Tm and made it less flexible

20 as determined by circular dichroism and differential scanning calorimetry. Functionally, less flexibly Tm resulted in a greater than 10-fold decrease in affinity for actin.

Interestingly, replacing Ala with residues that also have the capability of destabilizing

Tm restored flexibility and affinity for actin. Evolutionarily conserved Tm surface residues play a major role in directing the binding sites of Tm for actin as well as the overall shape of Tm [76, 77]. Taken as a whole, two prominent features of Tm are required for adequate actin binding: 1) local flexibility as dictated by repeating Ala clusters throughout the Tm molecule and 2) electrostatic interactions between actin and

Tm. Playing an important role in the overall architecture of continuous Tm strands are the

N and C-terminal domains.

1.5.3 N and C-terminal Domains

Both the N and C-terminus of Tm play an important role in forming the thin filament such that it allows for a continuous Tm strand for cooperative actin binding. The

N-terminus of Tm takes on the canonical coiled-coil structure and acetylation has been shown to be a requirement for proper overlap of Tm dimers and overall structure- function. Removal of the first nine residues of N-terminal Tm significantly reduced Tm head-to-tail association and actin binding, as well as Tn binding, and eliminated the regulatory capability of Tm [78]. Furthermore, bacterially expressed Tm known to lack

N-terminal acetylation also poorly binds actin and exhibits reduced polymerization compared to muscle isolated Tm [79]. Interestingly, replacement of un-acetylated N- terminal Tm with an Ala-Ser on the N-terminus restores Tm, functionally at least, such

21 that it reestablished head-to-tail polymerization, actin binding, and the inhibitory function on ATPase activity in the absence of the Tn complex [80].

Along the same line, the C-terminus of Tm is involved in the head-to-tail overlap of dimers, as well as with the Tn complex by means of TnT. Although not as deleterious as N-terminal loss, removal of Tm C-terminus exhibits a decrease in actin affinity and inability to form head-to-tail dimers [81]. While the N-terminal portion of Tm has well defined helical structure, the C-terminus has a less structured confirmation such that ends separate open to interlock with the N-terminus of another Tm molecule (N-terminus Tm1-

11 and C-terminus Tm274-284) as determined by solution NMR [82]. C-terminal Tm also binds to TnT and Mak and colleagues demonstrate that removal of the C-terminal 11 residues significantly reduced the affinity of Tm for TnT [81] further highlighting the functional role of Tm’s C-terminus.

1.5.4 TnT Binding

Troponin T of the Tn complex serves to anchor the Tn complex to the thin filament by means of binding to Tm. Specific regions of Tm said to interact with TnT are residues 175-190 and 258-284 with an interaction along a 18nm span with rod-like TnT region [24, 83]. Tm residues 190-284 are known to interact with the long, rod-like TnT

N-terminal region while C-terminal residues 258-276 are required to allow for Tn binding to promote higher affinity actin binding [84]. A central region of TnT consisting of residues 80-180 has been shown to interact with Tm and is essential in aiding in the high affinity interaction of actin and Tm [85]. Furthermore, it was determined that both the N

22 and C-terminal overlap of Tm dimers is required for proper association of the Tn complex, by means of TnT, to interact with Tm and allow for proper spacing along the thin filament [86-88]. Tm association with TnT appears to be a symbiotic relationship, in that specific regions of Tm interact with TnT to tie the Tn complex to the thin filament, and likewise TnT binding to Tm is necessary to enhance actin-Tm binding and proper cooperative function within the myofilaments.

1.5.5 Role in Cardiac Contraction and Cooperativity

Important to the ability of the myocardium to experience continuous and coordinated contraction and relaxation, a process termed cooperativity occurs through

Tm dimers. Cooperativity can be defined as a process “in which an action facilitates the same action in adjacent structures through an allosterically mediated change in protein conformation” [89]. Indeed, Tm participates in such a phenomenon as a result of the N and C-terminal overlap previously discussed. Following binding of a single myosin molecule to actin adjacent myosin binding sites are exposed through movement of Tm by myosin in transition from the “closed” to “open” state [25, 90]. Additionally, binding of myosin to the thin filament is capable of sensitizing non-Ca2+ bound TnC [89]. A study by Kad and colleagues demonstrates that cooperative activation of the thin filament can extend up to ~80nm, or two Tm molecules [91]; however, it was also shown that thin filament cooperativity could extend up to 1000nm, or 25 Tm molecules, depicting the wide range of potential cooperative activation of the thin filament [92]. One has to believe that the flexibility or rigidity of Tm can influence the distance of cooperative

23 activation and this is seen in a study in which a mutation of Tm increased flexibility and subsequently decreased the distance of cooperative activation [93]. PTM of Tm represents a mechanism allowing for the modulation of cooperative activation in the thin filament that proves to play an integral role in beat to beat function of the heart.

1.6 Modulation of Cardiac Output

How the heart functions at the organ level is described clinically as cardiac output

(CO). CO is “the amount of blood the heart pumps in one minute, and it is dependent on the heart rate, contractility, preload, and afterload [94]” (see Fig. 6). One way to increase

CO through is by increasing the rate at which the heart beats, keeping in mind that too large an increase in heart rate will eventually depress SV as insufficient chamber filling time results. Through alteration of stroke volume (SV), CO can be increased as needed through a variety of mechanisms. One method to enhance SV, and in turn CO, is through an increase in contractility. Contractility of the heart is independent of preload or afterload in the heart and represents the intrinsic capabilities of the muscle to amplify contraction [95]. Interestingly, cardiomyocytes appear to only function at ~25-50% of their maximal ability, thus harboring a reserve should there be a need for increased contraction yet remaining energy efficient when there is not. A major way to increase contractility to enhance CO through SV is either by altering crossbridges, Ca2+ handling, or the Tn complex. Through PTM of proteins responsible for Ca2+ handling and contraction such an enhancement can be achieved as detailed below.

1.6.1 β-adrenergic Signaling Pathway

One such example of PTMs to alter cardiac function is during a time of increased body demand for oxygen and nutrients. In response for such a demand the heart has a mechanism to rapidly increase CO as needed by means of increased force and frequency of contraction. This process shown in Figure 7 is called the β-adrenergic signaling pathway and is initiated through sympathetic neuronal signaling. The release of catecholamines from the adrenal medulla enters the circulatory system and upon reaching the destination of the heart binds to β-receptors on the surface of the cardiomyocyte membrane. β1 and β2 constitute approximately 70:30 of the receptors on the cell surface with epinephrine and norepinephrine acting as equal affinity ligands for activation [96].

Signaling occurs through the G-protein couple receptor with binding of β-agonists results in the dissociation of the stimulatory subunit (Gs) and subsequent activation of adenylyl cyclase to raise cytosolic levels of cyclic AMP (cAMP). cAMP acts as a secondary messenger to bind to protein kinase A (PKA) regulatory subunits resulting in dissociation of PKA catalytic subunits that then target an array of proteins involved in cardiomyocyte calcium signaling to contraction. All of the phosphorylations by PKA targeting Ca2+ handling proteins – LTCC, PLB, and RyR – and contractile proteins – troponin I (TnI) and MyBP-C – result in increased force to contribute to enhancement of CO [97].

1.6.2 Phosphorylation of Troponin I

The regulatory mechanism of TnI involves an intricate interaction of various structurally distinct regions with other components of the thin filament to regulate

25 sarcomeric contraction. One way to fine-tune the structure-function relationship is through PTM, which in the case of TnI is predominately phosphorylation. TnI exhibits a number of phosphorylation sites – the predominant sites being Ser-23, Ser-24, Ser-42,

Ser-44, Thr-143, and Ser-150 (for a complete view of all known and possible phosphorylation sites see Fig. 8). In this section specific phosphorylations pertinent to the studies presented below will be discussed, namely the pathway/kinase responsible, the site of phosphorylation on TnI, and how the phosphorylation of the specific site alters function in the thin filament, and ultimately the heart.

The primary myofilament mechanism utilized by the heart to meet the body’s demand for increased cardiac output is enhancement through the β-adrenergic signaling pathway [98, 99]. Playing a major role in this signaling is the cardiac TnI unique N- terminal phosphorylation at Ser-23/24 by PKA [100]. Ser-23/24 is predicted to be basally phosphorylated ~40% and modulating this baseline level of Ser-23/24 phosphorylation can act as a regulatory mechanism to alter cardiac function and utilize the cardiomyocyte’s contractile reserve [101]. While Ser-23/24 phosphorylation is commonly referred to together there is a difference in the ability to be phosphorylated. Treatment of alanine mutated Ser-23 or 24 TnI with PKA demonstrated that Ser-24 is phosphorylated more readily than Ser-23 and that both phosphorylations are required to induce canonical desensitization [102]. The bisphosphorylation requirement was additionally confirmed in cardiomyocytes using pseudophosphorylation of Ser-23 and 24 and further identified

~55% bisphosphorylated TnI needed to result in maximum Ca2+ desensitization [103].

Increased Ser-23/24 phosphorylation by PKA results in decreased myofilament Ca2+

26 sensitivity increasing relaxation rate, a mechanism believed to function through the extension of the N-terminal region of TnI and resultant weakened interaction with TnC

[4]. Additionally, increased crossbridge cycling has recently been implicated in the functional alterations resulting from TnI Ser-23/24 phosphorylation [41]. A combination of decreased Ca2+ sensitivity, a necessary response to allow for adequate diastolic filling of the heart during a period of increased cardiac demand, and enhanced crossbridge cycling helps to increase cardiac performance following β-adrenergic stimulation in conjunction with increased heart rate [104, 105].

Traditionally, AMP-activated protein kinase (AMPK) is considered to be the major metabolic regulator in the cardiomyocyte; however, recent work linked this energy sensor to functionally alter the myofilaments [106]. Initially identified as a substrate for p21-activated kinase 3 (PAK3) through MALDI-TOF mass spectrometry

[107], Ser-150 phosphorylation lies between the switch peptide and inhibitory peptide of

TnI. Sancho-solis and colleagues went on to further identify Ser-150 as a preferred target for AMPK cardiac TnI by mass spectrometry and immunological detection, both in isolated form as well as in complex with TnC and TnT [108]. Being that Ser-150 is located in such an important region of TnI, it is possible that incorporation of a negative charge in that of a phosphorylation could alter the structure of the Tn complex. Indeed, incorporation of phosphorylation at TnI Ser-150 can elicit structural changes. Such alterations on TnI-TnC association were investigated by means of FRET analysis in the context of steady-state Ca2+ binding and Ca2+ dissociation kinetics.

Pseudophosphorylation of TnI Ser-150 in reconstituted thin filaments resulted in a closer

27 association of the TnI switch peptide with TnC N-lobe, similar to that induced by strong crossbridges, to increase Ca2+ sensitivity with a concurrent decrease in Ca2+ dissociation kinetics [109]. It is speculated by Ouyang and colleagues that the mechanism by which

Ser-150 phosphorylation results in decreased intersite TnI-TnC distances to increase Ca2+ sensitivity occurs by means of the negative phosphorylation charge neutralizing a highly basic region between the IP and switch peptide, therefore promoting strong hydrophobic interactions with the TnC interface. Additionally, it has been previously shown that the region in which Ser-150 is located interacts with the N-terminal extension that harbors

Ser-23/24 phosphorylation sites [110]. This could have a profound functional impact as crosstalk between phosphorylation sites may alter function. Chapters 2 and 3 detail work carried out to investigate the potential impact of phosphorylation crosstalk between Ser-

150 and Ser-23/24 to alter the regulatory capacity of the thin filament.

1.6.3 Phosphorylation of Tropomyosin

As previously established, N-terminal acetylation of Tm is absolutely required for normal polymerization, high affinity actin binding, and proper myosin ATPase inhibition.

Initially identified in 1973 by Reddy and colleagues as an endogenous modification, phosphorylation of Tm represents a modulatory component of Tm structure and function with only αTm and not the β isoform capable of being phosphorylated [111, 112].

Through the use of column chromatography, an attempt was made at identifying the kinase responsible for phosphorylation of Tm; however, it was only demonstrated that a purified kinase was capable of Tm phosphorylation with the identity of said kinase

28 undetermined [113]. Recently it has been shown that PKC can phosphorylate smooth muscle Tm, albeit with an unknown site [114]. Mak and colleagues determined by enzymatic digestion and NMR that Ser-283 in the C-terminus of frog skeletal αTm, with an analogous C-terminal structure to cardiac Tm, was the site of phosphorylation and was absent in βTm [111]. While the site of Tm phosphorylation had been identified, the functional effect of this phosphorylation remained unclear.

One of the earliest studies investigating the functional effect of Tm Ser-283 phosphorylation was carried out by Heeley and colleagues examining actin binding affinities, viscosity, and myosin ATPase activity [115]. It was concluded that Ser-283 phosphorylation in αTm had little to no effect on altering Tm affinity for actin; however, it did result in an increase in viscosity suggesting increased head-to-tail polymerization.

Additionally, it was demonstrated that phosphorylated Tm elicited higher ATPase rates compared to that of non-phosphorylated Tm in a myosin S1-actin assay but when fully reconstituted with the Tn complex there was no difference between the two. It is speculated that Tm phosphorylation weakened binding with the Tn complex, presumably through TnT, and is possibly attributable to an inability of the long N-terminal TnT region to link overlapping Tm. Phosphorylation of Tm plays an integral part in the biochemistry of the thin filament, and when considering the in vivo implications the story does not change. For example, Tm phosphorylation allows for extension of cooperative activation and maintains tension for force generation in the sarcomere [116, 117].

Conversely, dephosphorylation of Tm may be able to exhibit a beneficial effect in the context of hypertrophic cardiomyopathy mutations associated with Tm as it was shown

29 that removal of Tm phosphorylation can rescue the FHC phenotype [118]. It is clear that phosphorylation of Tm is significantly contributes to cooperative activation of the thin filament and helps to enhance the magnitude and rate of cardiac contraction.

1.7 Cardiac Ischemia

Cardiac ischemia results from vessel (coronary) occlusion leading to hypoxia of the nearby tissue, a buildup of metabolic products, and failure of substrate delivery [119].

The end product of such damage to the heart is tissue necrosis resulting in limited perfusion and reduced pumping ability ultimately leading to death without prompt intervention. In this section, the molecular mechanisms underlying cardiac ischemia will be discussed while touching on how the myofilaments are affected resulting in dysfunction.

1.7.1 Metabolic Alterations and Decreased pH in Ischemia

In the well perfused heart, 95% of ATP generated in the cardiomyocyte comes from oxidative phosphorylation in the mitochondria, with the remaining percentage of

ATP coming from glycolysis. Furthermore, substrate utilization plays a role in dictating energy production as 60-90% of acetyl-CoA production results from fatty acid oxidation while 10-30% comes from pyruvate oxidation by means of glycolysis and lactate oxidation [120]. However, in the case of reduced blood flow during cardiac ischemia, even as little as ten seconds, cardiomyocytes consume the remaining oxygen with oxidative phosphorylation ending shortly after [121]. In an attempt to generate more 30 energy in condition lacking oxygen, the cardiomyocytes undergo rapid anaerobic glycolysis to generate lower than normal ATP levels, but ATP nonetheless.

Unfortunately, a byproduct of such glycolysis is lactate production and hydrogen ions which have a profound effect on pH showing a rapid drop in intracellular pH from ~7.2 to as low as 6.5 in an extreme case [122]. Indeed, the effect of excess lactate production has been seen as early as 1880 by Gaskell as he demonstrated that the perfusion of the heart with a lactic acid solution brought the heart to a standstill in a state of complete relaxation [123]. It was speculated that the cessation of contraction was a result of myofilament dysfunction as there are no observable changes in the Ca2+ transient during ischemia; however, in later studies it was demonstrated that acidosis resulted in a fast response in which myofilaments are immediately affected and a slow response in which

Ca2+ transient decreases [124, 125]. Acidic conditions in the heart results in numerous processes that ultimately results in increased energy consumption and decreased energy production. Luckily, the heart has a mechanism in place to sense this alteration and attempt to correct such imbalances.

1.7.2 Role of AMPK

As previously discussed, an ischemic episode in the heart can result in decreased oxygen, and in turn, a lower output of ATP. As more and more ATP is consumed, the balance of energy being produced and energy being consumed shifts toward a higher consumption rate altering the AMP to ATP ratio. To monitor the metabolic changes in the heart the cardiomyocytes utilize AMP-activated protein kinase (AMPK), the energy

31 sensing protein, to counter metabolic stress and restore ATP levels. AMPK consists of three domains α, β, and γ with the first two existing in two isoforms and the last with three; however, the predominant isoform in the healthy heart is the α2β2γ1 holoenzyme

[126]. The γ subunit is responsible for AMP and ATP binding and elicit the “energy sensing” capabilities of AMPK with one non-exchangeable AMP bound and two other

AMP binding sites that can out-compete ATP when AMP levels are high enough [127].

Of additional importance is the catalytic α domain as it contains an activation loop comprised of Ser/Thr residues that can be phosphorylated to activate the protein, the most prominent marker of activity being Thr-172 [128]. Upstream kinases target Thr-172 as the major site for phosphorylation to activate the enzyme with AMP binding aiding in the protection from dephosphorylation, and thus, deactivation of the enzyme [129].

Serine/Threonine Kinase 11, also known as liver kinase B1, is the major kinase responsible for phosphorylation and activation of AMPK with Ca2+-calmodulin- dependent protein kinase kinases playing a lesser role. The unique aspect of the latter kinase is that it can be activated irrespective of AMP levels as it becomes activated by elevated intracellular Ca2+ levels [130, 131].

It is well documented that in cardiac ischemia, AMPK becomes activated and sustains activity during the period of metabolic stress in which it aids in the increase import of glucose to shift the balance of metabolism from fatty acids to glycolysis [132,

133]. This process is beneficial in the short term as the small amount of ATP generated from anaerobic glycolysis is necessary to maintain processes that help keep the cardiomyocytes viable. AMPK is traditionally viewed as a metabolic sensor and an

32 enzyme that regulates anabolic/catabolic processes associated with ATP but there is evidence that AMPK is associated with the myofilament lattice, specifically TnI.

Troponin I has been shown to be a suitable substrate for AMPK resulting in the phosphorylation of Ser-150 in the inhibitory peptide/switch peptide region [108]. AMPK activation during global ischemia phosphorylates TnI at Ser-150 to increase cardiomyocyte shortening [106]; however the molecular mechanisms underlying modification remain unclear. Phosphorylations of proteins are not the sole mechanism of

PTM to modulate function in the heart. As a result of ischemia-induced byproducts in the cardiomyocyte, highly reactive free radicals also offer an approach to alter protein function.

1.7.3 Generation of Free Radicals During Reperfusion

Under normal physiological conditions, free radicals occur in low abundance and serve as signaling mechanism; however, in periods of pathological stress such as reperfusion following ischemia, levels of free radicals, termed reactive oxygen species

(ROS) or reactive nitrogen species (RNS), can become significantly elevated. The most

·- common free radical present during reperfusion is the superoxide anion (O2 ) formed by addition of an additional electron to elemental oxygen. Such molecules are produced by multiple sources however two serve as a substantial foundation for ROS production in the heart – 1) oxidative enzymes such as xanthine oxidase (XO), NADPH oxidase, and nitric oxide synthase and 2) mitochondrial electron transport chain dysfunction [134]. In addition to ROS, the formation of the RNS peroxynitrite (ONOO-) occurs following a

-. reaction of O2 and the vasodialating compound nitric oxide [135, 136]. RNS as well as

ROS are short lived and pose a significant threat to cardiomyocytes as they are unstable and highly reactive with proteins and lipids.

Myofilament proteins are highly susceptible to modification by free radical molecules formed during reperfusion to alter their function [137]. ROS have been shown to alter cardiac function in vitro at the level of the myofilament as demonstrated in experiments examining the effect of oxidizing agents on skinned donor left ventricular fibers. It was demonstrated that sulfhydryl oxidation of myofibrillar proteins decreased the maximal Ca2+-activated tension, the Ca2+ sensitivity, and the steepness of the hill coefficient [138]. Furthermore, it was shown that exposure of skinned fibers to hydrogen peroxide reduced peak maximal force without altering Ca2+ sensitivity [139] suggesting potential different mechanisms of free radical modification of the myofilaments to alter function. Additional in vivo studies demonstrated that XO played a major role in myofilament protein oxidation as chronic inhibition of XO significantly eliminated protein oxidation [140]. While less extensively studied, RNS have been shown to alter myofilament function as well. Mihm and colleagues showed that upon treatment of cardiac trabeculae with ONOO-, tyrosine nitration of sarcomeric proteins occurred resulting in weakened force production at the sarcomeric level [141]. Moreover, it has been confirmed in a rodent model that ONOO- mediated nitration of tyrosine is increased in post-ischemic hearts [142]. ROS and RNS represent an alternative method of protein modification both in normal physiology and pathophysiology. An intricate balance of the products required to form free radicals is integral in dictating whether the free radicals

34 will assist in redox-mediated signaling or detrimentally affect protein functions in the heart.

1.8 Overall Goal of Dissertation

The goal of chapter 2 was to determine the ability of AMPK to phosphorylated

TnI at Ser-150 and the effect said phosphorylation has on the function of canonical TnI

Ser-23/24 phosphorylation. The working hypothesis states phosphorylation of TnI at Ser-

150 will alter the function of TnI Ser-23/24 phosphorylation. The goal of chapter 3 was to determine what happens to levels of TnI Ser-150 and Ser-23/24 phosphorylation during cardiac ischemia and how these phosphorylations crosstalk with each other under ischemic-mimetic conditions to uncover possible mechanisms of such phosphorylations during ischemia. The working hypothesis states phosphorylation of TnI at Ser-150 will aid in reducing the thin filament desensitizing effect of acidic pH. The goal of chapter 4 was to determine the effect of Tm Ser-283 phosphorylation on the dynamic effect of myofilament regulation in the muscle. The working hypothesis states incorporation of pseudophosphorylation at tm Ser-283 will alter kinetic properties of myofilament contraction and relaxation. Lastly, the goal of chapter 5 was to determine the effect of Tm nitration on structure and function while also identifying specific nitration sites of Tm.

The working hypothesis states nitration of Tm will result in altered structural organization leading to changes in protein function.

The overarching goal of the dissertation work described below is to expand on the role of PTM of myofilament proteins, both in the context of normal physiology as well as

35 in a diseased state. In particular we are highly interested in the integrated crosstalk capability of TnI phosphorylation. In the diseased heart a multitude of cellular changes occur, either as a compensatory mechanism in response to modifications brought on as a result of cardiac disease or a direct result of the pathophysiology. One such avenue for uncovering the molecular mechanisms underlying cardiac disease and their functional changes is to study PTM of proteins. While extensive work has been done characterizing phosphorylation of cardiac contractile regulatory proteins, this work has been conducted investigating the modifications in isolation. Despite the fact that a single phosphorylation site may be sufficient to alter function, the additive functional effect of multiple integrated phosphorylation sites to crosstalk inter-molecularly and change function differently than that evoked by a single phosphorylation must be taken into consideration.

Figure 1. Schematic representation of excitation-contraction in the cardiomyocyte.

Membrane depolarization results in an increase of activating intracellular Ca2+ entering the cell

(as shown by green arrows). CICR release additional Ca2+ to activate the thin filament and result in contraction. Ca2+ is subsequently removed from the cell to allow for relaxation (as shown in red arrows) by means of reuptake into the sarcoplasmic reticulum, extrusion by the sodium- calcium exchange channel and Ca2+-ATPase, or to a lesser extent the mitochondria. Figure adapted from [2]. LTCC, L-type Ca2+ channel; NCX, Na+-Ca2+ exchanger; RyR, ryanodine receptor; PLB, phospholamban; ATP, ATPase.

Figure 2. Schematic representation of distinct regions across two sarcomeres.

A single sarcomere is defined between two dark bands on an electron micrograph known as the

Z-line or Z-disc. The M-line, located at the center of the sarcomere, is responsible for organizing and stabilizing the myosin molecule. The I-band is the region of thin filament actin that is not overlaid with thick filament myosin. The A-band is comprised of the entire thick filament myosin length; however there is some overlap with the thin filament actin. The H-zone represents the region of thick filament that is not overlaid with thin filament.

Figure 3. Five-step schematic representation of crossbridge cycling.

Beginning in the dissociated state, ATP hydrolysis occurs to put the myosin head into a cocked state (1). Ca2+ enters the cell and allows for the interaction of myosin with its binding sites on actin (2). Release of inorganic phosphate produces a myosin powerstroke in which the myosin slides the thin filament to induce contraction (3). ADP is release from the myosin molecule resulting in a rigor bound state of myosin binding to actin (4). ATP rebinds to myosin resulting in the dissociation of myosin from actin to repeat the cycle (5).

Figure 4. Schematic representation of the TnI in the thin filament

Within the TnI molecule six important regions are denoted: 1) the cardiac-specific N-terminal extension; 2) a region that interacts with the C lobe of TnC; 3) a region which binds to a portion of TnT to form the IT arm; 4) the inhibitory peptide; 5) the switch peptide; and 6) a C-terminal actin binding region. Figure adapted from [143].

Figure 5. Schematic representation of Tm dimer structure and interactions.

Necessary residue heptad repeat interactions required for proper formation and shape of the Tm molecule. Hydrophobic residues are represented in green, ionic residues in red, and actin binding residues in blue.

Figure 6. Parameters of cardiac output.

Cardiac output is defined by the heart rate and stroke volume. Factors that can enhance stroke volume, and in turn cardiac output, are contractility and preload while afterload negatively alters cardiac output. Figure adapted from [95].

Figure 7. Schematic representation of β-adrenderic stimulation pathway.

Stimulation of β-adrenderic receptors by catecholamines (E = epinephrine; NE = norepinephrine) results in the activation of PKA by binding of cAMP. Subsequent activation results in the phosphorylation of various Ca2+ handling and myofilament proteins to alter function as shown in red. Figure adapted from [2]. LTCC, L-type Ca2+ channel; NCX, Na+-Ca2+ exchanger; RyR, ryanodine receptor; PLB, phospholamban; ATP, ATPase; β-AR. Β-adrenergic receptor; AC, adenylyl cyclase; PKA, protein kinase A; cAMP, cyclic AMP.

Figure 8. Schematic representation of possible phosphorylation sites in TnI.

Shown in red are the known phosphorylation sites as identified by multiple reaction monitoring mass spectrometry while predicted phosphorylation sites are shown in blue. Numbering of sites corresponds to the human TnI molecule including the N-terminal methionine. Figure adapted from [144].

Chapter 2: AMP Activated Protein Kinase Phosphorylates Cardiac Troponin I at Ser-150 to Increase Myofilament Calcium Sensitivity and Blunt PKA Dependent Function

2.1 Introduction

AMP activated protein kinase (AMPK) is a serine/threonine protein kinase that functions as a central physiological regulator of energy-generating pathways in multiple organ systems [145, 146]. By responding to various metabolic changes, AMPK regulates key enzymes in metabolic pathways to maintain cellular energy stores. Classical AMPK activation results from the increase in cellular AMP/ATP ratio-induced AMP binding during periods of high energy demands. This ability of AMPK to “sense” decreased ATP levels and its subsequent activation to increase ATP production have led AMPK to be termed the “master switch” for maintaining cellular energy levels [145]. In the heart,

AMPK signaling to increase ATP during elevated metabolic demand is necessary to maintain cardiac contractility. Recent evidence has demonstrated AMPK is activated by other stimuli in addition to AMP binding (see [147, 148]), suggesting AMPK signaling may function to regulate other systems in addition to regulating metabolism. In the absence of clear downstream signaling targets the role of AMPK signaling directly at the myofilament is unclear.

Troponin I (TnI) plays a critical role in the regulation of muscle contraction [37].

The phosphorylation of cardiac TnI (cTnI) at Ser-23/24 through the -adrenergic signaling pathway represents one of the primary mechanisms to regulate cardiac

45 contractile dynamics (for review see [35]). Cardiac TnI can also be phosphorylated at a number of other residues through different signaling pathways; however the physiological relevance of the majority of these phosphorylations is not clearly understood. Recently Oliveira et al. [149] reported AMPK can phosphorylate cTnI at Ser-

150 in vitro and Sancho Solis et al. [108] demonstrated the kinase domain of AMPK was sufficient to phosphorylate cTnI at Ser-150 in the myofilament lattice. Serine 150 is located directly within the TnI switch peptide, a key element in the Ca2+ regulation of muscle contraction. Evidence supporting Ser-150 phosphorylation as functionally relevant was recently demonstrated by Ouyang et al. [109] who reported cTnI pseudo- phosphorylation altered the interaction of cTnI with troponin C (TnC) to affect thin filament Ca2+ regulation. To date the phosphorylation of cTnI Ser-150 in vivo and its functional effect on contraction are not known.

To determine the role of AMPK as a common signaling molecule between cardiomyocyte cellular metabolism and contractile function, we investigated the role of

AMPK to phosphorylate cTnI at Ser-150 and its effect on cardiac contraction. Consistent with previous findings we demonstrate the AMPK holoenzyme phosphorylates cTnI at

Ser-150 in vitro as well as within the muscle lattice. We further demonstrate that cTnI is endogenously phosphorylated at Ser-150 in the heart. Through the exchange of cardiac troponin (cTn) containing a pseudo-phosphorylated cTnI into cardiac skinned fibers we demonstrate cTnI Ser-150 phosphorylation significantly increases cardiac muscle Ca2+ sensitivity. Importantly, this cTnI Ser-150 phosphorylation crosstalks within cTnI to blunt the functional effects of -adrenergic induced cTnI Ser-23/24 PKA

46 phosphorylation. Our findings support AMPK as a signaling molecule that links cardiac myocyte metabolic needs to the direct enhancement of the myofilament contractile response through uncoupling the thin filament -adrenergic response.

2.2 Materials and Methods

2.2.1 cDNA constructs

All cTnI residue numbers presented in this manuscript are presented according to the native human sequence including the first methionine. The human cTnI Ser-150 to

Asp (cTnI S150D), Ser-23/24 to Asp (S23/24D) and Ser-23/24/150 to Asp

(S23/24/150D) pseudo-phosphorylation mutant cDNA were generated by site-directed mutatgenesis (Quick Change II kit, Agilent) according to the manufactures direction and resultant constructs were verified by DNA sequencing.

2.2.2 Proteins

The individual recombinant human cTn subunits were expressed in E. coli and purified to homogeneity as previously described [150]. Troponin used for fiber exchange and kinase experiments contained human cardiac TnT (TnT) with an N-terminal myc tag.

Our lab and others have previously demonstrated the presence of this myc tag on TnT does not affect myofilament function [151, 152]. Troponin used in Ca2+ binding experiments consisted of native human TnT, cTnI and human cardiac TnC with the

T53C, S35/84C mutations [153]. Cardiac Tn complexes were reconstituted by sequential dialysis and column purified as previously described [150]. Column fractions containing pure cTn were dialyzed against exchange buffer (200 mM KCl, 5 mM MgCl2, 5 mM

EGTA, 1 mM DTT, 20 mM MOPS, pH 6.5) and aliquots stored at -80 C until use.

Myofibrils were prepared as described previously [152] and endogenous cTn partially exchanged for exogenous cTn as previously described [150, 152]

2.2.3 Kinase treatments

Purified cTn or exchanged myofibrils were treated with purified PAK, purified bovine protein kinase A catalytic subunit (Sigma) or active AMPK holoenzyme complex composed of 1/ 1/ 2 or 2/ 1/ 2 subunits (SignalChem). Kinase reaction conditions were 200 mM KCl, 10 mM MgCl2, 1 mM DTT, 20 mM MOPS, pH 7.0 in the presence of

2.5 mM EGTA or 0.25 mM CaCl2. The reaction was initiated by the addition of 1mM

ATP and carried out at 37 C for varying time points before stopping the reaction with an equal volume of 2x urea sample buffer consisting of 8M Urea, 2M thiourea, 75 mM DTT,

50mM Tris-HCl, pH 6.8 containing 3% SDS and 0.05% bromophenol blue. Samples were stored at -80 C until use.

2.2.4 Protein electrophoresis, staining and Western blot

Proteins (purified, myofibrils or fiber bundles) were solubilized in denaturing sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue and 10%

48 glycerol), heated for 5min at 80 C and clarified by centrifugation for 5 min. Resultant protein was separated by SDS-PAGE on cooled 8 x 10 cm (Hoefer) 12% (29:1) acrylamide gels by methods previously described [40]. Gels were either stained for phospho-proteins, total protein or transferred to membranes for Western blot as described in the figure legends. Phospho-proteins were identified by ProQ Diamond phosphoprotein staining (Invitrogen) and imaged on a Typhoon 9410 imager (GE Healthcare) with an excitation of 532nm and an 580BP30 emission filter according to the manufactures protocol. Total protein was visualized by Commassie and digitized or

Sypro Ruby (Invitrogen) staining imaged on a Typhoon 9410 (GE Healthcare) with an excitation of 457nm and a 610BP30 emission filter [40]. Western blot was conducted by wet transfer (Hoefer) of proteins to 0.45uM PVDF at 10 C for 90 min at 90 volts by as previously described [40]. Cardiac TnI Ser-150 phosphorylation was identified with the rabbit pTnI 150 antibody, detected by a Horseradish Peroxidase conjugated rabbit secondary and developed with enhanced chemiluminescent plus (GE Healthcare) on

Hyperfilm (GE Healthcare). Subsequently, total cTnI was detected following blocking by re-probing the same membrane with the mouse cTnI antibody C5 (Fitzgerald), detected with an alkaline phosphatase anti-mouse secondary antibody incubated in 5-bromo-4- chloro-3-indolyl-phosphate and nitro blue tetrazolium (BCIP/NBT). This sequential development using different primary/secondary combinations and development methods is critical to avoid signal bleed from the first Western to the second. Alternately, myofibril gels probed for pTnI 150 were transferred to 0.22 m low fluorescence PVDF

(GE Healthcare) for fluorescent Dylight secondary (Jackson ImmunoResearch

Laboratories, Inc.) detection on a Typhoon 9410 imager (GE Healthcare).

2.2.5 2-D isoelectric focusing

Cardiac TnI was analyzed by 2-D isolelectric focusing on 18 cm 7 – 11 IPG strips

(GE Healthcare) by methods previously described [40], except the first dimension of focusing was carried out on an Agilent 3100 OffGel fractionator (Agilent) run in the “in- gel” method. Resultant strips were separated in the second dimension on 18 x 8 cm

(Hoefer) 12% (29:1) gels.

2.2.6 Exchange of recombinant cardiac troponin into skinned mouse cardiac fiber bundles

Left ventricular papillary muscles were dissected from hearts of mice anaesthetized with sodium pentobarbital as previously described [154]. Following dissection muscles were cut in uniform strips of no greater than 150 uM in diameter and treated with 1% (v/v) Triton X-100 in relax buffer (41.89 mM potassium proprionate, 10 mM EGTA, 10 mM creatine phosphate, 6.22 mM ATP, 6.57 mM MgCl2, 5 mM NaN3 and 100 mM N,N-bis[2-hydroxyethyl]-2-aminoethane sulfonic acid (BES), pH 7.0) at

4 C for 4 hours to extract membranes (skinned). The endogenous cTn from the resultant fiber bundles was then exchanged for exogenous cTn by overnight incubation at 4 C in

13 M recombinant cTn [150]. Exogenous and endogenous TnT migrate at different mobility when separated by SDS-PAGE, therefore, the percent of exogenous cTn 50 exchange to total fiber cTn was determined in each fiber by western blot for TnT with the mouse TnT monoclonal antibody CT3 (Developmental Studies Hybridoma Bank) as previously described [150]. Animal care and use was performed in accordance with the guidelines of the Institutional Animal Care and Use Committee at the University of

Illinois at Chicago and The Ohio State University.

2.2.7 Measurement of isometric tension

The measurement of steady state isometric tension as a function of free [Ca2+] was conducted as previously described [154]. Fiber measurements to determine skinned fiber mechanical parameters were conducted at a sarcomere length of 2.2 m as determined by laser diffraction while fiber measurements to determine length dependent activation were conducted at sarcomere lengths of 1.9 m and 2.2 m in the same fiber

[47, 155]. Fiber bundles were activated over a range of free [Ca2+] to determine steady- state isometric tension. Only muscles that maintained greater than 85% maximal tension were included for analysis. Following the mechanical experiment each bundle was briefly dried and stored frozen for biochemical analysis.

2.2.8 Measurement of thin filament steady-state Ca2+ binding to TnC

Steady-state thin filament Ca2+ binding to TnC was measured as previously described [153]. Briefly, thin filaments were reconstituted with various cTnI in the presence of 2-(4’-iodoacetamidoanilo)naphthalene-6-sulfonic acid (IAANS) labeled

C35S, C84S and T53C TnC and IAANS fluorescence measured at various free [Ca2+] as indicative of Ca2+ binding to TnC.

2.2.9 Data processing and statistical analysis

Tension-Ca2+ relationships and steady-state Ca2+ binding were fit to a modified

Hill equation to determine 50% maximal binding and Hill coefficient. PKA phosphorylation of cTn over time was fit with a single exponential to determine time to

50% maximal phosphorylation. Results of skinned fiber force measurements, length dependent activation and Ca2+ binding were compared by ANOVA with the Bonferroni post-hoc evaluation. Cardiac Tn phosphorylation was compared by Student’s t-Test. A

P<0.05 was considered statistically significant. Data are presented as mean SEM.

2.3 Results

2.3.1 The AMPK holoenzyme complex phosphorylates cTnI at Ser-150.

As a tool to establish the relevance of cTnI Ser-150 phosphorylation we generated a custom antibody that specifically recognizes cTnI only when phosphorylated at Ser-150

(pTnI 150). To investigate the role of the AMPK holoenzyme to phosphorylate cTnI, we first sought to determine the effect of the functional AMPK holoenzyme (composed of

1/ 1/ 2 subunits) to phosphorylate cTnI Ser-150 in purified recombinant human cTn in vitro. Western blot with the pTnI 150 antibody demonstrates the time dependent

52 appearance of a single 25 kDa band following AMPK treatment that was of similar mobility to controls phosphorylated at Ser-150 with PAK (Fig. 9A). Subsequent re- probing of the membrane with a total cTnI antibody imaged by varied detection methods demonstrates the 25 kDa band is cTnI. Coomassie staining of an identical gel further demonstrates similar loading and integrity of the cTn complex. Next we conducted

AMPK treatment over an extended time course to characterize AMPK holoenzyme phosphorylation of cTnI Ser-150 to saturation. Results in Figure 9B demonstrate the phosphorylation of cTnI Ser-150 reached a maximum at 6 h without additional increased phosphorylation upon further addition of the AMPK holoenzyme. Similar to the 1/ 1/ 2

AMPK holoenzyme, the 2/ 1/ 2 holoenzyme also induced the time dependent Ser-150 phosphorylation of cTnI (data not shown). These findings demonstrate cTnI Ser-150 in isolated cTn is a target of the AMPK holoenzyme.

To establish the significance of cTnI Ser-150 phosphorylation in the normal heart we conducted Western blot of rabbit cardiac tissue separated by two dimensional isoelectric focusing (2-D). Two dimensional fractionated Western blot of rabbit ventricular myofibrils with the pTnI 150 antibody demonstrates 3 cTnI species with Ser-

150 phosphorylation (Fig. 10A). Following stripping, the membrane was re-probed with a total cTnI antibody. Differential detection of the total cTnI antibody identified 4 cTnI species (Fig. 10B). Alignment of the membranes demonstrates the pTnI 150 antibody solely recognized only the 3 most acidic spots (P1-P3) and not the most basic, unphosphorylated spot (U). Using the maximally Ser-150 phosphorylated sample from our

AMPK treatment time course as a standard, we investigated the extent of cTnI Ser-150

53 phosphorylation in rabbit myofibril samples. By this method we determined normal rabbit myofibrils are Ser-150 phosphorylated at about 3% of total cTnI. These results demonstrate cTnI is endogenously phosphorylated at Ser-150 in the normal rabbit heart.

Next we sought to determine if the AMPK holoenzyme would phosphorylate cTnI at Ser-150 in the myofilament lattice. Employing similar methods described above, we first determined Ser-150 phosphorylation accounts for approximately 3% of the total cTnI in myofibrils from the normal rat heart. Endogenous cTn of rat myofibrils was then partially exchanged with purified, non-phosphorylated recombinant human cTn and treated with the AMPK holoenzyme complex (consisting of 1/ 1/ 2 subunits). Western blot with a total cTnI antibody demonstrates exogenous recombinant human cTnI migrates slightly faster than the endogenous rat cTnI (Fig. 11B). Taking advantage of this migration difference, quantification of the two cTnI species demonstrates myofibril exchange resulted in 46% recombinant human cTn that was similarly loaded in both sham (Control Tn MF) and AMPK treated cTn exchanged myofibrils (AMPK Tn MF).

Sequential differential detection of Ser-150 phosphorylation with the pTnI 150 antibody demonstrates non-exchanged rat myofibrils (Rat MF) exhibit endogenous cTnI Ser-150 phosphorylation that was decreased following the exchange protocol (Control Tn MF)

(Fig. 11A). Following incubation with AMPK, endogenous rat cTnI Ser-150 phosphorylation was increased by 3.4 times while exogenous human cTnI Ser-150 phosphorylation was also increased (Fig. 11A). Differential fluorescent Western imaging allowed for simultaneous detection of the pTnI 150 and total cTnI antibodies. Merge of the two antibody signals demonstrates pTnI 150 (green) and total cTnI (red) identified the

54 same band (yellow) in the AMPK Tn MF sample (Fig. 11C). Note that AMPK phosphorylated cTnI exhibits a slightly slower migration similar to that previously demonstrated to result from PAK Ser-150 phosphorylation [107]. The AMPK cTnI Ser-

150 phosphorylation in myofibrils was further verified by Western blots probed solely with the pTnI 150 antibody detected by enhanced chemiluminescence (data not shown).

These findings demonstrate cTnI Ser-150 is endogenously phosphorylated in the rat heart and AMPK further phosphorylates Ser-150 in the native muscle lattice.

2.3.2 cTnI Ser-150 phosphorylation increases myofilament Ca2+ sensitive force development and blunts PKA dependent Ca2+ desensitization.

To determine the effect of cTnI Ser-150 phosphorylation on the regulation of force development, we exchanged the endogenous cTn in mouse skinned cardiac fiber bundles with either recombinant wild-type (Tn WT) or Tn containing Ser-150 pseudo- phosphorylated cTnI (Tn S150D). Exchange efficiency of each fiber bundle following force measurements was evaluated by Western blot for exogenous human TnT. The results demonstrate recombinant cTn exchange averaged 70 5 % (Fig. 12). This percent exchange was the same for all the cTn exchange groups in the study. Following exchange, fiber bundles were subjected to force-Ca2+ measurements at a sarcomere length of 2.2 m. The results in Figure 12A demonstrate fibers exchanged with Tn S150D exhibit a significant 0.51 M increase in EC50 compared to that of fibers exchanged with

Tn WT (EC50; Tn WT = 1.27 0.03, Tn S150D = 0.76 0.02; Figs. 12A, 12C and Table

1). This change in Ca2+ sensitivity occurred in the absence of altered maximal tension 55 development or Hill coefficient (Table 1). Thus cTnI Ser-150 pseudo-phosphorylation significantly increases the ability of sub-maximal Ca2+ to activate the thin filament resulting in increased force development at a given sub-maximal Ca2+ concentration.

Previously we demonstrated that single amino acid residue modifications within cTn can alter the effects of cTnI PKA phosphorylation function [150, 156]. To investigate Ser-150 phosphorylation crosstalk on the effect of cTnI PKA phosphorylation, we exchanged mouse cardiac fiber bundles with human recombinant cTn containing cTnI pseudo-phosphorylated at the PKA sites (cTn S23/24D) or cTnI pseudo-phosphorylated at both the PKA and Ser-150 sites (cTn S23/24/150D). As expected, exchange of cTn S23/24D decreased Ca2+ sensitivity by 1.04 M compared to

Tn WT (Figs. 12B, 12C and Table 1) [150, 157]. Upon combination with S150D, the

Ca2+ sensitivity of Tn S23/24/150D exchanged fibers was increased by 0.88 M compared to Tn S23/24D exchange alone and was not different from Tn WT (EC50; Tn

WT = 1.27 0.03, Tn S23/24D = 2.31 0.07, Tn S23/24/150D = 1.43 0.09; Figs. 12B,

12C and Table 1). The combined Tn S23/24/150D fibers exhibit normal maximal tension development and Hill coefficient (Table 1). These data demonstrate cTnI Ser-150 phosphorylation blunts the Ca2+-desensitizing effects of PKA-dependent phosphorylation

2.3.3 cTnI Ser-150 phosphorylation increases Ca2+ sensitivity by altering Ca2+ binding to TnC.

To investigate the mechanism of the cTnI Ser-150 phosphorylation induced increase in Ca2+ dependent force and its combined effect to blunt PKA dependent Ca2+

56 desensitization we measured Ca2+ binding to TnC in reconstituted thin filaments. Similar to force development, thin filaments reconstituted with Tn S150D increased Ca2+ affinity compared to reconstitution with Tn WT (EC50; Tn WT = 2.5 0.1, Tn S150D =

1.03 0.16; Fig. 13 and Table 2). As expected, Tn S23/24D reconstitution decreased Ca2+ binding to TnC. This PKA induced decrease in Ca2+ binding was blunted upon combination with Ser-150 pseudo-phosphorylation such that Ca2+ binding of Tn

S23/24/150D was no longer different from Tn WT (EC50; Tn S23/24D = 8.3 0.6, Tn

S23/24/150D = 3.7 0.8; Fig. 13 and Table 2). These data demonstrate Ser-150 phosphorylation increases Ca2+ dependent force development and blunts the Ca2+- desensitizing effects of cTnI PKA dependent force desensitization through a mechanism directly influencing Ca2+ binding to TnC in the thin filaments.

To validate our findings using the pseudo-phosphorlyated cTnI we treated recombinant human cTn separately with either PKA or AMPK prior to reconstituting thin filaments and the determination of Ca2+ binding to TnC. Representative Western blot of treated filaments following Ca2+ binding measurements demonstrate PKA treatment of cTn for 20 min (Tn PKA Tx) induced significant cTnI Ser-23/24 native phosphate incorporation (Fig. 14A) with minimal native Ser-150 phosphorylation (Fig. 14B).

Alternately, cTn AMPK treatment for 4 h (Tn AMPK Tx) induced significant levels of both Ser-150 and Ser-23/24 native phosphate incorporation (Figs. 14 and B). Calcium binding to TnC in filaments reconstituted with PKA treated cTn containing native Ser-

23/24 phosphate demonstrate decreased Ca2+ affinity compared to filaments containing sham incubated cTn as expected (EC50; Tn Sham Tx = 2.74 0.10, n=4; Tn PKA Tx =

7.03 1.39, n=4; p<0.05; Fig. 14C). Importantly, Ca2+ binding affinity of thin filaments reconstituted with AMPK treated cTn containing native phosphate at Ser-23/24/150 was not different from that of Sham Tn (EC50; Tn AMPK Tx = 3.72 0.35, n=4; Fig. 14C and

Table 2). These data from experiments employing authentic phosphorylation of cTnI sites confirm findings with the pseudo-phosphorylated cTnI.

2.3.4 cTnI Ser-150 phosphorylation alone does not alter length dependent activation but blunts cTnI PKA dependent induced length dependent activation.

The Ca2+ sensitivity of force development is altered by sarcomere length [47]. To further investigate cTnI Ser-150 phosphorylation induced Ca2+ sensitization we measured the Ca2+ regulated force development at short (1.9 m) and long (2.2 m) sarcomere lengths in the same fiber bundle. At the short sarcomere length of 1.9 m, the Ca2+ sensitivity of fibers exchanged with Tn S150D remained increased compared to Tn WT

(EC50 at 1.9 m; Tn WT = 1.53 0.03, Tn S150D = 0.92 0.01; Fig. 15A, 15C and Table

1). Similar to the 2.2 m measurements, the exchange of Tn S23/24D decreased Ca2+ sensitivity at 1.9 m compared to WT (EC50 at 1.9 m; Tn S23/24D = 2.82 0.05; Fig.

15B, 15C and Table 1). However, unlike at 2.2 m, Ca2+ sensitivity at 1.9 m of the combined Tn S23/24/150D exchange was slightly decreased from that of Tn WT (EC50 at

1.9 m; Tn S23/24/150D = 1.80 0.09; Fig. 15B, 15C and Table 1). Importantly, cTn

S23/24/150D Ca2+ sensitivity was increased compared to Tn S23/24D exchange, however this blunting of the PKA effect was less effective at 1.9 compared to at 2.2 m

(difference in EC50 from Tn WT to Tn S23/24/150D; at 1.9 m = 0.27 M, at 2.2 m =

0.16 M). Another way to evaluate the effect of sarcomere length on Ca2+ sensitivity is through length dependent activation by calculating the difference in half-maximally

2+ activating free Ca from 1.9 m to 2.2 m sarcomere lengths ( EC50) [47, 155]. In the case of length dependent activation, the exchange of Tn S150D by itself did not alter

EC50, while exchange of the Tn S23/24D PKA pseudo-phosphorylation increased

EC50 by 2-fold compared to Tn WT ( EC50; Tn WT = 0.26 0.04, Tn S150D =

0.16 0.06, Tn S23/24D = 0.52 0.08; Fig. 15D and Table 1). The combination of cTnI

Ser-150 and PKA pseudo-phosphorylation blunted this PKA induced length dependent effect such that the EC50 of Tn S23/24/150D fibers was not different from that of Tn

WT ( EC50; Tn S23/24/150D = 0.38 0.04; Fig. 15D and Table 1). These differences in the ability of the combined Tn S23/24/150D to blunt PKA dependent Ca2+ desensitization at varied lengths suggests cTnI Ser-150 phosphorylation effects myofilament Ca2+ sensitivity by a mechanism that includes more than just Ca2+ binding to TnC.

2.3.5 cTnI Ser-150 phosphorylation does not alter cTnI PKA phosphorylation.

Finally we sought to investigate if cTnI Ser-150 phosphorylation affects upstream PKA function by affecting the ability of PKA to phosphorylate cTnI when in the cTn complex. Troponin containing WT or S150D cTnI was incubated with the catalytic subunit of PKA and the reaction stopped at varied time points. The resulting time dependent phosphorylation of cTnI detected by ProQ Diamond phospho-protein staining normalized to total cTnI demonstrates no significant difference in the rate that 59

PKA phosphorylates either Tn WT or Tn S150D (Time to 50% maximal phosphorylation; WT = 19.57 1.17, Tn S150D = 18.43 3.56; P>0.05) (Fig. 16). This finding demonstrates cTnI S150D pseudo-phosphorylation does not affect the ability of

PKA to phosphorylate cTnI when incorporated into the cTn complex.

2.4 Discussion

The major findings of our studies include: 1) The AMPK holoenzyme phosphorylates cTnI at Ser-150. 2) Cardiac TnI Ser-150 is endogenously phosphorylated in normal rat and rabbit hearts. 3) The phosphorylation of cTnI Ser-150 increases myofilament Ca2+ sensitivity and blunts the Ca2+ desensitization induced by PKA Ser-

23/24 phosphorylation of cTnI. 4) Cardiac TnI Ser-150 phosphorylation alone does not affect length dependent activation but blunts the amplification of length dependent activation by PKA phosphorylation of cTnI Ser-23/24.

2.4.1 AMPK cTnI Ser-150 phosphorylation.

Recent work demonstrated that AMPK associates with cTnI and further that an

AMPK fragment phosphorylates this protein at Ser-150 [108, 158]. To date the ability of the AMPK holoenzyme to phosphorylate cTnI has been unknown. AMPK is a heterotrimeric serine/threonine protein kinase with the physiologically active AMPK holoenzyme consisting of a catalytic subunit in complex with regulatory and

60 subunits (for review see [146, 159]). The subunit contains regulatory nucleotide-binding motifs, while the subunit exhibits scaffold-like properties potentially localizing the

AMPK catalytic subunit. Solis et al. [108] demonstrated that the isolated AMPK subunit kinase domain fragment was sufficient to phosphorylate cTnI at Ser-150. Our current data significantly extends this finding demonstrating that the physiologically relevant AMPK holoenzyme phosphorylates cTnI Ser-150 in vitro (Fig. 9), as well as within the myofilament lattice (Fig. 11). These findings establish the role of the intact

AMPK subunit as a signaling molecule that can phosphorylate cTnI Ser-150 when in complex with the regulatory and subunits. The cTnI Ser-150 residue is known to be phosphorylated in vitro following kinase treatment with PKA [160], PAK [107] and

AMPK [108, 158]. Of these kinases, AMPK is likely the most physiologically relevant kinase to phosphorylate this site in vivo. Cardiac TnI Ser-150 represents a poor substrate for PKA (Fig. 14; [107, 108]. Likewise, PAK phosphorylates cTnI in vitro, however in vivo PAK overexpression leads to the de-phosphorylation of cTnI rather than direct phosphorylation [161]. While the ability of native AMPK to phosphorylate cTnI in vivo is currently unknown, Ser-150 phosphorylation is increased in a mouse cardiac hypertrophy model [162] and isolated perfused hearts treated with the AMPK activating drug AICAR

[163]. These results support a direct role for AMPK as a signaling molecule to phosphorylate cTnI Ser-150 in cardiac muscle.

2.4.2 The effects of cTnI Ser-150 phosphorylation on Ca2+ regulation of the thin filament.

Cardiac TnI is central to regulation of the myosin interaction with actin, myocyte force development and therefore cardiac contractile dynamics (for review see [35]). The

TnI C-terminus surrounding Ser-150 is comprised of two structural regions, the TnI inhibitory peptide (roughly comprised of residues 129-148 [164]) and the TnI switch peptide (residues 148-164) [55]. In the absence of Ca2+ binding to TnC, the TnI C- terminus has a low probability of binding to the TnC N-terminus, allowing the inhibitory peptide to bind actin contributing to the inhibition of myosin’s interaction with actin

[164, 165]. Upon Ca2+ binding to TnC and exposure of the N-terminal binding site for

TnI, the TnI inhibitory peptide is drawn from actin favoring binding of the switch peptide to TnC and stabilization of the TnC open, Ca2+ bound conformation [166]. The balance between TnI inhibitory peptide binding to actin and switch peptide binding to TnC is critical to TnC Ca2+ binding and therefore regulation of myosin’s interaction with actin

[153]. Cardiac TnI Ser-150 is located in close proximity the inhibitory peptide leading us to hypothesize that the incorporation of a negatively charged phosphorylation at this site will weaken cTnI inhibitory peptide binding to actin, shift the cTnI C-terminus away from its interaction with actin and increase Ca2+ dependent force development. This hypothesis is supported by data demonstrating that the presence of cTn containing the negatively charged S150D pseudo-phosphorylated cTnI in skinned fibers significantly increases Ca2+ sensitivity of force development (Fig. 12) and Ca2+ binding to TnC (Figs.

13 and 14). These findings are in agreement with others who have demonstrated that the

62 treatment of skinned fibers with PAK [107] or cTnI S150E pseudo-phosphorylation exchange [109] increased thin filament Ca2+ sensitivity.

To probe the molecular mechanism of cTnI Ser-150 phosphorylation increased

Ca2+ regulated force development we investigated the Ca2+ binding to TnC. The binding of Ca2+ to TnC in the reconstituted thin filament is a direct measurement of Ca2+’s ability to activate the filament in the absence of myosin. The increase in TnC Ca2+ binding of Tn

S150D (Fig. 13) and native phosphate at Ser-150 (Fig. 15) demonstrates the negatively charged phosphate at Ser-150 directly affects the thin filament structure/function to alter

Ca2+ binding in the absence of the thick filament. This direct effect of cTnI Ser-150 phosphorylation on thin filament Ca2+ binding occurs either through enhancement of switch peptide binding to TnC stabilizing the TnC active state or by shifting the balance of the cTnI C-terminus binding away from actin. Data demonstrating Ser-150 pseudo- phosphorylation shortens the apparent distance between the TnI C-terminus and cTnC

[109, 167] supports the latter indicating Ser-150 phosphorylation shifts the cTnI inhibitory domain from actin towards that of TnC.

Previously we demonstrated the TnC Gly-159 to Asp mutation by itself did not alter Ca2+ regulation of force but blunted the cTnI PKA dependent phosphorylation induced Ca2+ desensitization, establishing the role of Tn modifications to feedback on cTnI PKA function [150]. At the molecular level the PKA dependent phosphorylation of the cTnI Ser-23/24 residues alter the interaction of the cTnI N-terminus with TnC, destabilizing the TnC open conformation and desensitizing the thin filament to Ca2+ [38,

168]. Cardiac TnI Ser-150 is located in close proximity to Ser-23 and Ser-24 in the cTn

63 complex [110] and therefore may modulate the effect of cTnI PKA phosphorylation. Our results demonstrate cTnI Ser-150 phosphorylation crosstalks within cTnI to blunt PKA induced Ca2+ desensitization (Figs. 12, 13 and 14), through a thin filament mediated mechanism (Figs. 13 and 14). In the myofilament, Ca2+ sensitive force development is further altered by length [47]. Unlike activation of the reconstituted thin filament that solely depends upon Ca2+ binding to TnC, the force change resulting from increased length dependent activation entails other mechanisms of the myofilament. The finding that cTnI Ser-150 pseudo-phosphorylation is more effective in blunting PKA dependent

Ca2+ desensitization at 2.2 m compared to 1.9 m (Fig. 15) suggests that in addition to its effects on TnC Ca2+ binding (Figs. 12, 13 and 14), cTnI Ser-150 phosphorylation also effects the myofilament Ca2+ sensitivity response to length. The differential effect of cTnI

Ser-150 phosphorylation activation of the myofilament verses reconstituted thin filament is of great interest and deserves further future investigation.

2.4.3 AMPK signaling and the modulation of myofilament cardiac contractility.

The role of AMPK to regulate energy substrate availability as a modulator of heart function has been established [146]. We demonstrate for the first time that the classical metabolic role of AMPK is directly coupled to a mechanical effect on contraction at the level of the myofilament through the phosphorylation of cTnI Ser-150.

In the cardiac myocyte AMPK signaling induces a number of metabolic changes to increase energy and maintain cardiac contraction [146]. During cardiac stress that results in decreased ATP availability, the increased AMP/ATP ratio activates the AMPK

64 signaling pathway. Correspondingly in the non-stressed heart the level of cTnI Ser-150 phosphorylation resulting from AMPK would be predicted to be low, but increased following stress induced alteration of the AMP/ATP ratio. While we suggest cTnI Ser-

150 phosphorylation is relatively low in the normal heart, cardiac stress can nearly double

Ser-150 phosphorylation [162] and AMPK treatment increased Ser-150 phosphorylation in the muscle lattice by about 3.5 times (Fig. 11). Similar magnitude changes of cTnI

PTMs have previously been demonstrated to exhibit a significant effect on cardiac contractile function [61, 169]. Such coupling of AMPK increased energy substrate availability to a cTnI Ser-150 enhanced myofilament response to Ca2+ (Figs. 12, 13 and

14) is beneficial to maintaining enhanced contractility and cardiac output during periods of cardiac stress without the enhanced energetic cost of increased Ca2+ cycling [170].

While the physiological role of AMPK induced cTnI Ser-150 phosphorylation remains to be demonstrated, it is clear this pathway can constitute a significant myofilament level contractile regulatory mechanism.

The effects of AMPK signaling to blunt cTnI PKA dependent function and enhance contraction would likely be counterproductive to the normal and necessary cTnI induced Ca2+ desensitization of beta-adrenergic stimulation. The localized regulation of the AMPK and PKA signaling pathways is therefore necessary to avoid such AMPK induced blunting of PKA dependent myofilament desensitization. Although our data demonstrates cTnI Ser-150 phosphorylation by itself does not directly alter the ability of

PKA to phosphorylate cTnI (Fig. 16), a number of other regulatory mechanisms are likely in place to differentially regulate these two signaling pathways. Of significance,

PKA stimulation in adipocytes directly decreases AMPK activation [171, 172]. Although

PKA regulation of AMPK signaling has not been investigated in the cardiomyocyte, such mediated regulation would inhibit AMPK signaling induced blunting of the cTnI PKA desensitization. In addition to Ser O-linked phosphorylation, cTnI Ser-150 can also be modified by -N-acetyl-D-glucosamine which decreases myofilament Ca2+ sensitivity

[173]. Such glycosylation of Ser-150 would block O-phosphorylation at this residue and enhance PKA dependent Ca2+ de-sensitization. To date the differential regulation of the

PKA and AMPK signaling pathways in the heart has not yet been thoroughly investigated, however it is likely there are mechanisms that differentially regulate these pathways at the myofilament.

2.5 Conclusions

Our data demonstrate cTnI Ser-150 is endogenously phosphorylated in the normal heart, likely by AMPK. This cTnI Ser-150 phosphorylation significantly increases myofilament Ca2+ sensitivity and crosstalks within cTnI to blunt myofilament PKA dependent functional effects resulting in uncoupling of the myofilament beta-adrenergic response from other non-myofilament beta-adrenergic functional effects (i.e. Ca2+ handling). We propose the AMPK signaling pathway functions directly at the level of the myofilament to link the metabolic response of the cardiomyocyte to myofilament level function.

Table 1. Mechanical characteristics of exchanged fiber bundles.

1.9 m 2.2 m

Tn Fmax EC50 Hill Fmax EC50 Hill EC50 Exchanged WT (7) 21.2 1.0 1.53 0.03 2.85 0.10 21.6 1.3 1.27 0.03 2.84 0.12 0.26 0.04 *(P,PP,PPP) *(PP,PPP) *(P,PP) *(PP,PPP) *(PP) S150D (7) 22.8 2.2 0.92 0.01 2.50 0.10 24.8 2.4 0.76 0.02 2.65 0.17 0.16 0.06 *(W,PP,PPP) *(PP,PPP) *(W,PP,PPP) *(PP,PPP) *(PP,PPP) S23/24D (8) 20.5 2.6 2.82 0.05 4.47 0.17 26.3 4.5 2.31 0.07 3.85 0.12 0.52 0.08 *(W,P,PPP) *(W,P) *(W,P,PPP) *(W,P) *(W,P) S23/24/150D 23.7 2.1 1.80 0.09 4.18 0.29 30.7 4.0 1.43 0.09 3.87 0.18 0.38 0.04 (9) *(W,P,PP) *(W,P) *(P,PP) *(W,P) *(P)

Mechanical characteristics of skinned mouse cardiac fiber bundles exchanged with human cTn containing wild-type (Tn WT), Ser-150 pseudophosphorylated cTnI (Tn S150D), Ser-23/24 pseudo-phosphorylated cTnI (Tn S23/24D) or combined Ser-23/24 and Ser-150 pseudophosphorylated cTnI (Tn S23/24/150D).

Values are mean ± SEM. Fmax, maximal tension development in mN/mm2; EC50, the Ca2+ concentration in

μM at 50% maximal force; Hill, slope of the tension-Ca2+ plot; ΔEC50, the difference in the calcium concentration at 50% maximal force from 1.9 µm to 2.2 µm sarcomere length; n, number of fibers in each group; * ANOVA = P<0.05; W, significantly different vs. Tn WT; P, significantly different vs. Tn

S150D; PP, significantly different vs. Tn S23/24D; PPP, significantly different vs. S23/24/150D.

Table 2. Thin filament TnC Ca2+ binding characteristics.

Thin Filament Tn EC50 Hill n Tn WT 2.52 0.10 1.24 0.08 10 *(P,PP) *(PP,PPP) Tn S150D 1.03 0.16 1.87 0.04 9 *(W,PP,PPP) *(PP,PPP) 5 Tn S23/24D 8.33 0.60 1.34 0.10 *(W,P,PPP) *(W,P) Tn S23/24/150D 3.69 0.80 1.37 0.13 4 *(P,PP) *(W,P)

Binding characteristics of Ca2+ to TnC in thin filaments reconstituted with human cTn containing wild-type (Tn WT), Ser-150 pseudo-phosphorylated cTnI (Tn S150D), Ser-23/24 pseudo- phosphorylated cTnI (Tn S23/24D) or combined Ser-23/24 and Ser-150 pseudo-phosphorylated cTnI (Tn S23/24/150D). Values are mean ± SEM. EC50, the Ca2+ concentration in µM at 50% maximal binding; Hill, slope of the binding-Ca2+ plot; n, number of fibers in each group; *

ANOVA Bonferroni P<0.05; W, significantly different vs. Tn WT; P, significantly different vs.

Tn S150D; PP, significantly different vs. Tn S23/24D; PPP, significantly different vs.

S23/24/150D.

Figure 9. The AMPK holoenzyme complex phosphorylates cTnI Ser-150 in Tn.

Recombinant human cTn was incubated with the AMPK holoenzyme ( 1/ 1/ 2). (A) Tn incubated in the presence (+) or absence (-) of AMPK was stopped at 15 min intervals and identified by Western blot with the pTnI 150 antibody that only recognizes cTnI when Ser-150 is phosphorylated. Re-probing the same membrane with a cTnI antibody employing differential development and Coomassie staining demonstrates similar loading and integrity of the cTn samples. (B) Tn was incubated in the presence of AMPK and the reaction stopped at 1 h intervals, at 6 h 25% more AMPK was added and incubation continued for an additional 2 h followed by

Western blot with the pTnI 150 antibody. The plot of percent maximal pTnI 150 phosphorylation over time demonstrates Ser-150 reached maximal phosphorylation at 6 h and was not further increased by the addition of more AMPK.

Figure 10. Native rabbit cardiac muscle contains cTnI phosphorylated at Ser-150.

(A) Western blot of 2-D IEF separated rabbit ventricular myofibrils probed with the pTnI 150 antibody identified 3 cTnI species as containing Ser-150 phosphorylation (P1, P2 and P3). (B)

Sequential probing of the same membrane with a total cTnI antibody identified 4 cTnI species.

Alignment of the two membranes demonstrates the 3 most acidic, phosphorylated cTnI species were detected by the pTnI 150 antibody as containing Ser-150 phosphorylation without detection of the non-phosphorylated, basic species (U).

Figure 11. The AMPK holoenzyme phosphorylates cTnI Ser-150 in the cardiac muscle lattice.

Rat endogenous cTn in ventricular myofibrils was partially exchanged with recombinant human cTn and incubated in the absence (Control Tn MF) or presence of the AMPK holoenzyme

(AMPK Tn MF). (A) Myofibrils probed by Western blot with the pTnI 150 antibody. (B)

Simultaneous identification of total cTnI by differential detection identified two cTnI bands in exchanged myofibrils of similar size to endogenous rat and exogenous human cTnI at similar loading. (C) Merge of the pTnI 150 (green) and total cTnI (red) signals demonstrates the pTnI

150 band is identical in size to that of the total cTnI (overlap yellow).

Figure 12. Cardiac TnI Ser-150 phosphorylation increases myofilament Ca2+ sensitive force development and blunts cTnI PKA-dependent desensitization.

Skinned mouse cardiac fiber bundles were exchanged with human cTn containing either wild- type (Tn WT), Ser-150 (Tn S150D), Ser-23/24 (Tn S23/24D) or combined Ser-150 and PKA (Tn

S23/24/150D) pseudo-phosphorylated cTnI. (A) Average tension-Ca2+ measurements of fiber bundles at 2.2 m exchanged with Tn S150D (grey triangle, dashed line) compared to Tn WT

(black circles, dashed line) exchange. (B) Fiber bundles exchanged with Tn S23/24D (black square, solid line) or cTnI Tn S23/24/150D (grey diamond, dashed line) compared to Tn WT. (C)

The change in EC50 2.2 m of the various exchanged cTn from that of WT. *, ANOVA

Bonferroni EC50 P<0.05 vs. WT. Data in figure generated by A. Thawornkaiwong. 72

Figure 13. Cardiac TnI Ser-150 phosphorylation induced increase in sub-maximal force development results from increased Ca2+ binding to TnC.

Steady-state Ca2+ binding to TnC was determined by IAANS-labeled TnC fluorescence of thin filaments reconstituted with cTn containing either wild-type (Tn WT; black circle, black dashed line), Ser-150 (Tn S150D; grey triangle, grey dashed line), PKA (Tn Ser-23/24D; black square, black solid line) or combined Ser-150 with PKA (Tn S23/24/150D; grey diamond, grey solid line) pseudo-phosphorylated cTnI. Similar to Ca2+ regulated force development, Tn S150D reconstituted thin filaments exhibit increased Ca2+ binding to TnC compared to WT. Calcium binding in thin filaments containing Tn S23/24D was decreased, while the combined Tn

S23/24/150D filaments were not different from WT.

Figure 14. The incorporation of native phosphate at cTnI Ser-150 blunts Ser-23/24 decreased Ca2+ binding to TnC.

Recombinant human Tn was treated with PKA or AMPK, reconstituted into thin filaments to determine Ca2+ binding and cTnI phosphorylation was determined. (A) Representative Western blot of filaments with the pTnI 150 antibody. (B) Representative Western blot of filaments with the pTnI 23/24 antibody. (C) Steady state Ca2+ binding to TnC in filaments reconstituted with

PKA treated Tn (Tn PKA Tx; black square, black solid line) or AMPK treated Tn (Tn AMPK

Tx; grey diamond, grey dotted line) compared to sham treated filaments (Tn Sham; black circle, dashed black line). Mr Marker, molecular weight marker; Tn +150 cTnI Ser-150 phosphorylated positive control; Tn +23/24 cTnI Ser-23/24 positive control.

Figure 15. Cardiac TnI Ser-150 phosphorylation blunts the cTnI PKA dependent increase in length-dependent activation.

Skinned mouse cardiac fiber bundles were exchanged with human cTn containing either wild- type (Tn WT), Ser-150 (Tn S150D), Ser-23/24 (Tn S23/24D) or combined Ser-150 and PKA (Tn

S23/24/150D) pseudo-phosphorylated cTnI and Ca2+ dependent force development determined at

1.9 and 2.2 m in the same fiber. (A) Average tension-Ca2+ measurements at sarcomere lengths of 1.9 and 2.2 m of Tn S150D (grey triangle) exchanged fibers compared to Tn WT (black circle). (B) Average tension-Ca2+ measurements at sarcomere lengths of 1.9 and 2.2 m of Tn

S23/24/150D (grey diamond) exchanged fibers compared to Tn S23/24D (black square) exchange. (C) The change in EC50 of the various exchanged cTn from that of WT at 1.9 m. (D)

2+ Comparison of the change in half-maximal free activating Ca from 1.9 to 2.2 m ( EC50). *,

ANOVA Bonferroni P<0.05 vs. to WT. Data in figure generated by A. Thawornkaiwong. 75

Figure 16. Cardiac TnI Ser-150 phosphorylation does not alter cTnI PKA- dependent phosphorylation.

Recombinant human cTn containing wild-type (Tn WT) or Ser-150 pseudo-phosphorylated cTnI

(Tn S150D) was incubated with the PKA catalytic subunit for varied times before stopping the reaction. (A) Phosphorylation of cTnI detected by Pro-Q Diamond phosphoprotein specific stained gels demonstrates the time dependent increase in cTnI phosphorylation. Following Pro-Q

Diamond staining, gels were subjected to Sypro Ruby staining to determine total cTnI amount.

(B) Fit of cTnI phosphorylation normalized to total cTnI. Data in figure generated by J. Jin.

Chapter 3: Combined Troponin I Ser-150 and Ser-23/24 Phosphorylation Sustains Thin Filament Ca2+ Sensitivity and Accelerates Deactivation in an Acidic Environment

3.1 Introduction

Contraction of the heart results from the Ca2+ regulated interaction of myosin with the actin thin filament. The troponin (Tn) complex is an essential molecular switch that regulates this interaction of myosin with the thin filament. The binding of

Ca2+ to the troponin C (TnC) subunit of the Tn complex is the initiating step in activation of the thin filament to allow myocardial contraction while the dissociation of Ca2+ from

TnC is essential to relaxation (for review see [35, 165]). Tn PTM is a key cardiac mechanism to modulate this Ca2+ sensitive activation of the thin filament and therefore helps match myocyte contraction to meet altered cardiac demand [174, 175]. The β- adrenergic signaling pathway is a significant physiological mechanism utilized by the heart to match myocyte contraction to demand [174-176]. While the β-adrenergic signaling pathway modulates a number of cellular processes, protein kinase A (PKA) mediated troponin I (TnI) Ser-23/24 phosphorylation is a primary myofilament target of this signaling [177-179]. In the normal heart, basal TnI Ser-23/24 phosphorylation comprises approximately 40% of the total TnI with increases or decreases of this phosphorylation central to the modulation of cardiac function [101, 177, 178, 180, 181].

This phosphorylation of TnI at Ser-23/24 functions to alter contraction by desensitizing the myofilament to Ca2+. Thus, while TnI Ser-23/24 phosphorylation desensitization 77 decreases Ca2+ sensitive force production, desensitization also induces an acceleration in the rate of muscle relaxation necessary to allow adequate diastolic filling and maintained cardiac output during elevated heart rates [104, 177, 178].

In various pathological states of the heart, contractile dysfunction results in an impaired ability of the heart to meet demand. One such pathological state occurs during cardiac ischemia when a reduction in blood flow to the heart results in decreased ATP production and diminished contractile function [122]. While reduced blood flow affects a number of cardiac processes, a hallmark of ischemia is a decrease in intracellular pH.

During global cardiac ischemia the intracellular pH of the cardiomyocyte can drop from

7.0 to ~ 6.2 [182]. This decrease in pH directly affects cellular processes including an altered Ca2+ regulation of the thin filament. Ischemia induced acidosis decreases the Ca2+ sensitive activation of the thin filament contributing to decreased myocardial force production and exacerbating hypoxia-induced contractile dysfunction [183].

In chapter 2 we characterized the AMP-activated protein kinase (AMPK) phosphorylation of TnI at Ser-150 and its effect on TnI Ser-23/24 phosphorylation function. Our findings, and those of others, demonstrate that TnI Ser-150 phosphorylation increases myofilament Ca2+-sensitive force production and blunts TnI PKA-dependent desensitization of skinned fibers and the reconstituted thin filament [106, 184].

Consistent with the AMPK phosphorylation of TnI Ser-150, Oliveira et al. demonstrated an elevation of TnI Ser-150 phosphorylation during the increased cardiac demand of ex vivo cardiac ischemia [106]. To date, the effects of TnI Ser-150 phosphorylation on the

78 depressed cardiac contractile function that occurs during myocardial ischemia and how

Ser-150 interacts with TnI Ser-23/24 PKA phosphorylation are unknown.

In this chapter we sought to investigate the integrated role of TnI Ser-23/24 and

Ser-150 phosphorylation combination on cardiac thin filament contractile regulation under acidic conditions similar to those occurring in ischemia. Towards this end we quantified changes in TnI Ser-150 and Ser-23/24 phosphorylation following in vivo myocardial ischemia and investigated the combined effects of TnI pseudo- phosphorylation on thin filament regulation at acidic pH. Our findings demonstrate myocardial ischemia increases both TnI Ser-150 and Ser-23/24 phosphorylation. We demonstrate TnI Ser-150 pseudo-phosphorylation in isolation blunts pH mediated thin filament Ca2+ desensitization while its combination blunts Ser-23/24 Ca2+ desensitization with a minimal effect on Ser-23/24 induced acceleration of thin filament Ca2+ dissociation. These data support a role for ischemia-induced TnI Ser-150 and Ser-23/24 phosphorylation to maintain Ca2+ regulated force production while accelerating relaxation. The concurrent phosphorylation of TnI Ser-150 and Ser-23/24 may therefore play an adaptive role in sustaining cardiac contraction during the acidic conditions of an ischemic event without delaying relaxation.

3.2 Materials and Methods

3.2.1 In vivo myocardial ischemia

In vivo left ventricular myocardial infarction was achieved via left coronary ligation in C57BL/6J mice (Jackson Laboratories, Bar Harbor, Maine) at 4 months of age as previously done [185]. Briefly, mice were anesthetized with ketamine (55 mg/kg) plus xylazine (15 mg/kg). Animals were intubated and ventilated (tidal volume 250 µl, 150 breath/min) with a mouse respirator (687, Harvard Apparatus). Body temperature was maintained at 37°C using a heating blanket (TC-1000, CWE). Through a left thoracotomy, we ligated the left coronary artery 1 to 2 mm below the border of the left atrial appendage. Ischemia was confirmed by pallor distal to the occlusion and by ST elevation on ECG. At 30 minutes after ligation, the heart was removed, the left ventricular free wall quickly dissected free and flash-frozen in liquid nitrogen. All animal protocols and procedures were performed in accordance with National Institutes of Health guidelines and approved by the Institutional Laboratory Animal Care and Use Committee at The Ohio State University.

3.2.2 Protein electrophoresis and Western blot

Myofibrils from sham or ischemic left ventricle free wall were solubilized in denaturing buffer (2% SDS, 0.1% bromophenol blue, 10% glycerol and 50 mM Tris-HCl, pH 6.8), heated for five minutes at 80 C and clarified by centrifugation for five minutes.

SDS-PAGE and western blot were carried out as previously described [40]. Briefly, 80 cardiac TnI Ser-150 phosphorylation was quantified by incubation with a custom rabbit anti-phosphorylated TnI Ser-150 antibody that we previously demonstrated as specific to the detection of TnI only when phosphorylated at Ser-150 [184]. The Ser-150 phosphorylation antibody was followed by incubation with a Dylight fluorescent secondary antibody (Jackson ImmunoResearch Laboratories, Inc.) and visualized on a

Typhoon 9410 imager (GE Healthcare). Subsequent quantification of total cardiac TnI was quantified by re-probing the same membrane with a mouse anti-cardiac TnI antibody

(Fitzgerald; clone C5), detected and visualized as above. Sequential development using different primary/secondary combinations allows for quantification of phosphorylated and total TnI species in the same membrane [40, 186]. The phosphorylation of TnI Ser-23 and Ser-24 was conducted by incubation with the anti-phosphorylated TnI Ser-23/24 antibody (Cell Signaling Technology, Inc.) detected and visualized as above.

3.2.3 cDNA constructs

All cardiac TnI residue numbers presented in this manuscript are presented according to the native human sequence including the first methionine. The human cardiac TnI Ser-

150 to Asp (S150D), Ser-23/24 to Asp (S23/24D) and Ser-23/24/150 to Asp

(S23/24/150D) pseudo-phosphorylation mutant cDNA were generated by site-directed mutagenesis (Quick Change II kit, Agilent) according to the manufacturer’s direction and resultant constructs were verified by DNA sequencing as previously described [184].

3.2.4 Proteins

The individual recombinant human cardiac Tn subunits were expressed in E. coli and purified to homogeneity as previously described [187]. Tn used in Ca2+ binding experiments consisted of native human cardiac TnT, cardiac TnI and cardiac T53C labeled 2-(4’-iodoacetamidoanilo)naphthalene-6-sulfonic acid (IAANS) TnC containing the C35/84S mutations. Cardiac Tn complexes were reconstituted by sequential dialysis as previously described. Thin filaments were reconstituted by sequential incubation of actin first with a stoichiometric amount of tropomyosin followed by incubation with Tn as previously described [184].

3.2.5 Measurement of Tn steady-state Ca2+ binding to TnC

Steady-state Ca2+ binding to TnC in the thin filament or isolated Tn was measured at

15°C in 150 mM KCl, 3 mM MgCl2, 2 mM EGTA and 200 mM MOPS as previously described [184]. Briefly, Tn or thin filaments containing various TnI pseudo- phosphorylations were reconstituted at pH 7.0 or pH 6.5 and the change in IAANS labeled T53C TnC (C35/84S) fluorescence was monitored at various free [Ca2+] as indicative of Ca2+ binding to TnC.

3.2.6 Measurement of thin filament and Tn Ca2+ dissociation from TnC

Isolated Tn or reconstituted thin filaments were prepared at either pH 7.0 or 6.5.

Following reconstitution, 0.2 mM Ca2+ was added to saturate Tn or the thin filament. Ca2+ saturated thin filament or Tn was rapidly mixed with EGTA buffer and Ca2+ dissociation 82 was monitored by the change in IAANS fluorescence at 15°C as previously described

[188, 189].

3.2.7 Measurement of myosin S1 dissociation from the thin filament

Reconstituted thin filaments were prepared as previously described at either pH 7.0 or

6.5. Following reconstitution EGTA was added to 5 mM and myosin S1 added at a ratio of 7:4 (actin:myosin S1). Rigor bound myosin S1 thin filaments were then rapidly mixed with ATP (2 mM final) and the dissociation of myosin monitored by the change in

IAANS fluorescence at 15°C as previously described [188, 189].

3.2.8 Data processing and statistical analysis

Steady-state Ca2+ binding plots were fit to the Hill equation to determine 50% maximal binding. Ca2+ dissociation data were fit using a program (by P. J. King, Applied

Photophysics Ltd.) utilizing the nonlinear Levenberg-Marquardt algorithm where each

Ca2+ dissociation event represents an average of at least three separate experiments.

Results of Ca2+ binding and Ca2+ dissociation were compared by 2-way ANOVA with

Tukey’s post-hoc test. In vivo sham vs. ischemic cardiac TnI Ser-150 and Ser-23/24 phosphorylation was compared by Student’s t-Test. P<0.05 was considered statistically significant. Data are presented as mean SEM.

3.3 Results

3.3.1 In vivo myocardial ischemia increases both TnI Ser-150 and Ser-23/24 phosphorylation.

While the phosphorylation of TnI Ser-150 is low in the normal, non-stressed heart, its phosphorylation becomes elevated during myocardial ischemia [106]. In chapter

2 we demonstrate that TnI Ser-150 pseudo-phosphorylation increases Ca2+ sensitivity and blunts TnI S23/24D Ca2+ desensitization [184]. To investigate when the combination of

TnI Ser-150 and Ser-23/24 phosphorylation would be relevant to modulate cardiovascular function we determined TnI phosphorylation levels during myocardial ischemia. Western blot of left ventricular tissue from 6 mice subjected to in vivo ischemia for 30 minutes demonstrates the phosphorylation of TnI Ser-150 increased 31% and TnI Ser-23/24 phosphorylation increased 34% compared to that of 7 sham mice (Fig. 17). This simultaneous increase in both phosphorylations demonstrates their significance to cardiac contractile function during the pathological state of myocardial ischemia.

3.3.2 TnI Ser-150 and Ser-23/24 phosphorylation alters the acidic effects of thin filament regulation.

It is well established that an acidic environment, such as that which occurs in the cardiomyocyte during myocardial ischemia, depresses Ca2+-dependent force production at the level of the myofilament [183]. To determine the effect of Tn containing TnI Ser-

150 phosphorylation, Ser-23/24 phosphorylation, and their combination on myofilament regulation at ischemic pH we measured TnC Ca2+ binding at normal (7.0) and ischemic 84

(6.5) pH in the reconstituted thin filament. As demonstrated in Figure 18, lowering the pH to 6.5 decreased Ca2+ binding to TnC in all thin filament groups compared to their

Ca2+ binding at normal pH. Additionally, the Tn type also affected Ca2+ binding at pH 6.5 such that thin filaments containing TnI S150D remained sensitized, TnI S23/24D were desensitized and TnI S23/24/150D were similar to WT (Fig. 18 and Table 3).

Importantly, the effect of acidic pH to decrease Ca2+ binding in filaments containing TnI

S150D was blunted by 39% compared to WT filaments. Therefore the combined effect of pH with TnI S150D resulted in a pCa50 at pH 6.5 that was not different from that of WT filaments at pH 7.0 (Fig. 18D and Table 3).

3.3.3 The phosphorylation of TnI Ser-150 and Ser-23/24 affect function through different thin filament interactions.

To investigate the mechanism by which TnI Ser-150 phosphorylation blunts pH mediated Ca2+ desensitization, we employed a reductionist approach. To minimize the interactions within the thin filament responsible for the effects of TnI phosphorylation, we determined Ca2+ binding to TnC in isolated Tn devoid of actin and tropomyosin interactions at pH 6.5. Isolated Tn containing S150D increased Ca2+ binding to TnC compared to WT Tn (Fig. 19A and Table 4) preserving the TnI S150D function observed in reconstituted thin filaments and skinned fibers [184]. Unexpectedly, isolated

Tn containing TnI S23/24D did not alter TnC Ca2+ binding compared to WT (Fig. 19B and Table 4) differing from the previously observed effect of TnI Ser-23/24 phosphorylation in the reconstituted thin filament [184]. The effect of S150D to increase

Ca2+ binding was retained when combined with S23/24D such that TnI S23/24/150D Tn

Ca2+ binding was not different from that of S150D (Fig. 19C and Table 4). These findings demonstrate TnI Ser-150 and Ser-23/24 phosphorylation function through different Tn interactions at acidic pH. To determine if the TnI Ser-150 phosphorylation difference in Tn Ca2+ binding resulted from decreased pH or the phosphorylation itself,

Tn Ca2+ measurements were repeated at normal pH. While elevating the pH to 7.0 increased Ca2+ binding of all Tn’s, Ca2+ binding to TnC in the isolated Tn containing TnI

S150D and S23/24/150D remained increased and TnI S23/24D was not affected (Fig.

19D and Table 4). Thus the phosphorylation of TnI Ser-150 and Ser-23/24 is responsible for alteration of thin filament Ca2+ regulation through different Tn interactions.

3.3.4 The combination of TnI Ser-150 with Ser-23/24 phosphorylation retains accelerated thin filament deactivation.

The rate at which Ca2+ can be removed from TnC represents a critical event in thin filament deactivation and myocardial relaxation [165]. To investigate the effect of

TnI Ser-150 phosphorylation on the Ca2+ component of thin filament deactivation, we measured Ca2+ dissociation from TnC in the thin filament at acidic pH. The rate of Ca2+ dissociation was determined by monitoring the change in IAANS-labeled TnC fluorescence following rapid mixing with EGTA by stopped-flow fluorimetry [189].

Consistent with our previous Ca2+ sensitivity findings [184], thin filaments containing

TnI S150D slowed the rate of Ca2+ dissociation to half of WT filaments while S23/24D filaments accelerated Ca2+ dissociation 3.1-fold compared to WT (Fig. 20A; Table 5).

We next sought to investigate whether crosstalk between TnI Ser-150 and Ser-23/24 phosphorylation would affect TnC Ca2+ dissociation. Upon combination, TnI

S23/24/150D retained a 2.1-fold accelerated Ca2+ dissociation rate compared to that of

WT (Fig. 20B; Table 5). Thus, while the Ca2+ dissociation of the combined TnI

S23/24/150D was marginally slowed by the presence of S150D compared to that of TnI

S23/24D alone, this combined S23/24/150D dissociation was still dramatically faster than that of WT. These findings demonstrate the functional outcome of TnI Ser-150 phosphorylation to modulate the Ca2+ regulated component of thin filament deactivation at acidic pH is dependent upon its combination with TnI Ser-23/24 phosphorylation.

To determine if this unique effect of combined TnI S150D with S23/24D resulted from a pH effect or was an intrinsic property of phosphorylation itself we determined the rate of Ca2+ dissociation from TnC at normal pH. The results in Figure 20 demonstrate that increasing the pH to 7.0 did not affect the Ca2+ dissociation rate of thin filaments containing any Tn compared to that at pH 6.5. Additionally, thin filaments containing TnI

S23/24/150D retained a 2.4-fold accelerated Ca2+ dissociation rate compared to WT at pH 7.0 (Fig. 20C; Table 5). The similarly increased rate of TnI S23/24/150D thin filament Ca2+ dissociation at both pH 6.5 and 7.0 demonstrates this increased Ca2+ dissociation is dependent upon the phosphorylation of TnI and is not a pH-dependent effect.

In addition to Ca2+ dissociation from TnC, deactivation of the thin filament in the muscle also requires myosin dissociation from the actin filament. To investigate the effect of TnI Ser-150 phosphorylations on the myosin component of thin filament

87 deactivation, we measured myosin dissociation from actin in the thin filament at acidic pH. The rate of myosin S1 dissociation was determined by monitoring the change in

IAANS-labeled TnC fluorescence following rapid mixing with ATP by stopped-flow fluorimetry [189]. Thin filaments reconstituted with Tn containing either TnI S150D alone, TnI S23/24D alone or the combination of both all marginally slowed the rate of myosin S1 dissociation compared to WT by about 25% (Fig. 21; Table 6). Since both the individual and combined TnI phosphorylation similarly decrease myosin S1 dissociation at pH 6.5 we sought to determine if this change in S1 dissociation resulted from the phosphorylation of TnI or the acidic pH. Repetition of the myosin S1 dissociation rate measurements at pH 7.0 demonstrates the dissociation of S1 from thin filaments containing TnI S150D, S23/24D or their combination were not different from those at pH

6.5 (Fig. 21C; Table 9). These data demonstrate the phosphorylation of TnI at Ser-150,

Ser-23/24 or their combination similarly functions to marginally slow the myosin component of thin filament deactivation regardless of pH.

3.4 Discussion

The goal of this chapter was to determine the integrated role of TnI Ser-150 and

Ser-23/24 phosphorylation on thin filament regulation in an acidic environment similar to that of ischemia. The major findings from our study demonstrate: 1) The phosphorylation of TnI Ser-150 and Ser-23/24 are concurrently increased in response to myocardial ischemia. 2) TnI Ser-150 phosphorylation blunts the pH-dependent decrease in Ca2+

88 binding to TnC to sustain Ca2+ sensitivity at pH 6.5 identical to WT at pH 7.0. 3) TnI Ser-

150 and Ser-23/24 phosphorylation affect Ca2+ regulation of the thin filament through different Tn interactions. 4) Tn phosphorylation regulation of the thin filament is dependent upon whether TnI phosphorylation occurs alone or in combination. In isolation

TnI Ser-150 phosphorylation increases thin filament Ca2+ sensitivity and slows Ca2+ dissociation while isolated TnI Ser-23/24 phosphorylation decreases Ca2+ sensitivity and accelerates Ca2+ dissociation. Upon the combination of TnI Ser-150 with Ser-23/24 phosphorylation thin filament Ca2+ sensitivity remains similar to wild-type while Ca2+ dissociation is greatly accelerated.

3.4.1 Myocardial ischemia simultaneously increases phosphorylation of both TnI

Ser-150 and Ser-23/24.

The observed simultaneous increase of both TnI Ser-150 and Ser-23/24 phosphorylation in the presence of myocardial ischemia demonstrates the significance of these phosphorylations to modulate myofilament contraction during cardiac stress. TnI

Ser-150 is normally present at low amounts in the unstressed heart and it is increased

31% in response to myocardial ischemia (Fig. 17), indicating a modulatory role [106].

Conversely, approximately 40% of TnI is basally phosphorylated at both Ser-23 and 24

[101]. While TnI Ser-23/24 was previously reported to be decreased by approximately

30% in the ischemic, non-perfused rat papillary muscle [190, 191], we demonstrate ischemia increases TnI Ser-23/24 phosphorylation by 34% in the mouse left ventricular free wall containing both perfused and ischemic tissue. The implications of these

89 different findings support a role of TnI Ser-23/24 phosphorylation to be differentially regulated in ischemic versus perfused tissue during myocardial infarction to invoke regional modulation of cardiac contraction. Regardless, the ischemia-induced increase in

TnI Ser-150 phosphorylation in the presence of high levels of TnI Ser-23/24 phosphorylation demonstrates a modulatory role of TnI Ser-150 phosphorylation, both in isolation and when combined with Ser-23/24 on the same TnI molecule.

3.4.2 TnI Ser-150 phosphorylation differentially modulates thin filament Ca2+ regulation.

In the normal perfused heart, TnI Ser-150 phosphorylation is low and its functional impact is therefore minimal [106, 184]. During myocardial ischemia, decreased oxygen availability leads to an impaired metabolism, ATP depletion and acidosis [192]. In addition to a number of effects, ischemia-induced acidosis directly depresses myofilament Ca2+ regulated force production [183] while ATP depletion activates AMPK to phosphorylate TnI at Ser-150 [106, 108, 184]. In the previous chapter we demonstrated TnI Ser-150 phosphorylation increases Ca2+ sensitive force production at normal pH [184]; however, its role during acidosis when its phosphorylation is elevated was unknown. We now demonstrate TnI S150D in isolation blunts the effect of acidosis-induced Ca2+ desensitization. Thus ischemia-induced elevation of TnI Ser-150 phosphorylation results in enhanced Ca2+ activation of the thin filament at pH 6.5 that is not different from that of WT filaments at pH 7.0 (Fig. 18). TnI Ser-150 phosphorylation in isolation therefore functions to oppose acidosis-induced myofilament desensitization.

With the high basal level of Ser-23/24 phosphorylation it is likely that ischemia- induced Ser-150 phosphorylation will occur in combination with phosphorylated Ser-

23/24 on the same TnI molecule. Our lab and others have demonstrated myofilament

PTMs, including TnI Ser-150 phosphorylation, can modulate the contractile effects of

TnI Ser-23/24 phosphorylation [98, 184, 193, 194]. Chapter 3 expands upon these previous findings demonstrating the ischemia induced pH effect on Ca2+ desensitization is dependent upon whether TnI Ser-150 and Ser-23/24 phosphorylation occurs alone or in combination. By itself TnI S150D blunts pH-dependent Ca2+ desensitization while

S23/24D by itself enhances pH desensitization; however, their combination results in a

Ca2+ sensitivity similar to WT at acidic pH (Fig. 18). While these studies were conducted in thin filaments containing 100% pseudo-phosphorylated TnI, we previously demonstrated similar results at pH 7.0 upon the combination of TnI Ser-150 with Ser-

23/24 at sub-maximal phosphate incorporation levels [184]. Based upon this previous finding we speculate that the ischemia elevated combination of TnI Ser-150 with Ser-

23/24 phosphorylation will result in a similar Ca2+ sensitivity effect to that of the 100% pseudo-phosphorylated TnI combinations. In the future it will be important to determine if ischemia preferentially induces TnI Ser-150 phosphorylation alone or in combination with Ser-23/24 phosphorylation.

The binding of Ca2+ to TnC is a necessary event in contraction; however, the heart functions such that the activating intracellular myocyte Ca2+ concentration is constantly changing and never reaches equilibrium. Ca2+ binding to TnC and activation of the thin filament is therefore dependent upon constantly changing amounts of Ca2+ such that Ca2+

91 binding to TnC is not in steady-state. Equilibrium, steady-state Ca2+ binding measurements therefore provide only a partial picture of the force contraction processes in the heart. In addition, deactivation of the thin filament and relaxation requires the dissociation of Ca2+ from TnC. We therefore determined the effect of TnI Ser-150 and

Ser-23/24 ischemia-induced TnI phosphorylation on TnC Ca2+ dissociation kinetics at acidic pH. Based upon the understanding that TnI S23/24D desensitizes and S150D sensitizes the thin filament to Ca2+ one would expect TnI S23/24D to accelerate Ca2+ dissociation and S150D to slow dissociation compared to WT, which is exactly what we observed (Fig. 20). This finding that TnI S150D significantly slows Ca2+ dissociation is supported by previous FRET findings at normal pH [109]. However, the combination of

S23/24/150D on the same TnI molecule retained wild-type like Ca2+ sensitivity but exhibited significantly accelerated Ca2+ dissociation compared to wild-type (Figs. 18 and

20). Thus, while TnI S150D alone dramatically slowed dissociation to half of WT, the combination of S150 with S23/24 phosphorylation (S23/24/150D) was only marginally slowed from that of S23/24D alone such that it remained 2.1-fold accelerated compared to WT. Interestingly, TnI phosphorylation had a negligible effect on the myosin S1 dissociation component of thin filament deactivation with all 3 phosphorylation combinations minimally slowing S1 dissociation by about 25% of wild-type. Consistent with these phosphorylation data, we have also reported similar differential effects of TnI and TnT disease mutations on Ca2+ sensitivity and dissociation rates [188, 195]. The combination effect of TnI Ser-150 and Ser-23/24 phosphorylation on dissociation was not the result of a pH effect but rather resulted from combination of the phosphate residues as

92 demonstrated by the identical dissociation rates of TnI S23/24/150D at both pH 6.5 and

7.0 (Fig. 21). This represents the first demonstration of Tn phosphorylation to both maintain Ca2+ regulated thin filament activation and accelerate thin filament deactivation.

We speculate this combination of TnI Ser-150 with Ser-23/24 phosphorylation is significant to contractile function during ischemia by maintaining Ca2+-dependent force while accelerating relaxation.

3.4.3 TnI Ser-150 and Ser-23/24 structure-function.

The balance between TnI regulatory inhibitory region binding to actin and switch peptide binding to TnC is a critical determinant of thin filament activation upon Ca2+ binding to TnC [35]. TnI Ser-150 phosphorylation directly alters TnI regulatory region binding [109, 167]; however, the mechanism for how its combination with Ser-23/24 phosphorylation differentially affects Ca2+ sensitivity and dissociation is unknown. The finding that TnI S150D alters Ca2+ binding in isolated Tn devoid of actin and tropomyosin interactions but TnI S23/24D does not (Fig 19) demonstrates TnI Ser-150 phosphorylation functions through Tn interactions not present in TnI phosphorylated at

Ser-23/24. Thus the effect of TnI Ser-23/24 phosphorylation to alter Ca2+ sensitivity and dissociation further requires additional actin and/or tropomyosin interactions. Previous reports support Tn modification differences in Ca2+ binding to TnC in thin filament versus isolated Tn [195]. The lack of such a regulatory effect in isolated Tn containing

TnI S23/24D demonstrates Ca2+ regulation of the thin filament can be modulated through more than one Tn interaction and these regulatory interactions are different between Ser-

150 and Ser-23/24 phosphorylation. Different mechanisms of function are further demonstrated by the similar Ca2+ sensitivity of Tn S23/24/150D to S150D. Future definition of the protein structure-function interactions responsible for the functional effects of these phosphorylations will be beneficial to targeted drug design modulation of

Ca2+ sensitivity and/or dissociation.

3.4.4 Functional consequences of ischemia-induced TnI phosphorylation.

Myocardial ischemia induces an increase in the Ca2+ sensitive force development of the heart muscle [196]. While a number of factors likely contribute to this increase,

TnI represents a prime effector of this sensitization. Genetic manipulation has demonstrated TnI as a significant molecule to modulate cardiac contraction in response to ischemia. Mutation of the cardiac TnI Ala-164 residue to its skeletal His counterpart confers contractile resistance to the ischemic effects of cardiac acidosis [66]. In addition to genetic manipulation, myocardial ischemia itself directly induces PTM of TnI to modulate the ischemic contractile response. Ischemia was previously shown to induce proteolysis of the 17 TnI C-terminal amino acids resulting in increased Ca2+ sensitivity and slowed myofibril relaxation kinetics [62, 196-199]. Our data now demonstrate myocardial ischemia further induces the post-translational phosphorylation of TnI Ser-

150 further contributing to ischemia increased Ca2+ sensitivity (Fig. 18). While expression of the Ca2+ sensitized slow skeletal TnI has been shown to be protective during ischemia [200], it is currently unknown if direct Ca2+ sensitization of contraction alone is sufficient to improve cardiac function and/or long-term protection from

94 myocardial ischemia. Future experiments are necessary to establish the ultimate role of integrated ischemia induced TnI PTMs and their significance to survival following myocardial ischemia.

3.5 Conclusions

Taken as a whole, we demonstrate that TnI Ser-150 and Ser-23/24 phosphorylation function through varied Tn interactions contributing to different thin filament regulatory modulation. While isolated TnI Ser-150 phosphorylation blunts the ischemic pH mediated Ca2+ desensitization, the combination of these phosphorylations in the same TnI molecule represents the first demonstration of Tn phosphorylation to maintain Ca2+ sensitivity and accelerate Ca2+ dissociation. We propose ischemic induced

TnI Ser-150 phosphorylation alters cardiac contraction during myocardial ischemia to improve recovery from ischemia.

Table 3. Steady-state Ca2+ binding to TnC in reconstituted thin filaments containing WT, S150D, S23/24D, or S23/24/150D Tn at pH 6.5 or pH 7.0.

pH 6.5 pH 7.0

Thin Filament pCa50 n pCa50 n

∆pCa50 Tn WT 5.35±0.01 3 5.79±0.04 3 0.44 *(W,P,PP,PPP,P’,PP’) *(P,PP,W’,PP’,PPP’) Tn S150D 5.82±0.04 3 6.09 0.01 3 0.27 *(PP,W’,P’,PP’,PPP’) *(W,PP,PPP,W’,P’,PP’,PPP’) Tn S23/24D 5.09±0.01 4 5.51 0.02 3 0.42 *(W,P,PP,PPP,W’,P’,PP’,PPP’) *(W,P,PPP,W’,P’,PP’,PPP’) Tn S23/24/150D 5.28±0.04 3 5.78 0.02 3 0.50 *(W,P,PP,PPP,P’,PP’,PPP’) *(P,PP,W’,PP’,PPP’)

2+ Values are mean SEM. pCa50, Ca concentration at 50% activation; n, number of thin filaments in each group. Two way ANOVA was performed and found a significant interaction of pH and phosphorylation type. * ANOVA = P<0.05; W, significantly different vs. WT pH 7.0; P, significantly different vs. S150D pH 7.0; PP, significantly different vs. S23/24D pH 7.0; PPP, significantly different vs. S23/24/150D pH 7.0. W’, significantly different vs. WT pH 6.5; P’, significantly different vs. S150D pH 6.5; PP’, significantly different vs. S23/24D pH 6.5; PPP’, significantly different vs. S23/24/150D pH 6.5.

Table 4. Steady-state Ca2+ binding to TnC in isolated Tn containing WT, S150D, S23/24D, or S23/24/150D TnI at pH 6.5 or pH 7.0.

pH 6.5 pH 7.0

Tn pCa50 n pCa50 n TnI WT 6.19±0.03 3 6.38±0.01 3 *(W,P,PP,PPP,P’,PPP’) *(P,PPP,W’,PP’) TnI S150D 6.40±0.03 3 6.56±0.01 3 *(P,PPP,W’,PP’) *(W,PP,W’,P’,PP’,PPP’) TnI S23/24D 6.16±0.01 3 6.32±0.02 3 *(W,P,PP,PPP,P’,PPP’) *(P,PPP,W’,PP’,PPP’) TnI S23/24/150D 6.45±0.01 3 6.53±0.01 3

97 *(P,PP,W’,PP’) *(W,PP,W’,P’,PP’)

2+ Values are mean SEM. pCa50, Ca concentration at 50% activation; n, number of Tn in each

group. Two way ANOVA was performed and found a significant interaction of pH and

phosphorylation type. * ANOVA = P<0.05; W, significantly different vs. WT; P, significantly

different vs. S150D; PP, significantly different vs. S23/24D; PPP, significantly different vs.

S23/24/150D.

Table 5. Ca2+ dissociation from TnC in reconstituted thin filaments containing WT, S150D, S23/24D, or S23/24/150D Tn at pH 6.5 or pH 7.0.

pH 6.5 pH 7.0

Thin Filament Koff (/s) n Koff (/s) n Tn WT 108.48±8.98 6 106.42 2.57 9 *(PP,PPP,PP’,PPP’) *(P,PP,PP’,PPP’) Tn S150D 42.13±0.27 6 44.80 0.28 16 *(PP,PPP,PP’,PPP’) *(W,PP,PPP,PP’,PPP’) Tn S23/24D 335.23±39.86 6 332.50 27.45 8 *(W,P,PPP,W’,P’,PPP’) *(W,P,W’,P’,PPP’) Tn S23/24/150D 228.68±17.69 9 256.48 10.78 16 *(W,P,PP,W’,P’,PP’) *(W,P,W’,P’,PP’)

2+ Values are mean SEM. Koff, rate of Ca removal from TnC per second; n, number of thin filaments in each group. Two way ANOVA was performed and found no significant interaction of pH and phosphorylation type. * ANOVA = P<0.05; W, significantly different vs. WT pH 7.0;

P, significantly different vs. S150D pH 7.0; PP, significantly different vs. S23/24D pH 7.0; PPP, significantly different vs. S23/24/150D pH 7.0. W’, significantly different vs. WT pH 6.5; P’, significantly different vs. S150D pH 6.5; PP’, significantly different vs. S23/24D pH 6.5; PPP’, significantly different vs. S23/24/150D pH 6.5.

Table 6. Ca2+ dissociation from TnC in reconstituted thin filaments containing WT, S150D, S23/24D, or S23/24/150D Tn and myosin S1 at pH 6.5 or pH 7.0.

pH 6.5 pH 7.0

Thin Filament + Koff (/s) n Koff (/s) n

Myosin s1 Tn WT 293.31.17±13.88 9 282.17 11.20 18 *(P,PP,PPP,P’,PP’,PPP’) *(P,PP,PPP,P’,PP’,PPP’) Tn S150D 184.52±8.79 9 191.48 7.55 18 *(W,W’) *(W,W’) Tn S23/24D 231.18±5.98 9 223.02 7.84 18 *(W,W’) *(W,W’) Tn S23/24/150D 219.95±9.95 8 198.78 8.83 18 *(W,W’) *(W,W’)

Values are mean SEM. Koff, rate of myosin S1 dissociation from thin filament per second; n, number of thin filaments in each group. Two way ANOVA was performed and found no significant interaction of pH and phosphorylation type. * ANOVA = P<0.05; W, significantly different vs. WT pH 7.0; P, significantly different vs. S150D pH 7.0; PP, significantly different vs. S23/24D pH 7.0; PPP, significantly different vs. S23/24/150D pH 7.0. W’, significantly different vs. WT pH 6.5; P’, significantly different vs. S150D pH 6.5; PP’, significantly different vs. S23/24D pH 6.5; PPP’, significantly different vs. S23/24/150D pH 6.5.

Figure 17. Western blot of TnI Ser-150 and Ser-23/24 phosphorylation in ischemia cardiac tissue.

Left ventricular free wall myofilament preparations from mice that underwent 30 minutes of left coronary artery occlusion or sham operation were subjected to SDS-PAGE and subsequent western blot to detect levels of TnI Ser-150 (pTnI 150) or Ser-23/24 (pTnI 23/24) phosphorylation following regional ischemia (sham, n=7; ischemia, n=6). (A) Representative western blot and relative quantitation of TnI Ser-150 phosphorylation with a pTnI 150 specific antibody or total TnI antibody. (B) Representative western blot and relative quantitation of TnI

Ser-23/24 phosphorylation with a pTnI 23/24 specific antibody or total TnI antibody. *P<0.05 vs. sham.

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Figure 18. TnI phosphorylation steady-state Ca2+ binding in the reconstituted thin filament at ischemic and normal pH.

The change in IAANS fluorescence following the addition of various Ca2+ amounts in thin filaments containing IAANS labeled TnC and unmodified TnI at pH 7.0 (WT, open square) or pH

6.5 (WT, closed square) vs. pseudo-phosphorylated cTnI Ser-150 filaments (S150D, closed circle) (A), pseudo-phosphorylated cTnI Ser-23/24 filaments (S23/24D, closed diamond) (B) or pseudo-phosphorylated cTnI Ser-23/24/150 filaments (S23/24/150D, closed triangle) Tn (C).

* Comparison of thin filament pCa50 at pH 7.0 (black) and pH 6.5 (dark gray) (D). P<0.05 vs.

WT.

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Figure 19. TnI phosphorylation steady-state Ca2+ binding in isolated Tn at ischemic and normal pH.

The change in IAANS fluorescence following the addition of various Ca2+ amounts in isolated recombinant human cTn containing IAANS labeled TnC and unmodified Tn (WT, open square) vs. pseudo-phosphorylated cTnI Ser-150 (S150D, closed circle) (A), pseudo-phosphorylated cTnI

Ser-23/24 Tn (S23/24D, closed diamond) (B) or pseudo-phosphorylated cTnI Ser-23/24/150 Tn

(S23/24/150D, closed triangle) (C). Comparison of pCa50 between WT, S150D, S23/24D, and

S23/24/150D isolated Tn at pH 7.0 (black) and pH 6.5 (dark gray). (D). *P<0.05 vs. WT.

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Figure 20. Ca2+ dissociation kinetics of phosphorylated TnI in the reconstituted thin filament at ischemic and normal pH.

Ca2+ saturated thin filaments were rapidly mixed with EGTA and Ca2+ dissociation monitored as the change in IAANS fluorescence. Representative stopped-flow traces from Ca2+-saturated thin filaments containing WT Tn vs. S150D and S23/24D filaments (A) or S23/24/150D filaments

(B). Traces demonstrating the change in fluorescence over time are normalized and staggered for

2+ clarity. Comparison of the Ca dissociation rates (koff) from WT, S150D, S23/24D, and

S23/24/150D containing thin filaments at pH 7.0 (black) and pH 6.5 (dark gray). (C). *P<0.05 vs.

WT. Raw data in figure generated by S. Walton from the Davis lab at OSU.

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Figure 21. Myosin dissociation kinetics of phosphorylated TnI in the reconstituted thin filament with myosin S1 at ischemic and normal pH.

Myosin S1 bound thin filaments were rapidly mixed with EGTA and Ca2+ dissociation monitored as the change in IAANS fluorescence. Representative stopped-flow traces from Ca2+-saturated myosin s1 thin filaments containing WT Tn vs. S150D and S23/24D filaments (A) or

S23/24/150D filaments (B). Traces demonstrating the change in fluorescence over time are

2+ normalized and staggered for clarity. Comparison of the Ca dissociation rates (koff) from WT,

S150D, S23/24D, and S23/24/150D containing thin filaments at pH 7.0 (black) and pH 6.5 (dark gray). (C). *P<0.05 vs. WT. Raw data in figure generated by S. Walton from the Davis lab at

OSU.

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Chapter 4: Tropomyosin Ser-283 Phosphorylation Slows Myofibril Relaxation

4.1 Introduction

Tropomyosin (Tm) represents a critical myofilament protein in the Ca2+ regulation of myosin’s interaction with actin and therefore striated muscle contraction and relaxation [24, 69]. In the muscle, Tm forms a coiled-coil dimer spanning 7 actins.

The Tm dimer polymerizes in a head-to-tail fashion through interaction of 9-11 N- and C- terminal amino acids to form a continuous filament on actin [201]. The position of Tm on actin is critical to the Ca2+ regulation of myosin’s interaction with actin and thus contraction and relaxation [35, 165, 202-204]. In the absence of activating Ca2+, the Tm strand occupies a position on actin directly blocking myosin binding sites. In the presence of elevated intracellular Ca2+, Ca2+ binding to troponin C (TnC) is transmitted through the troponin (Tn) complex to allow movement of the Tm filament on actin and exposure of myosin binding sites. Once bound, myosin further moves the Tm filament on actin promoting a fully active myofilament state and contraction. Relaxation requires the dissociation of Ca2+ from Tn, the detachment of myosin from actin and the return of Tm to its myosin blocking position. The strength of the Tm dimer interaction at the Tm head- to-tail overlap is an important factor in the position of Tm along the actin filament and therefore the regulation of muscle contractile activation and relaxation [201, 205-208].

105

The PTM of myofilament proteins represents a critical mechanism to modulate the Ca2+ regulated contraction/relaxation of striated muscle [209]. Muscle Tm is primarily composed of or gene products that encode largely conserved 284 amino acid isoforms [69]. Both skeletal and cardiac muscle express varied amounts of the and

Tm isoforms. In the mammalian heart and fast skeletal muscles the Tm isoform predominates. In addition to varied isoform expression, Tm is also modified by phosphorylation at a single Ser-283 residue [111]. The location of this phosphorylated residue is significant as it is located directly within the C-terminal portion of the head-to- tail overlap critical to Tm transmission of the Ca2+ activation signal. The significance of

Tm phosphorylation to cardiac muscle contraction is demonstrated by its tight developmental regulation such that prior to birth the majority of Tm is phosphorylated with Tm phosphorylation decreasing to ~30% in the adult mammalian heart [210, 211].

Tm Ser-283 phosphorylation levels can also become altered in response to cardiac stress concurrent with altered contractile function [117, 186, 212]. At the molecular level, phosphorylation enhances head-to-tail interaction of neighboring Tm dimers and increases binding to Tn and troponin T [115, 213-215]. These phosphorylation-dependent alterations in protein-protein interactions are affiliated with increased cooperative activation [116], maximal Ca2+ activated ATPase activity [115, 215] and Ca2+ sensitivity of force development [216]. While the functional effects of Tm phosphorylation have been investigated at steady-state equilibrium, the speed of striated muscle contraction and relaxation are dynamic non-equilibrium events. The current lack of knowledge regarding the dynamic effects of Tm phosphorylation on myofilament regulation limits our

106 understanding of this functional role of Tm phosphorylation on striated muscle contraction and relaxation.

To determine the specific functional effects of Tm Ser-283 phosphorylation in the absence of other confounding PTMs we investigated the role of recombinant Tm. Native

N-terminal acetylated Tm (Ser-283), pseudo-phosphorylated Tm (Ser-283 mutated to

Asp) or phosphorylation null Tm (Ser-283 mutated to Ala) were expressed and purified from insect cells. Results employing these recombinant Tm’s demonstrate Tm pseudo- phosphorylation does not alter calcium binding to regulated thin filaments or equilibrium myosin binding to Tm decorated actin, however pseudo-phosphorylated Tm does decrease the dynamic dissociation of calcium from regulated thin filaments. Myofibrils exchanged with pseudo-phosphorylated Tm demonstrate slowed relaxation of dynamic

Ca2+ regulated force in the absence of altered contractile activation. These results demonstrate for the first time the specific role of Tm Ser-283 phosphorylation to alter relaxation kinetics of force as a mechanism to modulate striated muscle function.

4.2 Materials and Methods

4.2.1 Baculoviral Spodoptera frugiperda (Sf9) insect cell expression and purification of recombinant Tm.

cDNA encoding wild-type mouse Tm Ser-283 was mutated to Ala generating the phosphorylation null S283A encoding mutant or to Asp generating the pseudo-

107 phosphorylation encoding mutant by site directed mutagenesis (Stratagene) [184].

Authentic N-terminal acetylated mouse WT, S283A and S283D Tm were expressed by identical means in Spodoptera frugiperda insect cells (Sf9) employing the Bac-to-Bac

Baculovirus Expression System with slight adaptation of the manufactures direction

(Invitrogen). Baculovirus was generated from Tm cDNA containing an L21 nucleotide leader sequence [217] cloned into the pFastBac1 vector (Invitrogen) at the BamHI and

EcoRI restriction sites. The sequence verified construct was transformed into DH10Bac

E. coli (Invitrogen), resultant colonies streaked and recombinant bacmid purified from liquid culture by HiPure Plasmid DNA Miniprep (Invitrogen). Resultant PCR positive recombinant bacmid was transfected into healthy Sf9 cells by Cellfectin II Reagent

(Invitrogen) and 72 hours post transfection media collected as a viral stock. This initial viral stock underwent 2 subsequent amplifications and the resultant amplified stock used to optimize Tm expression conditions. Mouse Tm’s were expressed individually by inoculating 2x106 Sf9 cells / mL in 1 L of Sf-900 II SFM complete media (Gibco) supplemented with GlutaMAX (Gibco) and 10 g/mL gentamycin with Tm baculovirus.

Sf9 insect cells do not express detectable amounts of Tm. Following incubation in shaking flasks at 27 C for 72 hours, cells containing Tm were collected by centrifugation at 4,000 x g. Resultant mouse Tm’s were purified similar to that previously described

[218] with the following adaptations. The Sf9 cell pellet was lysed by freeze-thaw at -

80 C following suspension in (200mM NaCl and 50 mM Tris-HCl, pH 8.0 containing 2.5

g/mL Leupeptin, 1 g/mL pepstatin and 0.1 mM PMSF). Following centrifugation as above, freeze/thaw was repeated and the solution passed 10 times through a 25 g needle

108 before clarification at 20,000 x g for 10 min. Resultant supernatant was brought to 1 M

NaCl and Tm precipitated by decreasing pH to 4.58 for 30 min on ice. Tm precipitant was recovered by centrifugation at 6,000 g for 20 min and the pellet suspended in (1 M

KCl, 0.5 mM DTT and 10 mM MOPS, pH 7.0 containing 2.5 g/mL Leupeptin, 1 g/mL pepstatin and 0.1 mM PMSF). Following clarification at 6,000 x g for 10 min, the Tm precipitation was repeated. Resultant pellet from the second precipitation was suspended in 0.5 mM DTT and 10 mM MOPS, pH 7.0 and clarified. Tm containing supernatant was subjection to ammonium sulfate precipitation at 50% saturation and contaminant precipitated proteins removed by centrifugation at 11,000 x g for 30 min. The Tm containing supernatant was then precipitated by the addition of ammonium sulfate to 65% saturation, Tm recovered by centrifugation, the pellet dissolved in Buffer A (0.1 M NaCl,

0.5 mM DTT and 10 mM MOPS, pH 7.0) and dialyzed overnight at 4 C against Buffer

A. Resultant proteins were then fractionated on a Source 15Q media column (General

Electric) equilibrated in Buffer A and fractionated by elution with a 0.1 M – 0.5 mM

NaCl gradient. Fractions containing pure Tm were identified by SDS-PAGE, pooled, dialyzed against 1 mM ammonium bicarbonate and lyophilized.

4.2.2 Protein purification.

Actin was purified from rabbit fast skeletal muscle as previously described [184,

219]. Myosin utilized for ATPase and Ca2+ binding experiments was purified from rabbit fast skeletal acetone powder and the myosin S1 subfragement generated as previously described [218, 219]. Myosin S1 subfragment utilized for myosin binding to actin was

109 porcine cardiac and was prepared as previously described [219]. Rabbit fast skeletal Tn utilized in myofibril reconstitution was prepared as previously described [220].

Recombinant human cardiac troponin T, troponin I, TnC and TnC containing C35/84S and T53C mutations were expressed, purified and Tn complexes formed as previously described [184, 218].

4.2.3 Gel electrophoresis and Western Blot.

Proteins (E. coli, purified or myofibrils) were solubilized in denaturing sample buffer (50 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue and 10% glycerol) and resultant protein separated by SDS-PAGE on cooled 8 x 10 cm (Hoefer) 12% (29:1) polyacrylamide as previously described [40, 184]. Resultant gels were either coomassie stained or transferred to PVDF membrane for Western blot with the Tm specific monoclonal antibody CH1 (Iowa hybridoma bank) detected on film by ECLplus chemiluminescence (General Electric) as previously described [40, 184].

4.2.4 Myosin S1 ATPase in reconstituted thin filaments.

Thin filaments were reconstituted with rabbit skeletal actin, human cardiac troponin and mouse WT, S283A or S283D Tm and Ca2+ activated myosin S1 ATPase rate was conducted at sub-activating (pCa 10.0) and maximally activating (pCa 4.0).

Conditions were 6 M Actin, 0.5 M Myosin S1, 1 M Tm in 1.2 M Tn at 35mM NaCl,

5mM MgCl2, 14mM MOPS, pH 7.0. The reaction was initiated upon the addition of ATP to 1mM and conducted at 20 C [156]. 110

4.2.5 Ca2+ binding to TnC in reconstituted thin filaments.

Thin filaments were reconstituted with rabbit skeletal actin, human cardiac troponin containing 2-(4’-iodoacetamidoanilo)naphthalene-6-sulfonic acid (IAANS)- labeled T53C, C34/85S TnC and mouse WT, S283A or S283D Tm and steady state Ca2+ binding to TnC measured by monitoring the change in IAANS fluorescence.

Measurements were performed using a Perkin Elmer Life Sciences LS 55 fluorescence spectrometer at 15 C. IAANS fluorescence was excited at 330 nm and monitored at 450 nm as microliter amounts of CaCl2 were added to 2mL of each filament. Conditions were

4 M Actin, 0.5 M Tm and 0.3 M Tn in 150mM KCl, 3mM MgCl2, 0.5 mM DTT, 200 mM MOPS, pH 7.0 conducted at 15 C [153, 188].

4.2.6 Myosin S1 binding to Tm decorated actin.

Steady-state binding of myosin to Tm-coated actin filaments was monitored through quenching of pyrene-actin fluorescence. Actin was labeled by pyrene through established methods [221]. Actin filaments were polymerized in the presence of the individual Tm proteins by dialyzing in a polymerization buffer that included 100 mM

KCl, 1 mM MgCl2, 20 mM PIPES, pH 7.0. To follow the myosin S1 binding to the decorated actin filaments, the change in pyrene fluorescence upon addition of increasing concentrations of myosin S1 was measured on a Shimadzu RF5301PC spectrofluormeter using excitation and emission at 365 nm and 386 nm, respectively. The change in fluorescence was plotted as a function of myosin S1 concentration, and the concentration of myosin S1 required for half-maximal binding was reported.

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4.2.7 Ca2+ dissociation from TnC in reconstituted thin filaments.

Thin filaments were reconstituted with rabbit skeletal actin, human cardiac Tn containing IAANS-labeled T53C, C34/85S TnC and mouse WT, S283A or S283D Tm and the rate of Ca2+ dissociation from TnC measured as the change in IAANS fluorescence upon rapid mixing of 200 M Ca2+ saturated filaments with 10 mM EGTA.

Measurements were conducted on an Applied Photophysics model SX.20 stopped-flow instrument with a dead time of 1.4 ms at 15 C. IAANS fluorescence was excited at 330 nm and emission was monitored through a 510 nm broad band-pass interference filter

[189, 222].

4.2.8 Myofibril experiments: Preparation, Tm-Tn replacement, force and kinetic measurements.

Myofibrils were prepared by homogenization of glycerinated rabbit psoas muscles, as described previously [223]. All solutions were kept around 0 °C and contained a cocktail of protease inhibitors including 10 μM leupeptin, 5 μM pepstatin,

200 μM phenylmethylsulphonylfluoride, 10 μM E64, 500 μM NaN3 and 0.5 mM DTT.

Endogenous Tm and Tn were extracted and replaced into myofibrils with recombinant Tm (mouse WT, S283A or S283D Tm) and rabbit fast skeletal Tn as previously described [220, 224]. Briefly, myofibrils were washed (5 - 7 times) in a low ionic strength solution (2 mM Tris-HCl, pH 8.0) to remove native Tm and Tn. Extracted myofibrils were then washed in a 200 mM ionic strength rigor solution (100 mM KCl, 2 mM MgCl2, 1 mM EGTA, 50 mM TrisHCl, pH 7.0) and reconstituted with exogenous 112

Tm (5 μM) and Tn (2 µM) in a two steps protocol (0 °C, 2 hr incubation per step).

Reconstituted myofibrils were washed and stored in 200 mM ionic strength rigor solution at 4 °C, and used within 3 days. At each stage of the protocol, samples were retained from both supernatant and pellet fractions and then used to determine the extent of the

Tm-Tn extraction and replacement by SDS-PAGE analysis. The extent of the Tm-Tn extraction and replacement was 98% complete as assessed by 12% SDS-PAGE analysis

[220].

In the present experiments we used previously published techniques to measure and control the force and length of isolated myofibrils activated and relaxed by fast solution switching [223, 225]. Briefly, a small volume of the myofibril suspension was transferred to a temperature controlled chamber (15 °C) filled with relaxing solution (pCa

8.0) and mounted on an inverted microscope. Selected preparations (single myofibrils or bundles of few myofibrils, 30-70 m long, 1-3 m wide) were mounted horizontally between two glass microtools. One tool was connected to a length-control motor that could produce rapid (<1 ms) length changes. The second tool was a calibrated cantilevered force probe (1-3 nm nN-1; frequency response 2-5 kHz). Force was measured from the deflection of the image of the force probe projected on a split photodiode.

Average sarcomere length and myofibril diameter were measured from video images (ca.

1800 X). The initial sarcomere length of the preparations was set around 2.4 μm.

Myofibrils were activated and relaxed by rapidly translating the interface between two flowing streams of solution of different pCa (pCa 8.0, pCa 4.5) across the length of the preparation. The solution change took place with a time constant of 2-3 ms

113 and was complete in < 10 ms [225]. Release–restretch protocols were applied to the myofibril to measure the rate of tension redevelopment under steady-state conditions of maximal activation. The rate constant of tension development following maximal Ca2+- activation (kACT), the rate constant of tension redevelopment following release-restretch

2+ (kTR), and the rate constant of the final fast phase of relaxation following Ca removal

(fast kREL) were estimated from mono-exponential fits of the tension records. The rate constant of the early slow force decline (slow kREL) was estimated from the slope of the regression line fitted to the tension trace normalized to the entire amplitude of the tension relaxation transient; the duration of the slow relaxation phase was measured from tension traces from the onset of solution change at the myofibril to the intercept of the regression line with the fitted exponential [226, 227].

Activating and relaxing solutions, calculated as previously described [223], were at pH 7.0 and contained 10 mM total EGTA (CaEGTA/EGTA ratio set to obtain pCa 8.0

-fully relaxing solution- and 4.5 –maximally activating solution), 5 mM MgATP, 1 mM free Mg2+, 10 mM MOPS, propionate and sulphate to adjust the final solution to an ionic strength of 200 mM and monovalent cation concentration of 155 mM. Creatine phosphate

(10 mM) and creatine kinase (200 unit ml-1) were added to all solutions. Contaminant inorganic phosphate (Pi) from spontaneous breakdown of MgATP and CP was reduced to

< 5 M by a Pi scavenging system (purine-nucleoside-phosphorylase with substrate 7- methyl-guanosine) [223]. All solutions contained the cocktail of protease inhibitors mentioned above.

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4.2.9 Statistical analysis.

Ca2+ dissociation and myosin binding to actin were fit with a single exponential to

2+ determine Kd, koff, kACT, kTR and kREL. Steady-state Ca binding were fit to the Hill equation to determine a dissociation constant (kd) and Hill coefficient. Myofibril rate constants kACT, kTR and fast kREL were determined by fit to mono-exponential of tension records. The rate constant of the myofibril slow kREL was determined from the slope of the regression line fit to the normalized tension trace. All data is presented as mean

SEM. Statistical significance is defined as p < 0.05 as determined by ANOVA.

4.3 Results

4.3.1 Expression and purification of N-terminal acetylated Tm.

Full functionality of Tm’s ability to regulate the thin filament requires its N- terminal acetylation within the head-to-tail overlap region [228, 229]. Inasmuch as proteins expressed in E. coli are not modified by N-terminal acetylation, studies investigating the role of Tm phosphorylation to date have largely utilized Tm purified from tissue. Tissue purified Tm may contain unknown and/or potentially confounding

PTMs that complicate the interpretation of phosphorylation results. To obtain functionally active Tm protein of identical molecular composition, we expressed and purified recombinant authentic N-terminal acetylated mouse -Tm by baculoviral infection of eukaryotic Sf9 insect cells [228, 230]. The time course of protein expression

115 following infection with baculovirus encoding mouse -Tm demonstrates the appearance of a band at approximately 32 kDa (Fig. 22A). Transfer of a similarly loaded gel for

Western blot with the Tm specific CH1 antibody verifies the identity of this band as Tm.

The Western blot further demonstrates a lack of Tm expression in uninfected Sf9 cells

(Fig. 22A, 0 hours), suggesting that Sf9 serve as a good host to express and purify recombinant Tm. Pseudo-phosphorylated Tm has previously been used to investigate the biochemical effects of Tm phosphorylation and was demonstrated to mimic properties of native phosphorylated Tm [214]. Baculovirus encoding native mouse Tm (Tm WT), phosphorylation-null Tm containing Ser-283 mutated to Ala (Tm S283A) and pseudo- phosphorlyated Tm with Ser-283 mutated to Asp (Tm S283D) were generated and employed to express variant Tm in Sf9 cells. Resultant recombinant Tm were purified to homogeneity as demonstrated by coomassie stained gel and Western blot of the resultant purified proteins (Fig. 22B). Purified Tm’s subjected to N-terminal sequencing demonstrated a fully blocked N-terminus indicative of N-terminal acetylation and consisted of a single protein spot by two dimensional gel electrophoresis indicating a single Tm species (Fig. 23). These data demonstrate the expression of highly pure and molecularly identical N-terminal acetylated mouse Tm WT, S283A and S283D variants from Sf9 insect cells.

4.3.2 Pseudo-phosphorylated Tm increases maximal ATPase Activity.

Phosphorylated Tm purified from tissue demonstrates increased maximal calcium activated myosin S1 ATPase activity in reconstituted thin filaments [115, 215]. To 116 validate the regulatory functionality of Sf9 purified Tm we measured the myosin S1

ATPase rate of filaments reconstituted with Tm WT, Tm S283A or Tm S283D in the absence and presence of Ca2+. At sub-activating Ca2+ (pCa 10.0) the ATPase rates of filaments reconstituted with Tm S283A or S283D were not different from that of Tm

WT, while at maximally activating Ca2+ (pCa 4.0) the filaments reconstituted with Tm

S283D were increased by 1.45 fold compared to Tm WT. Filaments reconstituted with either Tm WT or Tm S283A were not different from each other at either Ca2+ (Fig. 24;

ATPase rate at sub-activating Ca2+ (s-1), Tm WT = 0.32 0.03, Tm S283A = 0.31 0.04,

Tm S283D = 0.35 0.03; ATPase rate at maximal activating Ca2+ (s-1), Tm WT = 0.65

0.05, Tm S283A = 0.67 0.05, Tm S283D = 0.93 0.07). These results demonstrate Sf9 purified Tm is functionally active and that Tm S283D pseudo-phosphorylation regulates thin filaments similar to that of phosphorylated Tm purified from muscle. Sf9 expressed pseudo-phosphorylated Tm therefore provides a unique model to study the effects of a single phosphorylation change on Tm’s regulatory function.

4.3.3 Tm pseudo-phosphorylation does not alter steady-state Ca2+ sensitivity of the thin filament.

Calcium binding to TnC is a necessary upstream step in the regulation of Tm’s position on actin and initiation of muscle contraction [35, 202]. To investigate the mechanism of Tm phosphorylation to alter ATPase activity we measured Ca2+ sensitive activation as the change in fluorescence upon steady-state Ca2+ binding to IAANS labeled

TnC in Tm and Tn reconstituted actin filaments [153]. The results in Figure 25

117 demonstrate filaments reconstituted with WT, phosphorylation null S283A or pseudo- phosphorylated S283D Tm all exhibit similar Ca2+ sensitivity of IAANS fluorescence

2+ (Fig. 25 and Table 7). Interestingly, while the kd of TnC Ca binding was unaltered, the

Hill coefficient of Tm S283D filaments trended to be increased (ANOVA p = 0.058), suggesting Tm pseudo-phosphorylation increased cooperative activation of the thin filament (Table 7). These results demonstrate Tm phosphorylation does not directly affect steady-state Ca2+ sensitivity of thin filament activation, but may alter the spread of this Ca2+ activation.

In addition to Ca2+, full activation of the myofilament and muscle force requires the downstream strong-binding of myosin to actin [35, 202]. To investigate the effects of

Tm phosphorylation on this downstream activation we measured the change in fluorescence upon steady-state myosin binding to Tm decorated pyrene-actin. Myosin induced quenching of pyrene-labeled actin fluorescence demonstrates the affinity of myosin S1 binding to actin was not altered by the presence of WT, phosphorylation null

S283A or pseudo-phosphorylated S283D Tm (Fig. 26; myosin concentration required to reach 50% maximal binding (nM); Tm WT = 18.9 2.3, Tm S283A = 17.8 1.8, Tm

S283D = 17.1 3.4). These results demonstrate Tm phosphorylation does not alter steady-state binding of myosin to actin differentially from that of WT, non- phosphorylated Tm.

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4.3.4 Tm pseudo-phosphorylation decreases the rate Ca2+ of dissociation from regulated thin filaments.

The kinetic of Ca2+ release from TnC is critical to regulate the position of Tm, cross-bridge cycling and thus the speed of muscle relaxation [165, 189]. To determine the effects of Tm phosphorylation on dynamic myofilament Ca2+ deactivation we measured the rate of Ca2+ dissociation from Tn and Tm reconstituted actin filaments as the change in Ca2+ bound IAANS-labeled TnC fluorescence following rapid mixing with EGTA

[189]. Results demonstrate Ca2+ dissociation from filaments containing Tm S283D was slowed by 13% compared to those containing Tm WT. Ca2+ dissociation of Tm WT did not differ from that of Tm S283A filaments (Fig. 27 and Table 7). Utilizing this rate of

2+ dissociation and the previously measured Kd, we calculated the apparent rate of Ca association. This calculated Ca2+ association rate was likewise decreased by 10% in Tm

S283D compared to Tm WT filaments (Table 7). Together these data demonstrate the pseudo-phosphorylation of Tm slows dynamic Ca2+ regulation of the myofilament.

4.3.5 Tm pseudo-phosphorylation slows muscle force relaxation.

To determine whether Tm S283D induced alteration of Ca2+ regulated thin filament dynamics would translate into altered dynamic contractile function we measured force changes upon rapid alteration of Ca2+ in Tm exchanged skeletal myofibrils [220,

224, 226]. Endogenous Tm and Tn in myofibrils were batch extracted and exogenous recombinant Tm and skeletal Tn were reconstituted by a 2-step procedure [220]. SDS-

PAGE comparison of sham, extracted and reconstituted myofibrils demonstrates

119 endogenous Tm and Tn removal upon extraction with the replacement of exogenous Tm and Tn upon reconstitution with Tm WT, S283A or S283D (Fig. 28A and B). Resultant reconstituted myofibrils were then mounted between a glass cantilevered force probe and length control motor for muscle kinetics determination. Kinetic force measurements were determined in an activation-relaxation cycle consisting of the rate force activation upon rapid solution switching from relaxing (pCa 8.0) to maximally activating Ca2+ (pCa 4.5)

(kACT), maximal steady-state tension (Po), the rate of tension redevelopment upon a rapid release-restretch length maneuver (kTR) and force relaxation upon rapid switch back to

2+ relaxing Ca (kREL). Representative activation-relaxation traces demonstrate myofibrils reconstituted with the varied Tm’s did not differ in maximal tension, the rate of activation nor the rate of tension redevelopment (Fig. 28C and Table 8). Analysis of normalized expanded time scale relaxation traces demonstrates Tm S283D myofibrils relaxation is impaired. Following rapid switch from maximally activating Ca2+ to relaxing Ca2+, Tm

S283D myofibrils demonstrate prolonged duration of slow phase and a decreased rate of fast phase relaxation compared to WT. Relaxation was not different between Tm WT and

S283A reconstituted myofibrils (Fig. 28D, 28E and Table 8). These results demonstrate

Tm pseudo-phosphorylation prolongs muscle relaxation and supports Tm phosphorylation as an important mechanism to modulate striated muscle relaxation of force.

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4.4 Discussion

The phosphorylation of tropomyosin at Ser-283 was first identified as a striated muscle PTM more than 30 years ago [111], yet its role in modulating muscle force remains unclear. To investigate the effects of Tm phosphorylation in the absence of other confounding PTMs we employ molecularly identical recombinant, N-terminal acetylated wild-type (S283), Ser-283 phosphorylation null (S283A) or Ser-283 pseudo- phosphorylated (S283D) Tm. Our main findings demonstrate pseudo-phosphorylated

Tm increases maximal myosin ATPase activity (Fig.24) without altering the steady-state

Ca2+ sensitive regulation of the myofilament (Fig. 25) similar to those previously reported for muscle purified Tm [115, 215]. Additionally we demonstrate for the first time that Tm pseudo-phosphorylation alters the dynamic relaxation of striated muscle force through altered thin filament dynamics (Figs. 27 and 28) without altering the rate of activation, maximal tension development (Table 8) or myosin binding to actin (Fig.

26). Together these findings expand our understanding of Tm as a modulator of dynamic striated muscle relaxation rather than steady-state, equilibrium contractile muscle function.

4.4.1 Pseudo-phosphorylated Tm functions as a model of Ser-283 phosphorylated muscle Tm.

Experiments to date investigating the effects of Tm phosphorylation have employed Tm purified from muscle. Muscle purified Tm is inherently prone to 121 complicating factors including the loss of phosphorylation over time, the presence of other muscle induced non-identified PTMs, the selective co-purification of different modifications, the potential of multiple amino acid residue phosphorylations and the non- specific effects of kinase/phosphatase treatment. These factors may contribute to a confounding interpretation of specific Tm Ser-283 phosphorylation functional effects. To investigate the sole role of Tm Ser-283 phosphorylation in the absence of these confounding factors we employed molecularly identical recombinant wild-type, phosphorylation null and pseudo-phosphorylated Ser-283 Tm. Tm requires N-terminal acetylation to be fully active [228, 229], however proteins expressed in E. coli are not acetylated. Native N-terminal acetylation is significant to investigations of Tm Ser-283 phosphorylation effects since both modifications are located in the functionally important

Tm head-to-tail overlap region [201, 205, 206, 208, 228]. We therefore chose to express and purify native N-terminal acetylated recombinant Tm in Sf9 insect cells to retain native N-terminal acetylated head-to-tail function. Similar to that previously demonstrated for Sf9 expressed Tm, our purified Tm’s are fully N-terminal acetylated

(Fig. 23) [230]. Purified Tm’s retain regulatory inhibition in the absence of Ca2+ (Fig.

24A), while Ser-283 pseudo-phosphorylated Tm increases maximal myosin ATPase (Fig.

24B) in the absence of altered Ca2+ sensitive activation (Fig. 25). These findings are similar to those demonstrated for muscle purified phosphorylated Tm [115, 215].

Together our results demonstrate the Ser-283 to Asp pseduo-phosphorylation of Sf9 expressed Tm serves as model to investigate the role of Tm phosphorylation on myofilament regulation in the absence of other confounding PTMs.

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4.4.2 Tm Ser-283 pseudo-phosphorylation does not alter steady-state myofilament regulatory function.

To date the role of Tm phosphorylation to affect Ca2+ activation of the myofilament is unclear. Initial biochemical studies demonstrated Tm phosphorylation did not alter skeletal thin filament Ca2+ regulation of myosin activity [115, 215]. More recently gelsolin extracted cardiac fiber preparations that were reconstituted with skeletal actin, cardiac Tn and phosphorylated Tm were shown to exhibit increased Ca2+ sensitivity of force production [216]. Our data demonstrates steady-state Ca2+ activation of skeletal actin and cardiac Tn thin filaments reconstituted with Tm S283D were not different from that of non-phosphorylated WT or S283A Tm (Fig. 25 and Table 7). This finding is further supported by the lack of altered steady-state myosin binding to WT versus S283D Tm decorated actin (Fig. 26). Correspondingly, we demonstrate that the exchange of skeletal myofibrils with Tm S283D did not alter stead-state tension production at maximal activating Ca2+ (Table 8). While it remains possible that the ability of Tm phosphorylation to alter Ca2+ sensitivity requires coordinated components of the cardiac muscle myofilament, our data supports a lack of Tm S283D to affect steady-state, equilibrium Ca2+ regulation of the myofilament.

4.4.3 Tm Ser-283 pseudo-phosphorylation slows dynamic muscle relaxation.

While steady-state, equilibrium measurements of myofilament activation are informative, measurements of the dynamic rate of myofilament activation and deactivation are important to determining the speed of contraction/relaxation. To date

123 there are no investigations into the effects of Tm phosphorylation on the dynamic properties of muscle activation/deactivation. Our data demonstrates that Tm phosphorylation slows Ca2+ deactivation of the thin filament (Fig. 27) resulting in prolongation of the slow phase duration and decreased rate of fast phase force relaxation

(Fig. 28D-F and Table 8). Previously we demonstrated a mutant TnC with decreased

Ca2+ dissociation similarly prolonged the duration of the slow phase of relaxation in the absence of other mechanical alterations [226]. Our similar finding upon Tm phosphorylation suggests the properties that determine slow phase duration are related to the rate of Ca2+ dissociation through Tm.

In addition to the 13% slowed Ca2+ dissociation in the Tm S283D thin filament we also calculated a concurrent 10% slowing of calculated Ca2+ association (Table 7).

Although we did not measure a statistical difference in Tm S283D contractile activation or the rate of force activation at the muscle level, both trended to be slowed (Table 8).

The similar slowing of both dissociation and association supports why Tm S283D does not alter steady-state Ca2+ sensitive activation as we observed (Fig. 25 and Table 1).

Finally, our data demonstrates Tm S283D strongly trends to increase the cooperative Ca2+ activation of the thin filament (ANOVA p = 0.058; Table 1), presumably as a result of phosphorylation induced increase in head-to-tail interaction strength [115, 214]. This finding is in agreement with data from Rao et al. who demonstrated phosphorylated Tm increased the myosin induced cooperative activation of the thin filament [116] and increased myosin cross-bridge lifetime [231]. Together these results support a mechanism of Tm phosphorylation induced strengthening of the head-

124 to-tail overlap to increase cooperative activation resulting in slowed relaxation of force by maintaining isometric force for a longer period during slow phase relaxation and slowing the rate of fast phase relaxation.

The work produced by striated muscles is regulated by factors including the rate of force relaxation, however Tm phosphorylation slowed relaxation may be of differential significance to cardiac and skeletal muscle function. Increased cardiac muscle function

(cardiac output) requires increased rates of relaxation to maintain adequate ventricular filling [232]. Tm phosphorylation induced slowing of cardiac relaxation may function to prolong the diastolic portion of the contraction cycle such that it could encroach on ventricular filling to decrease cardiac output. Tm phosphorylation prolongation of relaxation would function opposite to that of the well-described troponin I protein kinase

A phosphorylation induced increase in cardiac relaxation [37, 157, 233]. Alternately, skeletal muscle function may be improved upon Tm phosphorylation. The force produced by skeletal muscle is primarily regulated by fiber recruitment, however Tm phosphorylation induced prolongation of relaxation may decrease the threshold for skeletal fiber summation and increase force production. To date the organ level functional effects of Tm phosphorylation on cardiac and skeletal muscle function are unknown.

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4.5 Conclusions

Our data demonstrates Tm S283D pseudo-phosphorylation functions to slow deactivation of the thin filament and muscle force relaxation dynamics in the absence of dynamic and steady-state effects on muscle contraction. We propose the phosphorylation of Tm Ser-283 is therefore detrimental to cardiac output as the result of impeded ventricular filling. Future efforts are necessary to determine the ultimate contribution of this myofilament phosphorylation during adaptation to physiological and pathological cardiac stress and dysfunction.

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Table 7. Effects of Tm S283 WT, Tm S283A phosphorylation null or Tm S283D pseudo-phosphorylation reconstitution on thin filament calcium binding characteristics.

2+ 2+ nH 2+ Apparent Ca kon Thin Filament Ca k ( M) Ca k (/s) 6 -1 -1 d off (x10 M s ) Tm WT 1.71 ± 0.08 2.00 ± 0.06 237 ± 2 13.9 ± 0.7 Tm S283A 1.75 ± 0.08 2.31 ± 0.31 235 ± 4 13.4 ± 0.7 Tm S283D 1.65 ± 0.08 2.45 ± 0.07 # 206 ± 2 * 12.5 ± 0.6

2+ 2+ Values are mean SEM. kd, Ca concentration at 50% maximal activation; koff, rate of Ca

2+ dissociation from TnC; kon, rate of Ca binding to TnC calculated from kd and koff. ANOVA; * p

< 0.05, # p = 0.058.

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Table 8. Effects of Tn–Tm extraction and reconstitution with skeletal Tn and WT, phosphorylation null, or pseudo-phosphorylation Tm on rabbit psoas myofibrils tension kinetics.

Relaxation Tension Generation Slow Phase Fast Phase Myofibril P0 kACT kTR Duration kREL kREL Treatment (mN mm-2) (s-1) (s-1) (ms) (s-1) (s-1) Sham 443 ± 29* 6.86 ± 0.44 7.14 ± 0.25 62 ± 4 1.63 ± 0.16 1.6342 ± 0.16± 3 (9) Tm WT 364 ± 36 6.75 ± 0.40 7.15 ± 0.25 68 ± 8 1.70 ± 0.23 36 ± 3 (9) Tm S283A 353 ± 18 6.82 ± 0.36 6.68 ± 0.39 67 ± 3 1.81 ± 0.22 40 ± 2 (12) Tm S283D 324 ± 22 6.59 ± 0.27 6.61 ± 0.34 90 ± 5* 1.64 ± 0.21 29 ± 3* (8)

Each group of data are from different myofibril batches. All values are given as mean SEM with the number in parentheses representing the number of myofibrils. Sham, solution treated but not extracted or reconstituted, Tm WT, Ser-283 reconstituted; Tm S283A, phosphorylation null

Tm reconstituted; Tm S283D, pseudo-phosphorylation Tm reconstituted; P0, maximum isometric tension; kACT, rate constant of tension rise following step-wise pCa decrease (8.0-4.5) by fast solution switching; kTR, rate constant of tension redevelopment following release-restretch of maximally activated myofibrils; kREL, rate constant of tension relaxation for slow and fast phases of tension decrease following step-wise pCa increase (4.5–8.0). * p<0.05 (ANOVA). Data collected by the Pogessi lab.

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Figure 22. Baculoviral Tm expression and purification in Sf9 cells.

(A) Sf9 insect cells were infected with Tm WT encoding baculovirus and samples taken at various time point post infection. Coomassie stained SDS-PAGE gel and Western blot with the anti-Tm specific monoclonal antibody (CH1) demonstrate no expression of Tm in non-infected

Sf9 cells and time course of Tm expression. (B) Coomassie stained SDS-PAGE gel and Western blot with the anti-Tm specific monoclonal antibody (CH1) of purified variant Tm’s demonstrates their purity. Sf9+Tm, Sf9 Tm control; Mr Marker, molecular weight marker; Tm WT, Tm S283 wild-type; Tm S283A, Tm phosphorylation null Ser-283 to Ala mutant; Tm S283D, Tm pseudo- phosphorylation Ser-283 to Asp mutant. Data in figure generated by B. Biesiadecki.

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Figure 23. Sf9 expressed Tm is N-terminal acetylated.

Purified Tm fractions were subjected to N-terminal sequencing by Edman degradation to determine the extent of N-terminal acetylation. Representative chromatography traces demonstrating the signal from (A) the solution Blank and (B) a mixed amino acid Standard.

Chromatography traces for three consecutive cycles of Edman degradation of Tm; (C) Cycle 1,

(D) Cycle 2 and (E) Cycle 3. The lack of a detectable amino acid signal in each of the 3 rounds of

Edman degradation demonstrates the inability of the Edman process to liberate individual amino acid residues from the Tm polypeptide. Data in figure generated by B. Biesiadecki.

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Figure 24. Myosin S1 ATPase of Tm variant reconstituted thin filaments.

Thin filaments were reconstituted with actin, Tn and various Tms in the presence of sub- activating or maximally activating Ca2+ and the rate of myosin S1 ATPase measured. (A) Graph representing myosin ATPase activity at pCa 10.0. (B) Graph representing myosin ATPase activity at pCa 4.0. Tm WT, Tm S283 wild-type (black bar); Tm S283A, Tm phosphorylation null Ser-

283 to Ala mutant (blue bar); Tm S283D, Tm pseudo-phosphorylation Ser-283 to Asp mutant

(red bar); *, p<0.05. Data in figure generated by B. Biesiadecki.

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Figure 25. Ca2+ binding to TnC in reconstituted thin filaments.

Thin filaments were reconstituted with actin, Tn, variant Tm’s and Ca2+ binding to TnC measured as the percent normalized change in IAANS-labeled TnC fluorescence at various Ca2+ concentrations. Tm WT, Tm S283 wild-type (black solid line); Tm S283A, Tm phosphorylation null Ser-283 to Ala mutant (blue dashed line); Tm S283D, Tm pseudo-phosphorylation Ser-283 to Asp mutant (red dashed line).

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Figure 26. Myosin S1 binding to Tm decorated actin.

Pyrene-labeled actin was fully decorated with the varied Tms and the steady-state change in fluorescence upon the addition of varied myosin S-1 concentrations measured as myosin binding to actin. Tm WT, Tm S283 wild-type (black line); Tm S283A, Tm phosphorylation null Ser-283 to Ala mutant (blue dashed line); Tm S283D, Tm pseudo-phosphorylation Ser-283 to Asp mutant

(red dashed line). Data in figure generated by O. Ogut.

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Figure 27. Dynamic Ca2+ dissociation from TnC.

Thin filaments were reconstituted with actin, Tn, variant Tm’s and the rate of Ca2+ dissociation from TnC measured as the change in IAANS-labeled TnC fluorescence over time upon rapid mixing with EGTA. Data was fit with a single exponential equation to calculate the kinetic rates and represent an average of 3 to 5 individual traces. The data traces have been staggered and normalized for clarity. Tm WT, Tm S283 wild-type (black line); Tm S283A, Tm phosphorylation null Ser-283 to Ala mutant (blue line); Tm S283D, Tm pseudo-phosphorylation Ser-283 to Asp mutant (red line); V, volts; ms, milliseconds. Data in figure generated by S. Little of the Davis

Lab at OSU.

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continued

Figure 28. Tm exchanged myofibril tension kinetics.

Endogenous Tm and Tn was extracted from myofibrils and force kinetics measured upon rapid solution switching between sub-activating (pCa 8.0) and high (pCa 4.5) Ca2+. (A) Coomassie stained SDS-PAGE gel demonstrating the efficiency of endogenous Tm and Tn extraction from myofibrils and subsequent reconstitution with recombinant Tn and Tm variants. (B)

Representative normalized myofibril activation-relaxation force traces demonstrating the rate of activation and maximal tension development following rapid solution switching from low to high

Ca2+ and the rate of tension redevelopment following rapid change in length. (C) Representative 135

Figure 28 continued

normalized myofibril relaxation traces over an expanded time frame demonstrate Tm S283D myofibrils exhibit slowed relaxation following rapid solution switching from high to low Ca2+.

(D) Graph demonstrating the significant prolongation of Tm S283D myofibril slow phase relaxation duration. (E) Graph demonstrating the significant slowing of the Tm S283D myofibril fast phase relaxation rate. kREL, rate of relaxation; Rec, reconstituted; Tm WT, Tm S283 wild-type

(black); Tm S283A, Tm phosphorylation null Ser-283 to Ala mutant (blue); Tm S283D, Tm pseudo-phosphorylation Ser-283 to Asp mutant (red); *, p<0.05. Data in figure generated by members of the Pogessi lab.

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Chapter 5: Identification and Characterization of Tropomyosin Nitration

5.1 Introduction

Tropomyosin (Tm) is a key protein involved in the regulation of cardiac muscle contraction that may contribute to the contractile dysfunction of heart disease. Tm is a 32 kDa, alpha-helical protein forming a constant filament on actin through the head- to-tail overlap of Tm dimers. In the heart, Troponin (Tn) dependent Ca2+ binding directly regulates the position of Tm on actin to regulate the interaction of myosin with actin and therefore cardiac contraction [35]. As depicted in Figure 6, Tm’s primary amino acid sequence is comprised of heptad repeats commonly denoted as a-b-c-d-e-f-g. These heptad repeats are significant to Tm’s structure-function regulation of cardiac contraction

[45]. Residues located in positions a and d are typically comprised of hydrophobic residues clustering at the Tm dimer interface, while charged residues commonly occupy the e and g positions providing dimer stability through opposite charge interactions between Tm monomers [30]. Detailed examination of the Tm amino acid sequence further demonstrated clusters of stabilizing hydrophobic residues interspersed with destabilizing polar residues [75]. The distribution of these stabilizing and destabilizing regions of Tm are critical to determine its flexibility, binding to actin and therefore its ability to regulate contraction [14, 45].

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- The superoxide anion (O2 ) is elevated in the myocardium during reperfusion

- following an ischemic event [136]. It has been shown that O2 readily reacts with nitric oxide (NO), also prevalent during reperfusion, to form peroxynitrite (ONOO-) [135].

Peroxynitrite is a strong nitrating compound that demonstrates a high propensity to induce PTM of proteins by reacting with tyrosine (Tyr) residues to form 3-nitrotyrosine

(3-NT) at physiological pH [142, 234]. The negatively charged incorporation of the 3-NT

PTM directly alters protein structure-function [235, 236] and the nitration of cardiac proteins occurs in both aging and cardiac pathology [237-239]. In the case of heart disease, reduced cardiac force has been implicated to, at least partially, result from protein Tyr nitration [240].

Tm contains six potentially nitrated Tyr residues. Five of these Tyr residues are located in either the a or d position implicating their function in stabilization of the hydrophobic dimer interface. Two of these Tyr residues (261 and 267) are further located in the region of Tm involved in binding to the Tn complex [24], and four Tyr residues

(60, 214, 221 and 261) are located in Tm-actin binding regions. The critical locations of these Tyr residues within these functionally relevant Tm domains highlight the potential of their nitration to structurally alter Tm’s cardiac contractile regulatory function. While the nitration of Tm Tyr-221 was demonstrated in aged myocardium by mass spectrometry, the validity of this mass spectrometry identification has been questioned

[241]. Furthermore, the quantification of nitration in diseased cardiac tissue remains a challenging task as the nitration modification characteristically occurs at low abundance.

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To date, the nitration and quantification of Tm Tyr residues as well as their effect on

Tm’s structure-function regulation of cardiac muscle contraction remains unclear.

To investigate the significance of Tm 3-NT modification we treated purified Tm with ONOO-. Results demonstrate Tm ONOO-treatment induces Tyr residue nitration to alter Tm stability. To further identify and quantitate specific Tm Tyr residue nitration we developed a highly versatile mass spectrometry approach. This novel target-driven LC-

MS/MS analysis was carried out on an LTQ-Orbitrap mass spectrometer and validated for Tm nitration. The application of this method on in vitro ONOO-treated Tm identifies the concentration specific selectivity of Tm Tyr residue nitration. Together our findings demonstrate the importance of ONOO-induced Tm nitration on Tm structure-function and establish the methodology necessary to discover challenging low abundance PTMs, including the nitration of Tm Tyr residues.

5.2 Materials and Methods

5.2.1 Expression of Tm

Authentic N-terminal acetylated mouse alpha Tm was expressed in Sf9 insect cells employing the Bac-to-Bac Baculovirus Expression System and purified to homogeneity as previously described [46].

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5.2.2 Nitration of Tm Tyrosine

Nitration of Tm was carried out using the (patho)physiologically relevant nitrating agent peroxynitrite (ONOO-) (Millipore). Purified Sf-9 expressed recombinant mouse alpha Tm was incubated with varying molar ratios of ONOO- to Tm ranging from 0:1 to

200:1 for five min to determine optimal concentration for Tyr nitration. The lowest dose to achieve maximal nitration was determined to be 150:1 molar ratio ONOO-:Tm. The inactive degraded ONOO- (Millipore) was used as a negative control.

5.2.3 Protein Electrophoresis And Western Blot

Identification of nitrated Tm was carried out by western blot as previously described [184]. Briefly, nitrated and native purified Sf-9 expressed recombinant mouse cardiac Tm were solubilized in denaturing sample buffer (50 mM Tris-HCl, pH 6.8, 2%

SDS, 0.1% bromophenol blue and 10% glycerol), heated for 5 min at 80 °C and centrifuged for 5 min. Clarified protein was separated by SDS-PAGE on 8 x 10 cm

(Hoefer) 12% (29:1) acrylamide gels and transferred to 0.2 μm Hybond LFP PVDF membrane (GE Healthcare). Western blot detection of 3-NT was achieved using a primary polyclonal rabbit antibody specific for 3-NT (clone 1A6, Millipore) that was detected with a 488 Dylight-conjugated secondary antibody (Jackson ImmunoResearch

Laboratories, Inc.). Images were collected on a Typhoon 9410 imager (GE Healthcare) with an excitation of 488 nm and 520BP30 emission filter. Similarly, total Tm was achieved using a primary monoclonal antibody specific for cardiac Tm (CHI, Iowa

Hybridoma) detected by a 649 Dylight-conjugated secondary antibody (Jackson

140

ImmunoResearch Laboratories, Inc.) with an excitation of 649nm and 670BP30 emission filter.

5.2.4 Circular Dichroism and Thermal Denaturation

Purified Sf-9 expressed recombinant mouse cardiac αTm was exposed to degraded (inactive) ONOO- or 150:1 molar excess ONOO- and diluted in CD Buffer

(500mM NaCl,10mM NaPO4, 1mM EDTA pH 7.5) at a final concentration of 5 µM and helical content was assessed by far UV circular dichroism (Aviv 62A DS CD spectrometer). For thermal denaturation, nitrated or non-nitrated Tm was diluted in CD

Buffer at a final concentration of 5 µM and ellipticity was monitored at 222nm in one degree intervals from 10 to 80°C using a thermoelectric temperature controller.

5.2.5 Measurement of nitrated Tm binding to isolated TnT or whole Tn

A modified solid-phase protein binding assay to assess 3-NT Tm association with

TnT and Tn will be carried out as previously described (Biesiadecki et al., 2004). Briefly, a 96 well plate (Greiner Bio One) will be coated with human TnT or Tn. Wells will be washed the following day and then blocked with BSA. Following blocking, wells will be washed three times. Nitrated or non-nitrated DyLight 649 NHS Ester (Pierce

Biotechnology) labeled purified Sf-9 expressed recombinant mouse cardiac αTm will be serially diluted three-fold, plated in triplicate, and incubated with TnT or Tn for two hours at 25°C. After completion of incubation, wells will be washed again and 8mM

Urea was added to each well to dissolve protein. Tm fluorescence was monitored 141 following excitation at 633nm using a GE Healthcare Typhoon 9410 Variable Mode

Imager with a 670nm bandpass 30 filter.

5.2.6 Steady-state Ca2+ binding in the reconstituted thin filament

Purified bovine cTm was treated with 40-fold molar excess ONOO-or degraded inactive ONOO-and exposed to 6mM DTT for five minutes at 80°C and then allowed to refold at room temperature for five minutes. Thin filament was then reconstituted first by incubation of 4μMactinand 0.57 μM treated or untreated Tm in 1X assay buffer (150mM

KCl, 10mM MOPS, 3mM Mg2+pH 7) for 15 minutes at room temperature. temperature.

0.3μM T53C IAANS human cardiac Tn was added and allowed to incubate for 15 minutes at room temperature. Before fluorescence measurement, thin filament was mixed

1:1 with 2X assay buffer (150mM KCl, 390mM MOPS, 3mM Mg2+, 4mM EGTA pH 7) and allowed to mix for a 10 minutes. IAANS fluorescence was monitored at 15°C using a

Perkin Elmer LS55 Luminescence Spectrometer at 450nm with an excitation wavelength of 330nm following the addition of calcium. Data was analysis was carried out using Fig.

P software.

5.2.7 Protein Digestion

Nitrated or non-nitrated Tm was dialyzed three times against 50 mM Ammonium

Bicarbonate (NH4HCO3) compatible with mass spectrometry analysis. After dialysis, in- solution tryptic digestion was carried out in 50 mM NH4HCO3 in combination with

0.25% (w/v) RapiGest (Waters Corp.) overnight at 37°C. The ratio of trypsin (Promega) 142 to purified Sf-9 expressed recombinant mouse cardiac Tm was 1:50 (w/w). Following overnight incubation, formic acid (EMD Chemicals) was added at a final percentage of

30% (v/v) to degrade RapiGest. To make the degradation completed, samples were heated at 37 °C for 1 hr and then placed at 4 °C for another hr. Subsequently, samples were centrifuged by 14,000 rpm at 4°C for 10 min. Supernatant containing the digested peptides was collected, dried completely by using a SpeedVac concentrator and redissolved in HPLC grade water at a final concentration of 500 ng/uL. Samples were stored at -80 °C until use.

5.2.8 LC-MS

Nitrated or non-nitrated purified Sf-9 expressed recombinant Tm were separated by reversed-phase HPLC (Dionex) and detected by ESI-Q-TOF mass spectrometer

(Waters). HPLC separation was carried out using a flow rate of 50 µL/min on a C18 column (1.0 mm × 150 mm). The elution gradient consisted of mobile phase A (0.1%

TFA in water) and mobile phase B (0.1% TFA in ACN), where mobile phase B linearly increased from 30% to 45% in 2 min, 45% to 60% in 20 min, and stayed at 60% for 4 min. Between each run, the column was washed to reduce sample carryover. A

Micromass LCT mass spectrometer with an orthogonal electrospray source (Z-spray) was coupled to the outlet of the HPLC. Tm samples were infused into the electrospray source at the flow rate 50 μL/min without splitting. ESI was performed at optimal condition

(capillary voltage = 3 kV, source temperature = 100 °C, cone voltage = 50 V). Data were acquired in continuum mode at the rate of 1 spectrum/sec. All spectra were obtained in

143 the positive ion mode. NaI was used for external mass calibration over the m/z range 500-

2500. Mass tolerance of TOF is ± 3Da.

5.2.9 LC-MS/MS

Nitrated or non-nitrated Sf-9 purified recombinant expressed αTm was run on an

SDS gel followed by subsequent in-gel trypsin digestion for two hours at 37°C or directly digested in-solution overnight at 37°C. Digested Tm was then resuspended in HPLC water at a final concentration 1 μg/μL. Peptides were separated over a 70 minute gradient consisting of solvent A (H2O and 0.1% formic acid) and solvent B (ACN and 0.1% formic acid). The resultant peptide elutions were identified by use of data-dependent acquisition LC-tandem MS on a Finnegan LCQ Deca with attached Dionex Ultimate

3000 liquid chromatography unit. Analysis of peptides and nitration modification were carried out using Mass Matrix Database search engine.

5.2.10 Selected Ion Monitoring (SIM)

Selected ion monitoring was administrated on the raw data files collected with

DDTRM method by the LTQ Orbitrap XL. Extracted ion chromatograms were constructed against MS1 spectra collected on the Orbitap mass analyzer. Thermo

Xcalibur Quan Browser was used to reconstruct ion chromatograms. The theoretical m/z value of each precursor ion was set as the center of mass tolerance window, mass precision: 4 decimals, mass tolerance: ± 10 ppm. The expected retention time window

144 was centered with the observed retention time at peak apex obtained from TRM experiment result, retention time window: ± 100 sec.

5.2.11 Data analysis

All the mass spectral data were searched against the MassMatrix database search program. The search parameters included the following variable modifications: nitration of Tyr, nitrosylation of Tyr, oxidation of methionine, ±10.00 ppm mass tolerance for precursor ions, ±0.80 Da mass tolerance for product ions, pp value of output ≥ 5.0, pptag of output ≥ 1.3. False discovery rates were estimated using the target-decoy strategy and subjected to manual validation [242]. Each of the tandem MS spectra matched by the database search was manually validated. Quantitative analysis was performed with

Thermo Xcalibur Quan Browser. Algorithm: genesis, retention time window: ± 100 sec, nearest retention time model, minimum peak height(S/N) 3.0. The selected m/z window was set to 0.8 m/z for transition ions.

5.3 Results

5.3.1 Western blot and structure-function analysis.

To establish the effect of ONOO- on Tm Tyr residues, purified recombinant expressed alpha Tm was incubated with varied amounts of ONOO- (molar ratios of

ONOO- to Tm = 0:1, 40:1, or 150:1). Figure 29 demonstrates the chemical reaction

145 between ONOO- and the Tyr amino acid residue. Following ONOO-treatment, western blot analysis for Tm 3-NT was then performed to identify Tm nitration. Figure 30 demonstrates Tm nitration to 3-NT by ONOO- and demonstrates 3-NT was increased following elevated ONOO-. Above the ONOO- to Tm molar ratio of 40:1, a second nitrated Tm band appeared indicating the development of multiple 3-NT Tm species.

Together these observations suggested the nitration of Tyr residues to 3-NT impacts Tm protein structure to cause a slower migration of Tm on the gel.

To investigate the effect of 3-NT modification to affect Tm structure, Tm helical content was assessed by circular dichroism. As demonstrated in Figure 31A, the exposure of Tm to ONOO- decreased overall helical content observed as a more positive ellipticity at 222 nm. Additionally, Tm protein stability following ONOO- exposure is decreased upon heat denaturation compared to Tm treated with inactive degraded ONOO- indicative of an increase in protein flexibility (Fig. 31B). Collectively, these results demonstrate tyrosine residues within Tm modified to 3-NT upon ONOO- treatment to induce a diminished structure helical content and decreased Tm stability (increased flexibility).

As a result of the observed change in Tm helical content, the effect of such structural alteration on protein function was first determined through solid-phase binding between nitrated or non-nitrated Tm and isolated TnT or whole Tn. Preliminary results in

Figure 32 demonstrate a marked decrease in of Tm binding to TnT and Tn as a result of

Tm nitration by ONOO-. Additionally, compared to TnT alone, nitrated Tm exhibited increased binding to whole Tn suggesting remaining subunits TnI and TnC play a role in

146 enhancing the binding of Tn to Tm. We then examined the functional effect of Tm nitration on Ca2+ sensitivity of the thin filament. Using reconstituted thin filaments containing nitrated or non-nitrated Tm a decrease in thin filament Ca2+ sensitivity results suggesting that nitration of Tm can alter the Ca2+ binding properties of the thin filament

(see Fig. 33). Additional efforts were made to determine the effect of Tm Tyr nitrosylation through the incubation of the NO-donors SNAP or GSNO. Efforts were aimed at determining the effect of nitrosylation on thin filament Ca2+ sensitivity; however, results were not obtained as the NO-donor themselves interfered with the

IANNS fluorescent signal rendering the experiment not possible to carry out.

5.3.2 Mass spectrometric identification of Tm tyr nitration.

While western blot analysis is highly sensitive, currently available total 3-NT antibodies do not provide information on the specific number of modified Tyr residues or their location. LC-MS is a useful technique to determine the overall modification distribution for a protein. By analyzing a protein’s MS data, the mass spectral peaks of different modified species provide clues to the modification type and number [243]. To confirm the incorporation of 3-NT following ONOO- treatment demonstrated by western blot, protein LC-MS was performed on Tm before and after ONOO- treatment. Figure 34 demonstrates a mass spectrometry peak at 32,728 Da (N-terminal acetylated mouse alpha

Tm) corresponding to unmodified Tm in the non-treated sample. Mass shifts of 29 Da

(nitrosylation) and 45 Da (nitration) were observed in ONOO- treated samples (molar ratios of ONOO- to Tm, 40:1 and 150:1). Multiple modifications were detected for Tm

147 nitration, Tm nitrosylation, and their combination. Tm protein nitration and nitrosylation levels were observed to increase as the molar ratio of ONOO-:Tm was increased with nitration being the predominate modification further confirming ONOO- dependent Tm modification.

We also sought to identify specific Tyr residues that were nitrated with our various ONOO- exposures utilizing LC-MS/MS. Following trypsin digestion of Tm into peptides, we observed excellent sequence coverage of 92% in Sf-9 expressed purified Tm exposed to 150:1 ONOO- (see Fig. 35). While there are various missed cleavages likely attributed to salt-bridge formation between charged residues known to disrupt tryptic cleavage of proteins, we were still able to specifically identify five peptides containing nitrated Tyr residues – 60, 162, 214, 221, and 261.

5.3.4 Quantification of nitrotyrosine and nitrosyltyrosine.

To quantify nitrotyrosine and nitrosyltyrosine changes on each modification site across all the treatments (the molar ratios of ONOO- to Tm, 0:1, 40:1, 60:1, 80:1, 100:1, and 150:1), triplicate TRM experiments were performed for each sample using an LTQ instrument. The mass spectral data were searched by MassMatrix database search program to identify peptide sequences. To improve selectivity, a pseudo-SRM data analysis was next performed. In this method only the first and second most abundant product ions (also referred to as transitions) are used for the quantitative analysis. The calculation of peak areas over selected ions was carried out by Thermo Xcalibur Quan

Browser. As a result, selectivity and accuracy were significantly improved for each

148 peptide as compared to TRM. To do absolute quantitative analysis, the full MS spectra obtained from DDTRM data was used to calculate the peak area using selected ion morning (SIM). SIM is a quantitation method in which the abundance of a selected precursor ion is used to represent the amount of an analyte in a matrix. Thermo Xcalibur

Quan Browser was used to calculate the elution peak area from the MS1 full scan for each precursor. The retention time used for each SIM calculation was obtained from

TRM data. The results of the SIM data analysis are shown in Figure 36 and Table 9.

5.4 Discussion

5.4.1 Characteristics of Tm nitration

Souza and colleagues have found that nitration of protein Tyr share similar circumstances that enhance nitration capability: 1) presence of acidic residues in close proximity to Tyr, 2) limited number of sulfur-containing residues to compete for reaction with ONOO- , and 3) the presence of turn-inducing residues such as proline or glycine

[244]. Both the primary sequence and secondary structure of Tm exhibit an acidic environment surrounding all Tyr; however, no clear pattern is present with regards to extent of acidic environment in relation to extent of nitration. Furthermore, no sulfur- containing residues are in close proximity to any Tyr to compete for reactivity with

ONOO-. While we cannot delineate a specific reasoning for preferential nitration of Tm

Tyr it is clear nitration of Tm does not occur in a random fashion as multiple experiments of dose-dependent Tm exposure to ONOO- produces the same preferential nitration 149 pattern (Tyr 60 > Tyr 214 > Tyr 261 > Tyr 221). One possible explanation for Tyr 60 being the most nitrated Tyr residue is its close proximity and polar contact with a glutamic acid (Glu) residue since as previously mentioned an acidic environment promotes nitration of Tyr. Furthermore, it was shown that the close proximity of a polar residue, in the case of Tyr 60 a serine residue, favors nitration of Tyr [245]. While the specific rationale for preferential nitration of Tm Tyr 60 remains unclear, it evident that there is a non-random, favorable targeting of specific Tyr residues for nitration of the Tm molecule in vitro. It is possible, however, in an in vivo setting Tm nitration could differ than that of in vitro nitration as interactions with neighboring proteins such as actin and

Tn could alter exposure and local environment of Tyr residues of Tm.

5.4.2 Tm nitration occurs in a specific, reproducible manner.

The mechanism underlying Tyr nitration does not occur in a random manner as protein Tyr abundance does not dictate the extent of nitration suggesting that other factors play a role in directing the nitration of Tyr [246]. One would predict that solvent accessibility would be a good indicator of nitrating potential as Tyr residues that are more exposed have been shown to have an increased reactivity with nitrating agents [247]. Of the four Tyr residues identified with LC-MS/MS, Tyr 60, the most nitrated residue, is

41% exposed followed closely by the second most nitrated Tyr 214 with 37% solvent accessibility. As expected, the least exposed Tyr residue Tyr 221 is only 3% solvent accessible. Interestingly, the third most nitrated Tyr residue, Tyr 261, has a solvent accessibility of 88% suggesting that exposure of the Tyr residue is not the sole

150 determining factor of ONOO- nitration susceptibility although increased solvent exposure does result in an increase in nitration.

5.4.3 Effect of Tm nitration on flexibility.

Tm plays an integral role in beat-to-beat function of the heart by allowing the interaction of actin and myosin, and thus cardiac contraction/relaxation, to occur. PTM

[111] [113] or mutation [248] [249] [250] of the Tm molecule has been shown to alter function of Tm [251] [115] and have potential to impact whole heart function. Critical to

Tm is its residue sequence, which imparts varying structural characteristics to allow for proper function. It was previously determined that in position a or d Tyr residues contributed to the stability of the Tm dimer while other residues such as Asp or Glu resulted in decreased stability, or increased flexibility, of Tm [252] [74] which is necessary for normal function. Incorporation of a negatively charged nitro group onto Tyr has similar electrostatic properties as that of phosphorylation [253]. Such an addition could elicit similar electrostatic effects to that of acidic residues which have been shown to alter function. Additionally, it has been shown that incorporation of a negative charge in a hydrophobic region, such as that of an aspartic acid, can decrease the stability of the

Tm dimer through electrostatic repulsion of monomers [254]. We hypothesize that the introductions of a negatively charged nitro adduct to Tyr residues results in increased destabilization, or increase flexibility, translating to altered function.

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5.4.4 In vivo implications of Tm nitration.

When considering the potential in vivo implications of Tm Tyr nitration, it is important to point out that Tyr residues lay within both actin and troponin T binding domains [24]. Oguchi and colleagues showed that increasing the destabilization within specific actin binding domains of Tm can alter the binding of actin as well as sliding velocity and force [255]. Furthermore, a decrease in Ca2+ sensitivity was observed in dilated cardiomyopathy mutations of Tm in similar regions [256]. Interestingly, Y60 is located within one of the domains that were altered resulting in decreased sliding velocity and force compared to wild-type Tm stressing the importance of modifications made to this region of Tm. Nitration of Y60 would likely induce similar destabilizing effects contributing to dysfunction with actin. Such complications at the whole heart level could result in systolic and diastolic dysfunction, for example decreased force production and increased relaxation time [257]. It is possible that nitration of other Tyr residues in Tm, albeit nitrated to a lesser extent than Y60, could also affect the normal function of Tm as

Y162, Y214, Y221 and Y267 are also found in actin binding regions of Tm. Additionally, Y261 is located in b position which is said to interact with actin [255] suggesting that nitration could alter the interaction of Tm and actin and therefore lead to dysfunction. As shown in

Figures 32 and 36, nitrations of Tm Tyr occurs in a dose-dependent manner in such a

- way that at lower concentrations of ONOO Y60 would initially be significantly nitrated and impart structural and functional alterations. Following an increase in ONOO- other

Tyr would be nitrated further contributing to structural and functional alterations. Further

152 investigation into the site-specific effects of nitrated Tyr in Tm is required to fully elucidate the biochemical and functional effects of Tm nitration.

5.5 Conclusions

The research goals for this project were 1) to determine the occurrence of ONOO- induced Tm Tyr nitration, 2) to evaluate if Tm nitration alters Tm structure-function, 3) to evaluate the distribution of nitration and nitrosylation of 6 Tyr residues on Tm, 4) to quantitatively measure the modification on each Tyr residue. Upon treatment of purified

Tm with ONOO- we demonstrate the concentration dependent incorporation of Tyr nitration. This Tm nitration was further shown to alter Tm structure and stability. To evaluate and quantitate specific Tm Tyr residue modification two targeted mass spectrometry methods were developed. First, LC-MS was employed to identify molecular weight additions associated with nitration or nitrosylation following exposure of Tm to

ONOO-. Secondly, we successfully identified five nitrated Ty residues by using LC-

MS/MS to provide a better understanding of the mechanism underlying Tm nitration.

The data described within provide the mechanistic and functional significance for

Tm Tyr nitration as well as establishing the means to detect and quantify low abundance protein modifications, such as nitration, induced during ischemic heart disease. Utilizing this approach will allow for greater understanding of the mechanisms underlying Tm nitration in cardiac disease. While this method has been applied to the detection of low abundance nitration of Tm, the true strength lies within the ability to detect other low 153 abundance protein PTMs not only in cardiac disease, but other pathophysiological systems as well.

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Table 9. Percent nitration and nitrosylation at specific Tm Tyr residues

Modification Modification percentage (×100)

60 YNO 0.00267 ± 0.00020 60 YNO2 0.22930 ± 0.01050 214 YNO 0.00088 ± 0.00002 214 YNO2 0.05167 ± 0.00972 221 YNO2 0.00710 ± 0.00118 261 YNO 0.00044 ± 0.00001 261 YNO2 0.03008 ± 0.00321

The data represent the percentage of nitration and nitrosylation on each Tyr site at the maximum treatment of ONOO- (molar ratio of ONOO- to Tm, 150:1). Triplicate experiments were performed for each method. Data were reported as mean ± standard error. Data in Table generated by J. You from the Freitas lab at OSU.

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Figure 29. Reaction of peroxynitrite (ONOO-) with Tyr.

Reaction of peroxynitrite (ONOO-) with Tyr results in the addition of a nitro- or nitrosyl- adduct at the three position to form 3-nitrotyrosine or 3-nitrosyltyrosine.

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Figure 30. Nitration of αTm by ONOO- occurs in a dose-dependent manner.

Sf-9 expressed recombinant alpha mouse cardiac Tm was treated with inactive degraded

ONOO- to act as a negative control and 40 or 150 molar excess ONOO- to Tm. BcTm

100:1 acted as a positive identification of nitration. Western blot detection of (A) 3-NT was achieved using an antibody specific for 3-NT (α3-NT) followed by a 488nm fluorophore-conjugated secondary antibody and (B) total Tm was achieved using an antibody for Tm (αcTm) followed by a 649 fluorophore-conjugated secondary anti- mouse antibody.

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Figure 31. αTm nitration results in altered structure and decrease in stability.

(A) Change in ellipticity at 10°C of recombinant αTm exposed to degraded ONOO- or 150:1

- molar excess ONOO at a final concentration of 5µM in 500mM NaCl, 10mM NaPO4, 1mM

EDTA pH 7.5 buffer. (B) Fraction folded of Tm exposed to degraded ONOO- or 150:1 molar

- excess ONOO following thermal denaturation monitored at 222 nm in 1 degree intervals from

10°C to 80°C.

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Figure 32. Nitrated Tm alters binding affinity of both isolated TnT and whole Tn.

(A) Representative fluorescent Tm-TnT solid-phase binding plate and schematic of technique.

Nitrated or non-nitrated DyLight 649 NHS Ester (Pierce Biotechnology) labeled Tm was incubated at serial dilutions with plated (B) isolated TnT or (C) whole Tn. After completion of

Tm-TnT incubation, 8mM Urea was added to each well to dissolve protein and Tm fluorescence was monitored following excitation at 633nM using a GE Healthcare Typhoon 9410 Variable

Mode Imager with a 670nm bandpass 30 filter.

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Figure 33. Steady-state Ca2+ sensitivity is reduced in thin filaments containing nitrated Tm.

The change in IAANS fluorescence following the addition of various Ca2+ amounts in thin filaments containing IAANS labeled TnC and either nitrated (40Tx, red open square) or non- nitrated (NoTx, black open square) Tm.

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Figure 34. LC-MS of nitrated or non-nitrated αTm.

Following exposure of recombinant αTm to degraded ONOO- or a low or high dose of ONOO-

αTm underwent 50:1 (w/w) in-solution trypsin digestion to generate peptides for mass spec identification. HPLC separation was carried out using a flow rate of 50 µL/min on a

1.0mm×150mm C18 column. Elution gradient consisted of mobile phase A (0.1% TFA in water) and mobile phase B (0.1% TFA in acetonitrile) where B linearly increased from 30 to 45% in 2 min, 60% in 20 min and was held at 60% for 4 min. Tm peptides were infused into the electrospray source at a flow rate of 50 µL/min without splitting. All spectra were obtained in the positive ion mode and nitration was identified as ± 3 Da.

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Figure 35. Sequence coverage of αTm exposed to ONOO- and identification of peptides with nitrated Tyr by LC-MS/MS.

Following treatment of αTm with ONOO- in-gel trypsin digestion was carried out and digest peptides were suspended in HPLC water and 0.1% formic acid at a final concentration at 1µg/uL.

(A) Sequence overage of nitrated Tm. Red indicates observed sequence while black indicates non-covered sequence. (B) Identified peptides and specific residues identified as nitrated.

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Figure 36. Selected ion monitoring experiment data of nitration and nitrosylation on each Tyr site.

Following exposure of αTm to varying molar ratios of Tm:ONOO- selected ion monitoring was carried out to selectively identify peptides of interest containing nitrated Tyr residues. SIM is a quantitation method in which the abundance of a selected precursor ion is used to represent the amount of an analyte in a matrix. Thermo Xcalibur Quan Browser was used to calculate the elution peak area from the MS1 full scan for each precursor. Data in figure generated by J. You from the Freitas lab at OSU.

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Chapter 6: Discussion

6.1 Principle Findings

In this section the major findings from each chapter will be summarized.

6.1.1 Chapter 2

1. The AMPK holoenzyme complex phosphorylates cTnI at Ser-150.

2. Large animal cardiac muscle exhibits endogenous cTnI Ser-150 phosphorylation.

3. Cardiac TnI Ser-150 phosphorylation increases calcium-dependent force generation.

4. The combined effect of cTnI Ser-150 phosphorylation blunts PKA-dependent desensitization.

6.1.2 Chapter 3

1. In vivo myocardial ischemia increases both TnI Ser-150 and Ser-23/24 phosphorylation.

2. TnI Ser-150 and Ser-23/24 phosphorylation alters the acidic effects of thin filament regulation.

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3. The phosphorylation of TnI Ser-150 and Ser-23/24 affect function through different thin filament interactions.

4. The combination of TnI Ser-150 with Ser-23/24 phosphorylation retains accelerated thin filament deactivation.

6.1.3 Chapter 4

1. Pseudo-phosphorylated Ser-283 Tm increases maximal ATPase activity.

2. Tm pseudo-phosphorylation does not alter steady-state Ca2+ sensitivity of the thin filament.

3. Tm Ser-283 pseudo-phosphorylation decreases the rate Ca2+ of dissociation from regulated thin filaments.

4. Muscle force relaxation is slowed by Tm Ser-183 pseudo-phosphorylation.

6.1.4 Chapter 5

1. ONOO- nitrates Tm in a concentration-dependent manner.

2. Exposure of Tm to ONOO- decreased overall helical content and thermal stability.

3. LC-MS/MS identified five nitrated Tyr residues on Tm following exposure to ONOO-.

4. LC-MS/MS identified preferential nitration of Tyr residues such that Tyr

60>214>261>221.

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5. Novel nitrosylation of Tm Tyr residues was identified but to a lesser extent than nitration.

6.2 Overall Implications and Therapeutic Potential

6.2.1 TnI Ser-150 and Ser-23/24 Phosphorylation

In the case of whole heart function it is well accepted that phosphorylation of TnI at Ser-23 and Ser-24 aids in enhanced relaxation and crossbridge cycling kinetics to contribute to the observed increase in force production [105, 258]. Given a baseline TnI

Ser-23/24 phosphorylation level of 40% [101] it appears likely that additional phosphorylation sites of TnI act as a fine-tuning mechanism to alter function of the heart as needed. We demonstrate at a biochemical level that TnI Ser-150 phosphorylation increases Ca2+ sensitivity and when in combination with TnI Ser-23/24 phosphorylation retains accelerated thin filament deactivation (i.e. relaxation) yet we must consider how these biochemical results translate to the function of the whole heart.

As a result of increased TnI Ser-23/24 phosphorylation, the myofilaments become

2+ desensitized and the cardiomyocytes have to bring in more Ca to obtain greater force at a lower Ca2+ sensitivity, an energetically taxing process on the heart. One can speculate then that the presence of TnI Ser-150 allow for sufficient force production while alleviating energetic costs for such force production as Ca2+ sensitizers have been shown to decrease energetic requirement for contraction [259]. In addition to the pro-energetic aspect of TnI Ser-150 phosphorylation it would be predicted that inducing TnI Ser-150 166 phosphorylation would increase contraction in the heart. Given that β-adrenergic stimulation induced PKA phosphorylation of TnI is associated decreased Ca2+ sensitivity and enhanced crossbridge cycling kinetics contribute to increased CO, one would speculate that incorporation of TnI Ser-150 phosphorylation under such a setting would further increase the rate of ventricular contraction, defined as dP/dTmax, as well as strength of contraction to enhance ejection fraction and in turn CO. It seems likely the mechanism underlying such functional responses in the heart by an increased Ca2+ sensitivity is an amplified number of activated Tn along the thin filament at a given period of time allowing for more myosin to cycle and in turn produce more force.

Alternatively we have demonstrated the ability of Tn containing Ser-150 and Ser-23/24 phosphorylation to exhibit accelerated deactivation similar to TnI Ser-23/24 alone it is reasonable to expect an increased rate of ventricular relaxation, or dP/dTmin, in hearts containing both phosphorylations while retaining normal-like Ca2+ sensitivity.

Collectively, under basal conditions it seems likely that TnI Ser-150 phosphorylation would increase cardiac performance through enhancement of ejection fraction and overall

CO while further increasing the β-adrenergic response without suffering from impaired relaxation.

In addition to the unique function of Ser-150 phosphorylation in conjunction with

Ser-23/24 phosphorylation, TnI Ser-150 phosphorylation imparts protection to thin filament regulatory function from acidic pH such that Ca2+ sensitivity is preserved and functions like WT thin filaments would at normal pH (7.0). We also observe the similar phenomenon of increased Ca2+ sensitivity with maintained elevated Ca2+ dissociation

167 kinetics at ischemic pH. Such a rapid drop in pH is commonly associated with cardiac ischemia and plays a large role in suppressing contractile function during a period of ischemia. Much work has been done detailing investigation of an acidic-resistant His residue inserted into cardiac TnI (see section 1.4.6) suggesting this portion of TnI is highly important in regulating function during acidosis. Located 12 residues from the His button, Ser-150 offers an attractive target for therapeutic intervention to preserve function following an ischemic insult. Further highlighting the importance of Ca2+ sensitivity during ischemia is work demonstrating that administration of the Ca2+ sensitizing drug levosimendan could improve contractile parameters in patients following acute ischemia and increase force production while reducing arrhythmias in an animal model of I/R injury [260, 261]. Phosphorylation of TnI at Ser-150 may offer an alternative target to increase force production following cardiac ischemia by activation of AMPK. One downside to such a Ser-150 phosphorylation therapeutic would be the traditional decrease in TnI Ser-23/24 phosphorylation associated with decreased heart performance. Early treatment with β-agonists proved detrimental to long-term health, likely as a result of such a high energetic demand induced through this signaling pathway. It is possible that administration of a β-agonist in combination with a TnI Ser-150 phosphorylation- inducing compound could prove energetically favorable and allow for improvement of impaired cardiac function in heart disease.

When considering the pros and cons of having TnI Ser-150 phosphorylation in the heart, at first glance it would appear that TnI Ser-150 phosphorylation to increase Ca2+ sensitivity and in turn enhance cardiac contraction would be a beneficial mechanism to

168 improve cardiac function. Furthermore, maintaining the ability to accelerate deactivation of the thin filament when in combination with TnI Ser-23/24 phosphorylation would appear advantageous as many myofilament sensitizers suffer from impaired relaxation.

Yet, one must be cautious when drawing such conclusions. During a period when the heart is under an immense amount of stress such as during ischemia, contraction of the cardiomyocytes could possibly pose more of a risk than a benefit. As the availability and utilization of oxygen is a key determinant in allowing for proper metabolic and contractile function of the heart, eliminating one of these components while enhancing the other could prove fatal. For example, when oxygen availability is at its lowest, ATP production would decrease drastically while existing ATP would be consumed rapidly.

By increasing cardiac contraction by TnI Ser-150 phosphorylation there is an increased need for ATP to carry out the crossbridge cycle and produce force such that this TnI phosphorylation could prove detrimental in the oxygen starved muscle. Conversely, in a period when the heart is not oxygen deprived yet functionally depressed such as that following an ischemic insult it would appear that TnI Ser-150 phosphorylation would prove to be beneficial as the heart can produce sufficient ATP for contraction but the force output is dampened as a result of injury. Collectively, the results demonstrated in this dissertation study represent an interesting pathway to modulate cardiac function; however, the complete role of TnI Ser-150 phosphorylation remains to be seen and requires in vivo studies to fully elucidate the advantageous or disadvantageous role in cardiac disease (as discussed in section 6.3).

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Outside of the cardiac muscle realm, another attractive therapeutic potential that phosphorylation of TnI Ser-150 offers is in neuromuscular disease where muscle weakness is a common characteristic associated with decreased quality of life.

Commonly investigated in cardiac therapeutic potential, Ca2+ sensitizers have gained significant ground as options for treatment of neuromuscular diseases such as muscular dystrophy and myasthenia gravis [262]. A recent report in Nature Medicine demonstrated such therapeutic targeting by activating fast skeletal Tn (fsTn) in a myasthenia gravis model of disease. By developing a compound that specifically activated fsTn an observed increase in Ca2+ sensitivity was seen producing enhanced force production at sub- maximal nerve stimulation. Additionally, a decrease in muscle fatigue was observed both in control rats and a myasthenia-gravis rat model of neuromuscular disease [263]. Similar to the fsTn activator used in the aforementioned study, phosphorylation at the corresponding cardiac TnI Ser-150 residue in fsTnI Ser-118 phosphorylation, presumably by AMPK activation, could provide a novel small compound therapeutic target to improve compromised muscle function in patients afflicted with neuromuscular disease.

6.2.2 Tm Phosphorylation

The functional effects of Tm phosphorylation at Ser-283 have been well established. Phosphorylation results in a number of functional alterations such as increased head-to-tail arrangements and an increased affinity but conceivably the most important result of Tm phosphorylation at Ser-283 is the ability to facilitate cooperative activation in the thin filament. Cooperative activation of the thin filament is an integral

170 component in ensuring efficient contraction of the sarcomere [27]. The capacity to elicit cooperative activation by phosphorylated Tm was shown in a study by Rao and colleagues in which they utilized an optical trap assay to monitor isometric stall force of multiple myosin moving actin filaments. It was concluded that phosphorylation of Tm was a requirement to induce cooperativity as non-phosphorylated Tm functioned similarly to bare actin [116].

The physiological requirement for Tm phosphorylation is apparent; however, the impact of Tm phosphorylation on disease is less clear. For example, decreasing phosphorylation of Tm was demonstrated to induce cardiac hypertrophy in one instance and in another study exhibited the ability to rescue a mutation-associated familial hypertrophic cardiomyopathy mouse [118, 264]. Furthermore, a decrease in Tm phosphorylation was demonstrated in a Tm Glu54Lys mouse model of dilated cardiomyopathy [265]. Interestingly, a recent study investigating the phosphorylation levels of Tm in diseased and healthy human hearts reports no significant alterations in phosphorylation status in healthy or diseased donor hearts [266] suggesting disparities between species and Tm phosphorylation. Based on these findings one could conclude that the function of phosphorylation as it stands is to increase the end-to-end arrangement of Tm dimers and facilitate cooperativity in the thin filament rather than playing a major role in human disease.

It has been reported that phosphorylation of Tm at Ser-283 is necessary for allowing for cooperative activation of the thin filament [116]. We demonstrate similar results in which Tm Ser-283 phosphorylation trended toward an increase in Hill

171 coefficient suggesting an increase in cooperative activation of the thin filament. Further confirming the importance of Tm phosphorylation in cooperative function in the heart is a study in which loss of such phosphorylation has been shown to result in reduced ejection fraction [117]. One could speculate then that the phosphorylation of Tm helps to increase head-to-tail organization to enhance transduction of cooperativity in thin filament to allow for increased myosin binding and subsequent amplification the amount of force produced. This mechanism appears to be put in place to allow for normal heart function; however, it is possible that through an induced increase in phosphorylation of

Tm heart function could be further enhanced providing an attractive target for therapeutic intervention where the heart cannot produce sufficient force.

6.2.3 Tm Nitration

Nitration of Tm has been implicated in cardiac disease in which excess free radical production occurs and has been shown to be associated with depressed cardiac contraction. When considering the functional consequences of Tm Tyr nitration, it is important to point out that Tyr residues are located in both actin and TnT binding domains [24]. Oguchi and colleagues showed that decreasing stabilization within specific actin binding domains of Tm can alter the binding of actin as well as sliding velocity and force [255]. Interestingly, Tyr 60 is located in the d position of Tm as well as within one of the domains that were altered resulting in decreased sliding velocity and force compared to wild-type Tm stressing the importance of modifications made to this region of Tm. In a recent study, a mouse model in which a highly conserved aspartic acid in the

172 d position of Tm was replaced with a stabilizing Leu residue resulted in decreased cardiac function with concomitant decrease in myofilament Ca2+ sensitivity induced by loss of

Tm flexibility [267]. These results highlight the impact of charged Tm residues in the d position to regulate Tm flexibility and function with nitration of Tyr 60 likely inducing similar destabilizing effects by incorporation of negative charge contributing to altered myofilament regulation.

It is possible that nitration of other Tyr residues in Tm, albeit nitrated to a lesser extent than Tyr 60, could also affect the normal function of Tm as Tyr 162, Tyr 214, Tyr

221 and Tyr 267 are also found in actin binding regions of Tm. Additionally, Tyr 261 is located in b position which is said to interact with actin [255] suggesting that nitration could alter the interaction of Tm and actin and therefore lead to altered function. We demonstrate nitration of Tm Tyr occurs in a dose-dependent manner, in such a way that at lower concentrations of ONOO- Tyr 60 would initially be significantly nitrated and impart structural and functional alterations. Following an increase in ONOO- other Tyr would be nitrated further contributing to structural and functional alterations. Further investigation into the site-specific effects of nitrated Tyr in Tm is required to fully elucidate the biochemical and functional effects of Tm nitration.

Effective therapeutics for the treatment of radical-mediated dysfunction remains elusive as antioxidant administration in humans has proved futile compared to the staggering success seen in animal models. One potential route to ameliorate nitration- induced cardiac dysfunction is identification and administration of a denitrase enzyme.

While not specifically pinpointed, denitrase activity largely exists in endothelial cells and

173 macrophages exhibiting an ability to remove nitration from a variety of proteins [268,

269]. Alternatively, if direct removal of nitration is unavailable the induction of a modification to negate the functional effects of nitration could prove useful. For example, preliminary data suggests that nitration of Tm in the reconstitute thin filament resulted in a decrease in Ca2+ sensitivity. It is possible that one could induce a Ca2+ sensitizing modification (e.g. TnI Ser-150 phosphorylation) to reverse such desensitization to improve function. It seems likely that the latter would be more probable for therapeutic intervention as identification and induction of a specific denitrase remains to be seen.

6.3 Future Directions

Significant understanding of functional crosstalk between Ser-150 and Ser-23/24 phosphorylation was gained carrying out the work described in chapters 2 and 3; however, questions remain as to how these phosphorylations structurally alter the thin filament regulation and the role these phosphorylations play in disease. To address these the following approaches should be taken: 1) determine structural alterations in Tn and the thin filament as a result of TnI Ser-150 and Ser-23/24 phosphorylation, 2) determine amount of TnI Ser-150 phosphorylation is required to elicit the effect demonstrated above

3) determine in vivo cardiac function in normal and diseased hearts containing transgenic

Ser-150 and Ser-23/24 phosphorylation, and 4) induce TnI Ser-150 phosphorylation in the setting of ischemia to determine benefit.

The use of FRET has been extensively utilized in investigating the structural alterations induced by thin filament protein phosphorylations. A strikingly similar

174 example is the study by Ouyang and colleagues that identified the structural changers between TnI and TnC as a result of Ser-150 phosphorylation in the reconstituted thin filament [109]. This same tactic could easily be employed to determine how the presence of both these phosphorylations alter TnI structure in the isolated Tn complex in the presence and absence of Ca2+ as well as in the reconstituted thin filament. While not immediately available, another route to answer the structural question is to carry out

NMR spectroscopy. Such technology has been demonstrated to be useful in identifying structural changes to TnI/TnC induced by Ser-23/24 bisphosphorylation making it likely a useful technique to determine what Ser-150 phosphorylation alone and in combination with Ser-23/24 phosphorylation does to the TnI/TnC interaction. Determining the structural alterations elicited by these phosphorylations may lead to the development of therapeutic targets to alter the structural interactions within the Tn complex and thin filament similar to one of the earliest Ca2+ sensitizing drugs EMD-57033 [143].

While the functional aspect of TnI Ser-150 phosphorylation demonstrated in this dissertation have proven interesting, the effects elicited by said phosphorylation occurred through the presence of 100% of Ser-150 being phosphorylated, a situation which likely wouldn’t occur in the heart under basal conditions. It has been reported that TnI Ser-

23/24 phosphorylation can cause a maximal functional change at ~55% bisphosphorylation [103]. A similar investigation into TnI Ser-150 phosphorylation would be beneficial as we previously reported ~9% basal phosphorylation at Ser-150 in cardiac tissue. Through incubation of skinned fibers and cardiomyocytes with varying concentrations of recombinantly expressed TnI S150D we could determine the minimal

175 percentage of Ser-150 phosphorylation required to elicit its increase in Ca2+ sensitivity.

An alternative method to obtain the same information would be to incubate fibers or cells for varying amounts of time with AMPK or PAK to induce phosphorylation. While it is possible that off-target effects other than Ser-150 could result it would still provide information with regards to amount of phosphorylation required. This would provide useful information with regards to therapeutic potential as a compound would potentially not have to induce phosphorylation of every TnI at Ser-150 rather just surpass a certain threshold to impart its functional alterations.

Possibly one of the most important aspects when considering whether or not phosphorylations are worth investigating for therapeutic development are the in vivo functional changes that result. Transgenic expression of TnI S150D and S23/24D alone and in combination to form TnI S23/24/150D would provide the best insight as to how these phosphorylations alter cardiac function. To determine the effects on the heart, baseline physiological examination by means of pressure-volume loops and associated hemodynamics would give details on how contractility is altered by these phosphorylations. Additionally, echocardiography would provide further insight in to contractile alterations as well as structural insight of possible ventricular remodeling as a compensatory mechanism in response to TnI Ser-150 and/or Ser-23/24 phosphorylation.

To elucidate the therapeutic potential, a coronary artery ligation model of myocardial infarction in transgenic animals would determine whether or not having these phosphorylations alone or in combination would prove beneficial. Again, pressure- volume loops would detail the functional changes following infarction and show

176 improvement, if any, as a result of the phosphorylations. Key to our understanding is the time at which we evaluate the animals post-infarct as acute recovery versus long-term recovery could yield varying results. Additionally, staining of cardiac tissue for fibrosis would provide an understanding as to how these phosphorylations may alter cardiac remodeling following infarction.

As with all therapeutics a modification of interest must be induced somehow either by gene expression or through modulation with compounds or small molecules.

Such a requirement is still needed as pharmacological AMPK activation through metformin or 5-Aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) has proven not as successful as hoped as a result of their off-target effects. Recently, a novel

AMPK activator was developed that has demonstrated the ability to activate AMPK a significantly lower concentrations than traditional AMPK activators, as well as prevent the production of anti-inflammatory interleukin-6 [270]; both processes which would prove beneficial to the heart during ischemia. As an initial screen to determine the therapeutic potential of this drug a dose-response curve would be carried out on cardiomyocytes and fractional shortening and calcium transients would be measured to determine the effect of the drug on Ca2+ handling and myofilament function. Upon determining the optimal concentration of drug use, movement into a higher order system such as trabeculae would prove beneficial as it would demonstrate the ability to possibly improve function in the muscle lattice rather than a single cell. Ultimately, administration of this drug before and/or after an ischemic insult to the heart would determine its clinical potential. Through the use of a catheter and balloon we would be able to determine the

177 effect of the AMPK-activating drug on whole heart function and hemodynamics before, during, and after coronary artery ligation mimicking a myocardial infarct. Demonstration of an AMPK activator to improve myocardial function following an ischemic insult is the ultimate goal preceded by the work demonstrated in this dissertation.

As a tangent to the cardiac muscle work done in our lab, the potential for TnI Ser-

150 (Ser-118 in skeletal TnI) phosphorylation to help correct skeletal neuromuscular disease offers an attractive route of investigation. It is well established that AMPK activation plays an important role in glucose utilization during contraction of the skeletal muscle [271]; however, the direct effect of AMPK on the myofilaments, as demonstrated in the heart in the work above, in skeletal muscle remains to be seen. To begin investigating such a phenomenon, administration of a traditional AMPK activator could be used with subsequent determination of skeletal muscle TnI Ser-118 phosphorylation by Western blot identification. Similar to the cardiac work done, exchange of pseudophosphorylated TnI at the corresponding Ser-118 site in the skeletal muscle would provide information regarding the Ca2+ sensitivity alterations. Should we see said increase in Ca2+ sensitivity, as would be beneficial in neuromuscular disease, we could attempt to induce TnI Ser-118 phosphorylation in the skeletal muscle in an animal model of neuromuscular disease, e.g. muscular dystrophy. Following administration of AMPK activator, a muscle fatigue protocol could be used to determine if TnI Ser-118 phosphorylation improves muscle function and strength with subsequent isolation of skeletal muscle for identification of TnI Ser-118 phosphorylation. This work could pave the way for translation of the work done in cardiac muscle to the skeletal muscle.

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While the main focus of the lab remains on Tn phosphorylation to alter structure and function of the thin filament, the nitration of Tm is lacking a critical step required to further the project. While extensive characterization and identification of specific Tm nitration sites has been carried out, two questions still remain: 1) how do specific Tyr nitrations alter the structure-function, and 2) can Tm nitration be clearly identified in cardiac disease tissue. To address the issue of specific Tyr nitration, a special expression system is available that allows for the incorporation of a nitrated Tyr residue into the Tm molecule at a location of our choice. Ideally, each individual site of possible Tyr nitration would be examined alone to determine how each contributes to the overall all change in structure and function. This would be carried out through the use of circular dichroism to determine overall helical content as we did with ONOO- treated Tm. Furthermore, protein-protein binding experiments as well as steady-state binding and kinetic Ca2+ measurements would be made to determine the effect on function. With regards to identification, further examination into the mass spectrometric identification of Tm Tyr nitration is required. While we were not able to identify nitration in diseased cardiac tissue this was only one experiment. Further modification of our novel mass spec technique as well as usage of alternate cardiac disease tissue (e.g. ischemia, cardiomyopathy, oxidative stress) could provide means for a better likelihood of successfully identifying nitration of Tm in disease.

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6.4 Final Remarks

The mechanisms underlying thin filament regulation of cardiac contraction are unique and intricate in that while a modification to the Tn complex may alter function in isolation the same function may not translate to a more complex system. The reductionist approach taken in the work described here provides information toward an understanding of the fundamental alterations made to the Tn complex and thin filament following phosphorylation of TnI. As is with human physiology multiple factors often feed in to one another resulting in a net process perceived as a single function, yet the underlying mechanisms of each individual component can often provide insight into just why things happen the way that they do. Investigating these mechanisms associated with the Tn complex and thin filament has been a rewarding experience with the overall implication – contraction and relaxation of the heart to sustain the human body – not being lost in translation. The work carried out previously by numerous gifted scientists in the field of muscle biochemistry and biophysics has provided a solid foundation to answer the current day questions at hand regarding the function of the heart and it is my hope that the work provided here will someday do the same for future scientists to come.

"If I have seen farther, it is by standing on the shoulders of giants." – Sir Isaac Newton

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