Relengthening RT50, Fig 3C-G)

Neuronal Nitric Oxide Synthase Signaling Contributes to the Beneficial Cardiac Effects of Exercise

DISSERTATION

Presented in Partial Fulfillment of the Requirements for

the Degree Doctor of Philosophy in the Graduate

School of The Ohio State University

Steve R. Roof

Biomedical Sciences Graduate Program

The Ohio State University 2012

Dissertation Committee:

Dr. Mark T. Ziolo, PhD Advisor

Dr. George E. Billman, PhD

Dr. Sandor Gyorke, PhD

Dr. Brandon Biesidecki, PhD

ABSTRACT

Exercise is beneficial to one’s health, reduces the risk of cardiomyopathies, and is utilized as a therapeutic intervention after disease [2-7]. This is due, in part, due to the beneficial chronic adaptations that enhance contraction and accelerate relaxation [8].

These intrinsic exercise-induced adaptations are observed at the level of the cardiomyocyte [9]. That is, ventricular myocytes from exercised (Ex) mice exhibit increased Ca2+ cycling and contraction-relaxation rates [6, 9-12]. Additionally, cardiac growth (physiological hypertrophy) and an increase in aerobic fitness (VO2max) are hallmark cardiac adaptations due to exercise training. The molecular mechanisms that explain how the heart adapts are not fully understood and studies examining signaling pathways are limited. A signaling molecule with a potential role in cardiac adaptations to exercise is nitric oxide (NO).

Nitric Oxide (NO) has been shown to be a key regulator of myocyte contractile function. NO, produced via the neuronal nitric oxide synthase (nNOS or NOS1), enhances basal contraction by increasing Ca2+ cycling through the sarcoplasmic reticulum

(SR) [13-15]. Data suggest that NOS1 signaling increases Ca2+ uptake by targeting the

SR Ca2+ ATPase (SERCA2a)/phospholamban (PLB) complex. NOS1 signaling also targets the SR Ca2+ release channel (ryanodine receptor - RYR2) to increase its open time probability [16]. Together, NOS1 signaling increases Ca2+ transient amplitudes, shortening amplitudes, and accelerates relaxation rates [14, 16-19]. These are similar

ii effects to exercise adaptations, but the role of NOS1 signaling on the beneficial effects of exercise on cardiac myocyte function has not been thoroughly investigated.

Thus, we have set up 5 specific goals. We want to 1) determine if exercise enhances NOS1 protein expression and NOS1-dependent NO bioavailability in murine ventricular myocytes, 2) determine if NOS1 contributes to the exercise-mediated enhancement of murine myocyte contraction, 3) determine the molecular mechanisms of the NOS1-mediated increase in murine contraction, 4) investigate the exercise-mediated cardiac adaptations (VO2max, physiological hypertrophy, and myocyte contraction) in the

NOS1 knockout (NOS1KO) mouse, and 5) determine if NOS1 signaling contributes to the exercise-mediated enhancement of contraction in a large mammal model. The results are as follows:

After an 8 week aerobic interval training program, Ex mice had a higher VO2max and a physiological hypertrophy compared to sedentary (Sed) wildtype (WT) mice.

Exercise induced an increase in NOS1 expression and nitric oxide production. Isolated ventricular myocytes from the Ex mice exhibited larger contraction and faster relaxation rates compared to Sed myocytes. Acute NOS1 inhibition with S-methyl-L-thiocitrulline

2+ 2+ (SMLT) resulted in a greater reduction in Ca transient amplitude, Ca transient RT50, shortening amplitude, SR Ca2+ load, and SR Ca2+ fractional release in Ex versus Sed. In fact, acute NOS1 inhibition normalized the Ex induced increase in contraction and Ca2+ decline rates to Sed levels. The NOS1 mediated effect on contraction was due to a shift in the kinase/phosphatase balance to increase PLB Serine16 phosphorylation (the PKA site).

Surprisingly, trained NOS1KO mice, did not exhibit any of the cardiac adaptations. That

iii is, Ex-NOS1KO mice did not have increased VO2max or hypertrophy compared to Sed-

NOS1KO mice. In fact, Ex-NOS1KO mice had depressed Ca2+ transient amplitude, SR

2+ 2+ Ca load, and slowed Ca transient RT50 compared to Sed-NOS1KO. Upon further investigation, this resulted from elevated reactive oxygen species levels that contributed to increase protein phosphatase activity and subsequently decrease PLB Serine16 phosphorylation to cause detrimental Ca2+ handling. Lastly, we observed a similar effect in an exercise-trained canine model. Specifically, NOS1 inhibition elicited a greater reduction in myocyte contraction in Ex versus Sed. These data strongly suggest a more universal role for exercise induced enhancement of NOS1 signaling in both large and small mammalian species

In conclusion, NOS1 signaling contributes to the adaptive cardiac effects of exercise. Specifically, exercise increases ventricular myocyte NOS1 expression and NO bioavailability, which is essential for aerobic fitness, hypertrophy, and enhanced contraction/relaxation. Hence, it may be possible to mimic the beneficial effects of exercise to the heart by enhancing NOS1 signaling. This pathway may provide a novel therapeutic for cardiac patients that are unable/unwilling to exercise.

DEDICATED TO THE WOMEN IN MY LIFE – PAM, AMANDA, & SIENA ROOF

ACKNOWLEDGMENTS

I remember where I was when the thought of combining my passion for exercise and medicine into a thesis project materialized. That is when ELIM (Exercise-Like

Induced Medicine) was born. From that point on, I’ve dedicated my time investigating how a normal heart is strengthened into an exercise-trained heart. With luck on my side, I met up with Dr. Mark Ziolo, and together, we found evidence that strongly suggest the

NOS1 is indeed necessary for the heart to adapt to exercise. This achievement did not come easy and there are plenty of people who deserve thanks.

I first want to thank my dissertation committee for their help and guidance on my project: Drs. George Billman, Sandor Gyorke, and Brandon Biesidecki. Next I would like to thank other laboratories and their members (graduate students and lab techs alike) in the department and college for the collaborations and insight into science: Drs. Jonathan

Davis, Paul Janssen, Peter Mohler and others.

It goes without question that a large amount of my thanks goes to my wife,

Amanda, for putting up with my long hours. Her sacrifices truly provided me with enough time to accelerate and complete my project.

Lastly, I have to thank the single most important person who deserves to be acknowledged for his efforts in helping me complete my Ph.D., and that is my advisor

Dr. Mark Ziolo. With very little lab experience prior to graduate school, Dr. Ziolo brought me in and allowed me to chase after my dream of seeing if ELIM could become

vi a reality. He dealt with my naive science immaturity and horrible speaking/writing skills in a very mentoring-like fashion. He kept me motivated and provided with nearly every tool needed. Without his outside of the lab friendship, my time spent in his lab would not have been as enjoyable. If it wasn’t for Dr. Ziolo, I would not have had such a successful

Ph.D.

vii

VITA

July 22nd, 1984...... Born, Canton, OH

2003-2008...... B.S. Biochemistry and Exercise Science,

Florida State University

2008-2012...... Graduate Research Associate, Department

of Physiology and Cell Biology, The Ohio State University

PUBLICATIONS

1. Kohr MJ, Traynham CJ, Roof SR, et al. cAMP-independent activation of protein kinase A by the peroxynitrite generator SIN-1 elicits positive inotropic effects in cardiomyocytes. J Mol Cell Cardiol. 2010;48(4):645-8.

2. Roof SR, Janssen PM, Ziolo MT, The Effects of Increased Systolic Ca2+ and/or Phospholamban Serine16 Phosphorylation on Ca2+ Transient Kinetics in Cardiac Myocytes, Am J Physiol Heart Circ Physiol 2011; 301(4):H1570-8.

3. Roof SR, Biesiadecki BJ, Davis JP, Janssen PM, Ziolo MT, Effects of increased systolic Ca2+ and β-adrenergic stimulation on Ca2+ transient decline in NOS1 knockout cardiac myocytes, Nitric Oxide, 2012 1;27(4):242-7.

4. Roof SR, Tang L, Ostler JE, Periasamy M, Györke S, Billman GE, Ziolo MT, Neuronal Nitric Oxide Synthase is Indispensable for the Adaptative Contractile Effects of Exercise, SUBMITTED

5. Traynham CJ, Roof SR, Wang H, Prosak RA, Tang L, Viatchenko-Karpinski S, Ho HT, Racoma IO, Catalano DJ, Huang X, Han Y, Kim SU, Gyorke S, Billman GE, Villamena FA, Ziolo MT, Di-esterified nitrone restores nitroso-redox balance and increases myocyte contraction via increased SR Ca2+ handling, IN PRESS, PLOS One.

6. Curran J, Tang L, Roof SR, Velmurugan S, Millard A, Shonts S, Santiago DJ, Ahmad A, Perryman M, Mohler P, Bers DM, Ziolo MT, Shannon TR, Nitric viii

Oxide Mediates Increased Diastolic Sarcoplasmic Reticulum Calcium Release in Response to Adrenergic Agents, SUBMITTED

7. Kohr MJ, Roof SR, Zweier JL, Ziolo MT, Modulation of Myocardial Contraction by Peroxynitrite, SUBMITTED

8. Jeyaraj SC, Roof SR, Unger NT, Mohler P, Ziolo MT, Chotani MA, Effects of Rap1a GTPase on cardiac myocyte function, IN PREPARATION

9. Roof SR, Ho T, Tang L, Haung X, Catalano DJ, Györke S, Ziolo MT, Elevated Reactive Oxygen Species Elicits the Detrimental Effects of Exercise in the Neuronal Nitric Oxide Synthase Knockout Mice, IN PREPARATION

10. Oaks JJ, Santhanam R, Walker CJ, Saddoughi S, Roof SR, Chen C-S, Ziolo MT, Levine R, Quintas-Cardama A, Ogretmen B, Perrotti D. The oncogenic Jak2V617F mutant and the phosphorylated form of the sphingosine analog FTY720 (FTY720-P) inhibit the tumor suppressor protein phosphatase 2A (PP2A) through a Jak2, SET and Nitric Oxide-dependent signaling pathway, IN PREPARATION

11. Peterson JM, Roof SR, Canan BD, Little SC, Gaw C, Davis JP, Ziolo MT, Janssen PML, Guttridge DC, Understanding the role of NF-kB in dystrophic cardiomyopathy, IN PREPARATION

12. Ostler JE, Maurya SK, Roof SR, Dials J, Ziolo M, and Periasamy M, Diabetic muscle atrophy in type 2 diabetic db/db mice precedes chronic hyperglycemia and is resistant to chronic exercise IN PREPARATION

FIELDS OF STUDY

Major Field: Integrated Biomedical Science Graduate Program

1. Emphasis: Cell, Organ Systems, and Integrative Biology

2. Cardiac Physiology

Table of Contents

Abstract ...... ii

Vita ...... viii

Publications ...... viii

Fields of study ...... ix

Table of Contents ...... x

List of Tables ...... xvi

List of Figures ...... xvii

Preface...... 1

Chapter 1: Introduction ...... 2

1.1 General Introduction ...... 2

1.2 Excitation-Contraction Coupling ...... 3

1.2.1 Cardiac action potential ...... 3

2+ 1.2.2 Cardiac myocyte [Ca ]i transient ...... 4

1.2.3 Myocyte Contraction ...... 5

1.3 Regulation of ECC ...... 6

1.3.1 Acute Regulation ...... 6

1.3.2 The force-frequency response ...... 6

1.3.3 The β-adrenergic receptor signaling pathway ...... 7 x

1.3.4 Kinase-Phosphatase Balance ...... 8

1.4 Reactive Nitrogen Species (RNS) ...... 9

1.4.1 Nitroso-Redox Balance ...... 11

1.4.2 Endogenous NO Production ...... 11

1.4.3 NOS1 signaling ...... 12

1.4.4 NOS3 signaling ...... 14

1.5 Chronic Regulation ...... 15

1.5.1 Beneficial Adaptations - Exercise ...... 15

1.5.2 Systemic Adaptations ...... 16

1.5.3 Cardiac Adaptations ...... 16

1.6 Detrimental Adaptations - Heart Failure...... 20

1.7 Fixing Heart Failure with Exercise ...... 22

1.7.1 Role of NO in Exercise Adaptations ...... 23

1.7.2 Systemic Adaptations ...... 23

1.7.3 Cardiac Adaptations ...... 25

1.8 Specific Aims, Objectives, and Rationales ...... 25

1.8.1 Determine if exercise enhances NOS1 protein expression and NOS1 dependent

NO bioavailibity in murine ventricular myocytes ...... 26

1.8.2 Determine if NOS1 signaling contributes to the exercise–mediated enhancement

of murine myocyte contraction...... 27

1.8.3 Determine the molecular mechanisms of the NOS1-mediated increase in murine

contraction...... 27

1.8.4 Investigate the exercise-mediated cardiac adaptations in NOS1KO mice ...... 28

1.8.5 Determine if NOS1 signaling contributes to the exercise-mediated enhancement

of contraction in a large mammal model ...... 28

Chapter 2: The Neuronal Nitric Oxide Synthase is Indispensable for the Cardiac Adaptive

Effects of Exercise ...... 31

2.1 INTRODUCTION ...... 31

2.2 METHODS AND MATERIALS ...... 32

2.2.1 Murine exercise protocol ...... 32

2.2.2 Canine exercise protocol ...... 33

2.2.3 VO2peak and VO2max testing protocol and training speeds ...... 34

2.2.4 Murine Cardiomyocyte isolation ...... 34

2.2.5 Measurement of myocyte Ca2+ transient and shortening ...... 35

2.2.6 Canine Cardiac Myocyte isolation ...... 35

2.2.7 Measurement of NO production with DAF-2 AM fluorescence...... 36

2.2.8 Force frequency response ...... 37

2.2.9 Post rest potentiation ...... 37 xii

2.2.10 SR Ca2+ load and SR Ca2+ fractional release ...... 37

2.2.11 Western Blot analysis ...... 37

2.2.12 Solutions and drugs ...... 38

2.2.13 Statistical Analysis ...... 38

2.3 RESULTS ...... 38

2.3.1 Exercise induced adaptations ...... 38

2.3.2 Exercise increases myocyte NOS1 protein expression, NO bioavailability, and

contraction/relaxation ...... 39

2.3.3 Effects of Ex and NOS1 signaling on SR Ca2+ cycling ...... 39

2.3.4 Effects of training NOS1 deficient mice ...... 41

2.3.5 Effects of Ex and NOS1 on canines ...... 41

2.3.6 Enhanced role of NOS1 and its downstream targets with Ex ...... 42

2.4 DISCUSSION ...... 42

2.4.1 The role of NOS1 signaling in the exercise induced beneficial cardiac adaptations

...... 43

2.4.2 Training NOS1 deficient mice results in Ca2+mishandling ...... 45

2.4.3 Beneficial effects of exercise ...... 46

3.1 INTRODUCTION ...... 62

3.2 METHODS AND MATERIALS ...... 64

xiii

3.2.1 Murine exercise protocol ...... 64

3.2.2 Cardiomyocyte isolation ...... 64

3.2.3 Measurement of myocyte Ca2+ transients and shortening ...... 65

3.2.4 Measurement of ROS Production with CM-H2DCFDA Fluorescence ...... 65

3.2.5 Western blot analysis ...... 66

3.2.6 Protein Phosphatase Activity ...... 66

3.2.7 VO2max testing protocol and training speeds ...... 67

3.2.8 Solutions and drugs ...... 67

3.2.9 Echocardiography...... 67

3.2.10 Statistical Analysis ...... 69

3.3 RESULTS ...... 69

3.3.1 Exercise decreases ROS production in WT and increases in NOS1KO mice ..... 69

3.3.2 Superoxide scavenger rescues myocyte contraction from trained NOS1KO ...... 70

3.3.3 Identification of the ROS source ...... 70

3.3.4 Mechanisms responsible for the Ca2+ mishandling ...... 71

3.3.5 Restoration of myocyte contraction with EMEPO ...... 72

3.4 DISCUSSION ...... 72

3.4.1 The role of NOS1 signaling in exercise induced adaptations ...... 73

3.4.2 Exercise-derived sources of ROS ...... 74

xiv

3.4.3 NOS1 plays a critical role in regulating the nitroso-redox equilibrium in response

to exercise ...... 76

Chapter 4: Future Directions ...... 87

List of References ...... 92

List of Tables

Table 1 Physiological hypertrophy and maximal oxygen consumption (VO2max). S...... 50

Table 2 LV function as assessed by echocardiography ...... 79

xvi

List of Figures

Figure 1 Excitation – contraction coupling in cardiac myocytes ...... 29

Figure 2 NOS1 and NOS3 localization and signaling pathways...... 30

Figure 3 Exercise increases myocyte NOS1 expression, nitric oxide (NO) production and enhances myocyte contraction, which is normalized by acute NOS1 inhibition...... 51

Figure 4 Exercise enhances the force frequency relationship, which is normalized by acute NOS1 inhibition...... 52

Figure 5 Exercise enhances SR Ca2+cycling and is normalized by acute NOS1 inhibition

...... 53

Figure 6 Exercise enhances post-rest potentiation, which is normalized by acute NOS1 inhibition ...... 54

Figure 7 Protein phosphatase 1 and 2a inhibition (okadaic acid, OA) and PKA inhibition

(PKI) normalized myocyte contraction ...... 55

Figure 8 Negative effects of exercising on Ca2+handling in NOS1 deficient myocytes. . 56

Figure 9 No enhancement of the force-frequency response or post-rest potentiation with exercise in NOS1 deficient myocytes...... 57

Figure 10 Exercise in a trained canine model elicited similar results with NOS1 inhibition on myocyte contraction...... 58

Figure 11 The role of a peroxynitrite decomposing catalyst (FeTPPS) on myocyte contraction...... 59 xvii

Figure 12 NOS1 and its downstream signaling targets have a greater effect with exercise

...... 60

Figure 13 VO2peak was measured to determine weekly training speeds...... 61

Figure 14 Exercise-trained NOS1KO myocytes have increased reactive oxygen species

(ROS) production and a superoxide scavenger a greater effect with on myocyte contraction...... 80

Figure 15 Effects of on myocyte contraction with a superoxide scavenger (MENO), on wildtype and NOS1KO sedentary (Sed) and exercise-trained (Ex) myocytes ...... 81

Figure 16 Effects of acute inhibition with allopurinol (xanthine oxidase), apocynin

(NADPH oxidase), and rotenone (mitochondrial complex 1) on Ca2+ handling from WT and NOS1KO sedentary (Sed) and exercise-trained (Ex) myocytes ...... 82

Figure 18 Decreased PLB Serine16 phosphorylation and increased phosphatase activity was rescued with a superoxide scavenger in Ex-NOS1KO...... 84

Figure 19 A synthesized compound used as a superoxide scavenger and nitric oxide donor (EMEPO) has a greatest effect in exercise-trained NOS1KO mice (Ex-NOS1KO)

...... 85

Figure 20 Effects of a compound that acts both as a superoxide scavenger and nitric oxide donor (EMEPO) on wildtype and NOS1KO sedentary (Sed) and exercise-trained (Ex) myocytes ...... 86

xviii

Preface

Numerous song lyrics depict how the heart is where “passion” resides or is commonly used as a metaphor for “strength/courage”. Heart is also sometimes referred to as the gateway to your “soul”. Scientifically this may not be accurate, but the analogues are not far off the truth. The heart supports the entire body by providing essential nutrients throughout the circulatory system. Its strong rhythmic beating is easily noticed during events when passion and courage are called upon. Its dynamic regulation and integrative connection with the body elevates its importance comparable to that of our inner spirit. Through numerous reasons, our life sustaining heart can fail us. When the function of the pump is compromised and heart failure takes over, death and the freeing of our soul may be on the horizon. But through strength and courage, exercise can save the day. By opening up ones heart and allowing the passion of vitality to be pumped through our circulatory system, self-motivation through daily exercise can rescue the dysfunction. Thus:

Open your Heart and use Heart to save your Heart

A simple motto, but our current lifestyles don’t promote exercise and therefore salvation may be in the form of an exercise pill. Thus, necessary research such as this dissertation is useful.

Chapter 1: Introduction

1.1 General Introduction

The cardiovascular system is a specialized network adapted to provide every organ and tissue with energy metabolites, an immune response, water, oxygen, and others. In addition, the cardiovascular system is also used for waste removal. This circulation is essential for survival and is highly dependent upon a healthy, strong, electrically stable heart. When the heart contracts, it forces blood out of the ventricles where it circulates throughout the body (based on the body’s current demands). If the heart can no longer meet the demands of the body, a person is at great risk. In fact, as the result of our western life style nearly 5 million people have been diagnosed with a cardiomyopathy and an estimated $37 billion dollars are spent directly or indirectly on heart failure (HF) each year [20]. In order to develop better treatments, basic science has been ruthlessly persistent. By better understanding how the heart functions, we are better equipped to tackle this problem. Thus, one large area of research is cardiac physiology at the level of the ventricular myocyte, which has dysfunction in HF animal models/patients

[21, 22]. The mechanism by how the myocyte contracts is called excitation-contraction coupling (ECC).

1.2 Excitation-Contraction Coupling

ECC is the process used to describe myocyte contraction [1]. In essence it explains how the electoral propagation (movement of ions across the plasma membrane) of an action potential can be coupled with chemical signaling and mechanical force. This process is shown in Figure 1 and further explained in much detail below.

1.2.1 Cardiac action potential

The cardiac action potential (AP) is initiated when the sinoatrial nodal cells spontaneously depolarize. This occurs via the slow inward current of Na+ and K+ ions.

Then, the propagation of the AP is transmitted through joined myocytes in the atria until the AP reaches the atrioventricular node. There the AV node slows the propagation of AP to allow the ventricles more filling time. Subsequently, the AP travels down the Bundle of His, where it branches to the left and right sides of the heart, and the purkinje fibers to electrically stimulate the ventricles via depolarization. This depolarization opens voltage- gated Na+ channels first. The greater extracellular concentration and negative internal resting membrane potential drives Na+ into the cell. This is referred to as phase 0 of the ventricular myocyte AP. Phase 1 occurs right after the peak of the AP’s upstroke as a brief efflux of K+ ions that creates an AP notch. The rush of Na+ inward depolarizes the cell and triggers both the voltage-gated Ca2+ (L-type) and K+ channels to open. Based on the electro-chemical gradient, the high extracellular Ca2+ concentration drives Ca2+ inward, whereas the high intracellular K+ concentration drives K+ out. These opposing currents elicit the “plateau phase” or phase 2 of the AP. To return to resting membrane potential, the further K+ efflux contributes to the repolarization of the myocytes (phase 3

3). The ions return to diastolic levels via the Na+/K+-ATPase and Na+/Ca2+ exchanger

(NCX) and await the next electrical propagation. During this process, intracellular Ca2+ triggers a cascade that eventually results in the contraction of the myocyte.

2+ 1.2.2 Cardiac myocyte [Ca ]i transient

Once the AP travels down the t-tubules and L-type Ca2+ channels open, “trigger”

Ca2+ enters the cell, binds to the ryanodine receptor (RyR2), and stimulates additional

Ca2+ to be released from the sarcoplasmic reticulum (SR – an internal Ca2+ storage organelle). This process is called Ca2+ induced Ca2+ release or CICR [23]. The dumping of Ca2+ into the cytosol raises the free Ca2+ (from ~100 nm to nearly 1 µM) and gives

2+ 2+ rise to the upstroke of the intracellular Ca transient [Ca ]i [24]. Increasing the amount of trigger Ca2+ and/or Ca2+ in the SR (SR Ca2+ load) can determine the amplitude of the

Ca2+ transient [25].

2+ 2+ The decline of the [Ca ]i is due to the resequestration of Ca into the SR via the

SR Ca2+-ATPase (SERCA)/phospholamban (PLB) complex coupled with the extrusion from the cell via the NCX. The proportion of Ca2+ cycled through the SR and extruded from the cell is dependent upon species. In small rodents, ~95% of the intracellular Ca2+ is pumped back into the SR. Whereas, SERCA only accounts for ~70% in large mammals

[1, 26]. Thus, NCX plays a greater role to extrude Ca2+ in large mammals (~30%) compared to small rodents (~4%). SERCA’s function is inhibited by PLB by decreasing the SR Ca2+ ATPase’s “pump” affinity for Ca2+ [27]. The PLB-mediated inhibition of

SERCA is relieved when PLB dissociates from SERCA. An increase in Ca2+ levels will

2+ cause this dissociation [28]. Thus, increasing the peak amplitude of the [Ca ]i transient 4

2+ will increase SERCA activity that then results in faster [Ca ]i decline rates [29, 30]. PLB is also a major phosphoprotein in the myocyte. PLB can be phosphorylated on Serine16 through protein kinase A (PKA) or on Threonine17 through Ca2+/calmodulin-dependent protein kinase (CAMKII) [31, 32]. Phosphorylation at either site also results in the dissociation of PLB from SERCA and results in faster Ca2+ decline and greater SR Ca2+ load. Consequently, dephosphorylation of PLB by protein phophatases PP1 and PP2a

[33] will increase PLB-mediated inhibition of SERCA and result in a slower Ca2+ decline and a lower SR Ca2+ load. At steady state NCX extrudes similar amounts of Ca2+ that entered the cell though the L-type Ca2+ channel to maintain Ca2+ homeostasis. In the forward mode, NCX extrudes 1 Ca2+ ion for every 3 Na+ ions [34]. This results in a slight net inward current. NCX can also function in the reverse mode. Under certain conditions

+ 2+ 2+ (ex: high [Na ]i), incoming Ca can act as “trigger Ca ” to induce a spontaneous CICR

[35]. Mitochondrial Ca2+ fluxes and the Ca2+ uniporter are also both known to play a

2+ 2+ small role in the Ca decline, contributing to ~1% [36, 37] in the fall of [Ca ]i.

1.2.3 Myocyte Contraction

The activation of myocyte contraction is dependent upon Ca2+ binding to the contractile proteins (myofilaments). The myofilaments are comprised of thick and thin filaments that are arranged in an anti-parallel fashion and form light and dark alternating bands (striations). Together, they interact and slide past one another to contract the myocyte. The thin filament is comprised of troponin C (TnC), troponin I (TnI), troponin

T (TnT), tropomyosin (Tm), and actin. The thick filament consists of mainly myosin.

2+ 2+ Once [Ca ]i rises above a threshold, Ca binds to TnC. This stimulates a conformational 5 structural change that pulls TnI off actin and subsequently moves Tm to expose the myosin binding domain on actin. Next, the myosin head binds to actin to form the crossbridge between the thick and thin filaments. With the hydrolysis of ATP, a power stroke

2+ is generated. As [Ca ]i declines, the reverse occurs and the myofilaments dissociate from each other giving way to relaxation.

1.3 Regulation of ECC

Since every cardiomyocyte contracts with every heartbeat, myocyte contraction is highly regulated. The heart has numerous ways to acutely modulate ECC and therefore, myocyte contraction on a beat-to-beat basis to match the changing demands of the body.

Furthermore, adaptations can occur over a period of time (months to years) that can be beneficial to enhance (chronic exercise or compensatory hypertrophy) or can be deleterious to depress (HF) ECC and myocyte contraction.

1.3.1 Acute Regulation

Acute regulation is executed by a large number of key regulators. For the sake of this project, the most relevant acute regulation are the force frequency response (FFR), β- adrenergic receptor (β-AR) signaling pathway, phosphatase/kinase balance, and reactive nitrogen species.

1.3.2 The force-frequency response

During exercise, when the demands of the body have increased, cardiac output

(HR x SV) is also increased. At peak exercise, the increase in HR may not be sufficient to meet the increased demand for oxygen at the tissue level (i.e. blood flow). The heart can 6 also modulate itself. Thus, further increases in cardiac output result from increases in SV; that is by increasing the force of contraction during each beat. The increase in contractile force at higher frequencies is called the FFR. The molecular mechanisms are not fully understood. However, evidence suggests that at higher frequencies there is less time for

Ca2+ efflux. This elevates diastolic Ca2+ levels and subsequently loads the SR with more

Ca2+ that can be released on the next beat. Data suggest that CaMKII activity increases as a consequence of elevating diastolic Ca2+ levels [38]. Activation of the SERCA/PLB complex provides a major player responsible for accelerating the Ca2+ decline and thereby, increasing SR Ca2+ load [39]. In vivo, the increased HR is usually accompanied with an increased catecholamine drive (epinephrine and norepinephrine) and activates the

β-AR signaling pathway that further contributes to greater force generation with every heart beat.

1.3.3 The β-adrenergic receptor signaling pathway

Activation of the β-AR signaling pathway can also modulate myocyte contraction by contributing to the positive inotropic (contraction) and lusitropic (relaxtion) response

[40]. This pathway is initiated when an agonist binds to the β-AR (mainly the β1- receptor). Activating the stimulatory G-protein (Gs). Gs, in turn, then activates adenylate cyclase increasing cyclic AMP (cAMP) levels. The elevated cAMP levels then activates protein kinase A (PKA) that phosphorylates different downstream targets involved in

ECC. Specifically, PKA phosphorylates L-type Ca2+ channel, RyR, PLB, myosin binding protein C (MyBP-C), and TnI (Figure 2). PKA dependent phosphorylation of the L-type

Ca2+ channel increases Ca2+ influx providing trigger Ca2+ that stimulates Ca2+ released 7 from the SR [41, 42]. This additional Ca2+ release augments myocyte contraction.

Although it is currently subject to debate, data suggest that PKA can increase SR Ca2+ release by phosphorylating RyR [43]. Upon PKA mediated PLB Serine16 phosphorylation, PLB dissociates from SERCA thereby, accelerating Ca2+ uptake into the

2+ SR. This ultimately has as a dual effect by 1) increasing the [Ca ]i decline (that promotes in relaxation) and 2) increases the SR Ca2+ load such that more Ca2+ be released on the subsequent beats thereby, enhancing contractile force. PKA dependent phosphorylation of the myofilaments also contributes to the positive lusitropic response. Phosphorylation of MyBP-C enhances the cross-bridge cycling kinetics [44]. Phosphorylation of TnI

Serine23/24 by PKA decreases myofilaments sensitivity and also contributes to the accelerated relaxation observed during β-AR stimulation [45]. It is also important to note that another key downstream target of the β-AR stimulating pathway inhibiting protein phosphatase activity [46]. Thus, β-AR stimulation, results in the activation of kinases and the inhibition of phosphatases, shifting in the kinase/phosphatase balance.

1.3.4 Kinase-Phosphatase Balance

Post-translational modifications (PTM) increase the functional range of a cell.

PTMs are the addition or subtraction of functional groups to proteins such as S- nitrosylation, glycosylation, hydroxylation, oxidation, S-gluthionylation, acetylation and many more. PTM phosphorylation mediated modulation of myocytes contraction is the most extensive investigated PTM process. Kinases add a phosphate group to a protein. In contrast, phosphatases remove the phosphate. As mentioned above, phosphorylation of key proteins (L-type Ca2+ channel, PLB, RyR2, TnI) during β-AR stimulation via PKA 8 contribute significantly to the enhanced contraction and relengthening of the myocyte. In addition, as phosphatases are inhibited, this indirectly enhances myocyte contraction by sustaining the phosphorylation status. The phosphorylation/dephosphorylation balance is critical for a healthy myocyte function. A shift in the phosphorylation status is necessary to increase myocyte contraction and accelerate relaxation. Detrimental shifts in the kinase-phosphatase balance (i.e., decrease kinase and increase phosphatase activity) are deleterious to the myocyte and contributes to the contractile dysfunction that occurs in heart disease [47]. Kinases and phosphatases are, in turn, regulated by many signaling pathways; including reactive species such as nitric oxide (NO) [48, 49].

1.4 Reactive Nitrogen Species (RNS)

The first evidence that nitrogen oxides were endogenously produced was in 1916

[50]. Since then, we have learned that NO is a soluble gas used as a signaling molecule. It is synthesized as the byproduct of the conversion of L-arginine to L-citrulline mediate by the action of enzymes called nitric oxide synthases (NOS) (molecular weight ranging from 110-160 kDa [51]). This conversion is a two step process involving the cooperation of five cofactors. The C-terminus (reductase domain) of the NOS enzyme binds nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD). The oxygenase domain (N-terminal) binds tetrahydrobiopterin (BH4), oxygen (O2), L-arginine, and contains a heme group for NOS dimerization [52, 53]. The NOS enzyme first transfers an electron from NADPH to FAD or FMN and then transfers an electron to the heme/BH4/L-arginine oxygenase domain.

This reduces and activates O2 at the heme to generate H2O. This electron transfer occurs twice for every L-arginine converted. N ω-hydroxyl-l-arginine formed as the intermediate compound after the first electron transfer cycle while L-citrulline and NO are the final key products. Binding of Ca2+/calmodulin can accelerate this reaction [54]. In addition, regulation of NOS-mediated NO production is modulated by phosphorylation status [55], shear stress [56, 57], various co-factors [58], heat shock proteins [59], and localization

[60].

Once NO is produced, it acts as a signaling molecule to regulate cellular processes. Currently, there are two classifications of signaling pathways; the cyclic guanosine monophosphate (cGMP)-dependent and cGMP-independent pathways. In the cGMP-dependent pathway, NO activates guanylate cyclase (GC) to increase cGMP levels that then activates protein kinase G (PKG) [61] and regulates phosphodieserases

(PDE) (stimulate PDE2 and inhibit PDE3) [62]. Once activated, PKG can target proteins involved in myocyte contraction, such as L-type Ca2+ channel [63] and TnI [64]. The cGMP-independent signaling pathway occurs primarily via S-nitrosylation [65] (a PTM that adds NO to the thiol group of a cystine residue). In addition, NO can react with local reactive oxygen species (ROS) to form other reactive nitrogen species (RNS). For example, the NO can rapidly combine with a superoxide anion radical and form peroxynitrite. By acting similar to a buffer, NO levels can directly effect ROS levels.

This is known as the nitroso-redox balance [66].

1.4.1 Nitroso-Redox Balance

The interaction between NO and ROS must be maintained for adequate myocyte contraction. It is generally thought that ROS is “bad,” but in fact, at physiological levels, it is needed to facilitate S-nitrosylation. NO and ROS tandem maintains physiological signaling pathways [67]. It is when the balance is shifted and ROS levels to a point that that this pathway is disrupted. This disequilibrium can result in pathophysiological conditions. One consequence is that superoxides oxidize cysteine residues that inhibit/block S-nitrosylation [68]. These alternate modifications are sometimes irreversible and detrimental to the cell. In fact, in animal models with cardiac disease and in human HF patients, this imbalance results in increased production of ROS [69-71] and/or disrupted location of NOS [72, 73]. There are three main sources of ROS; xanthine oxidase (XO), NADPH oxidase, and the mitochondria complex I. It is vital to determine the source of ROS during cardiomyopathies and in order to combat the ROS with antioxidants to inhibit or buffer the ROS production. However, this strategy has not always been completely successful [74]. Antioxidant therapy may not be beneficial as it only treats one part of the nitroso-redox balance. Indeed, it may be also necessary to return NO levels/location/activity to physiological values to completely restore nitroso- redox equilibrium [75]. Thus, alterations to the three main sources of NO may be necessary therapeutic targets.

1.4.2 Endogenous NO Production

Three NOS isoforms are currently known to exist in the cardiac myocyte; NOS1,

NOS2, and NOS3 [14]. NOS2 (or the inducible NOS or iNOS) is induced during immune 11 responses and can contribute to further pathophysiological conditions of the heart by reducing L-type Ca2+ channel as well as decreasing TnI phosphorylation through PKG mediated targeting [76, 77]. NOS1 (or the neuronal NOS or nNOS) and NOS3 (or the endothelial NOS or eNOS) are both constitutively expressed in the myocyte. Both NOS1 and NOS3 signaling play a unique role in modulating ECC, but their roles in signaling pathways, stimuli, localization and effects on myocyte contraction are different.

1.4.3 NOS1 signaling

NOS1 was first discovered in 1989 in the brain [78]. Since then, it has been discovered in other tissues including cardiac myocytes. NOS1 signals through the cGMP- independent signaling pathway (i.e – S-nitrosylation/formation of ONOO-). It is localized to the SR (Figure 3) and co-immunoprecipitates with RyR2 [13, 79]. NOS1 signaling regulates basal contraction by maintaining both the kinase-phosphatase and the nitroso- redox balance. NOS1 signaling alters kinase activity by activating PKA via peroxynitrite

[17, 18, 48]. Activated PKA phosphorylates PLB Serine16 to increase SR Ca2+ uptake

[48]. Moreover, acute NOS1 inhibition (with S-methyl-L-thiocitrulline (SMLT)) or

NOS1 knockout (NOS1KO) decreases PLB phosphorylation levels and SR Ca2+ uptake

[18]. In addition, myocyte-specific NOS1 overexpression results in increased basal PLB

Serine16 phosphorylation [80]. Thus, NOS1 signaling increases PLB Serine16 phosphorylation to accelerate Ca2+ decline, load the SR with Ca2+, and to increase myocyte contraction. NOS1 signaling also regulates phosphatase activity by regulating the ROS levels at the SR microdomain in healthy myocytes. Phosphatases, which are redox sensitive, are activated by increased oxidative stress [81] to dephosphorylate 12 proteins. Dephosphorylation of PLB by protein phosphatases PP1 and PP2a [33] increases PLB-mediated inhibition of SERCA resulting in slowed a Ca2+ decline and a lower SR Ca2+ load. Thus, maintenance of ROS levels at the SR microdomain is critical for both the maintenance of kinase-phosphatase balance and the regulation of myocyte function. The source of ROS has not been completely resolved. Studies have shown that the elevated ROS levels in the NOS1KO mouse is due to both NADPH oxidase and XO

[82-84]. Interestingly, similar to NOS1, XO is also localized to the SR [13] and it co- immunoprecipitates with NOS1 [85]. Thus, XO-derived ROS is a good candidate for the

ROS that interacts with NO at the SR. Moreover, NOS1-dervived NO plays a dual role in regulating ROS levels from XO. First, it acts to buffer superoxide and secondly it can directly inhibit XO activity [86, 87]. Together, these actions limit ROS production (as observed in the NOS1KO mouse), thereby, contributing to the maintenance of the nitroso-redox balance. In summary, NOS1 maintains the nitroso side of the nitroso-redox balance by synthesizing NO. The NO regulates the kinase balance by activating PKA.

NOS1 also regulates the redox side of the balance by buffering ROS. That, in turn regulates the phosphatase balance by reducing PP1 and PP2s activity at the SR.

Our, and other’s data suggest that NOS1 signaling can also signals through direct

S-nitrosylation of RyR [16]. This increases RyR activity and SR Ca2+ release, enhancing contraction. Although still a work in progress, we have preliminary data that suggest that

NOS1 may be activated by phosphorylation by protein kinase B (aka AKT) at Serine1412

[88]. Deactivation of NOS1 may be, in part, regulated by the phosphorylation of

Serine847 by CaMKII (as shown in other tissues [89]). In addition, NOS1 regulates the

FFR response through similar mechanisms [18]. The β-AR signaling pathway also can be regulated through NOS1 signaling [13]. Preliminary data suggest that the increased Ca2+ transients observed during β-AR signaling may be due to NOS1 signaling through

Ca2+/calmodulin kinase II (CaMKII) to increase RyR2 activity via phosphorylation at

Serine2814. NO-produced via NOS1 is not the only NO involved in the β-AR signaling pathway. NOS3 also regulates the β-AR signaling pathway, but through different targets and NOS3’s signaling results in a decrease in the β-AR response.

1.4.4 NOS3 signaling

NOS3 or the endothelial NOS (eNOS) has been heavily investigated as the enzyme responsible for producing the potent endothelium-derived relaxing factor in the form of NO in the vasculature [90-93]. NOS3 is also constitutively expressed in the heart; in both endothelial cells [94] and cardiac myocytes. It is localized to the plasma membrane and co-immunopreciptates with caveolin [13, 95]. NOS3 signals through the cGMP-dependent signaling pathway. Unlike NOS1, NOS3 signaling does not regulate basal myocyte contraction or the FFR [13, 15]. Similar to NOS1, NOS3 modulates the functional response during β-AR stimulation. However, NOS3 signaling decreases the functional response to β-AR stimulation [13, 96-98] by decreasing Ca2+ influx through the L-type Ca2+ channel in the caveolae [97], increasing K+ efflux [99], or decreasing myofilaments Ca2+ sensitivity by targeting TnI [100]. The reduction in β-AR stimulation and accelerated repolarization would tend to protect against cardiac arrhythmias. NOS3 has been shown to be protective in not only the heart, but the vasculature as well. In fact,

NOS3 has been extensively investigated in regards to its protective role following chronic exercise.

1.5 Chronic Regulation

Not only does the body have the means to acutely regulate nearly every cellular process, it can adapt to chronic changes. One of the chronic changes is the progression of

HF. Heart disease does not develop overnight, but is the consequence of months and years of continual negative stress on the heart. Not all chronic regulations are detrimental adaptations. One chronic regulation that is beneficial to heart is exercise.

1.5.1 Beneficial Adaptations - Exercise

Since we were young children, we have been taught to live a healthly lifestyle, “eat healthy and exercise regularly.” It is a common phrase that most of us choose to ignore.

Figuratively speaking, “exercise” has not always been “exercise,” but simply living. Our ancestors were faced with the burden of hunting/gathering food and avoiding predators.

This style of living provided an ample amount of physical activity. In time, lifestyles slowly evolved from agriculture to industry to what we now call our modern world.

Today, we have neglected the physical necessity of survival and now live a largely sedentary lifestyle. One of the consequences of this lifestyle choice is increased cardiovascular disease involving HF. Each year, 5 million patients are diagnosed with HF in America. Thus, a new therapeutic approach is of paramount importance. As effective therapies are lacking, it is now time to pursue more radical ideas. Valuable knowledge

15 can obtained by investigating the cardiac adaptations to exercise. This new knowledge may allow for the development of novel therapeutic treatments.

1.5.2 Systemic Adaptations

Countless studies have investigated the beneficial effects of exercise. Regular exercise is associated with a lower risk for both cardiovascular and non-cardiovascular disease and increases the quality of life [2, 3, 5]. Exercise training has a wide range of actions. Regular exercise increases energetic mitochondrial biogenesis in skeletal muscle

[101-103], improve blood cholesterol levels [104], prevents bone loss [105], reduce atherosclerosis [106, 107], and is anti-inflammatory [108]. In addition, the beneficial effects of exercise have been responsible for diabetics [109, 110], patients with chronic obstructive pulmonary disease [111], patients with renal dysfunction [112] and in patients recovery from cancer [113]. In addition to systems directly affected during acute exercise

(skeletal, vasculature, etc), prolonged exercise has even been shown to improve cognitive function [114, 115] and to reduce depression and anxiety [116-118]. Exercise training has also been shown to have major effects on the heart.

1.5.3 Cardiac Adaptations

Exercise training promotes beneficial cardiac adaptations [119-121]. These adaptations are clinically relevant as exercise has been shown to reduce the risk of cardiomyopathies [122, 123]. In addition, exercise is used as a rehabilitation tool for cardiac patients improving the quality of life and decreasing mortality, in part, through the direct adaptive responses of the heart [5, 124]. Exercise enhances ventricular

16 contraction and accelerates ventricular relaxation [4, 5]. These intrinsic exercise-induced adaptations can be observed even in individual cardiomyocyte [9]. For example, ventricular myocytes from Ex mice exhibit increased force production [125-127], myocyte contraction [10, 128-130], SR Ca2+ cycling and contraction-relaxation rates [6,

9, 11, 131, 132]. Although not always observed in early studies (possibly due to weak fluorescence dyes) [10, 133-135], recent data suggest that exercise increases Ca2+ transients amplitude compared to Sed rodents. Recently, studies have focused on investigating the exercise-induced changes in excitation-contraction coupling protein profiles [12, 136-138]. These changes have been attributed to enhancement of the

SERCA2a/PLB complex that increase Ca2+ uptake and the subsequent release of Ca2+ from the SR [11, 139]. More specifically, exercise has been shown to increase phosphorylation of PLB at Serine16 (PKA site) [137, 138] or Threonine17 (CaMKII site)

[12]. Altered phosphorylation status of PLB is suggestive of altered kinase-phosphatase activity that may positively effect myocyte contraction. Others have observed increases in

SERCA expression [12, 140]. However, the exercise-induced cardiac adaptations are present in SERCA knockout mice [141], suggesting that mechanisms other than SERCA must also contribute to the beneficial cardiac adaptations of exercise. Thus, the molecular mechanisms behind these cardiac adaptations are not fully understood and studies that have examined signaling pathways are limited.

Numerous systems support the transfer of atmospheric oxygen to individual cells for cellular respiration. Pulmonary diffusion, heart function, oxygen carrying capacity, blood volume, capillary density, muscle diffusion, mitochondrial oxidative enzyme, and

17 other factors contribute to the VO2max. VO2max or maximal oxygen consumption is determined by how much oxygen one can consume, transport, and utilized during maximal excursion. It is widely accepted that the VO2max provides the best measurement to determine one’s fitness. Dysfunction of any components of oxygen transportation/utilization can disrupt the entire system. However, under normal physiological conditions in trained subjects, oxygen supply is what limits the VO2max

[142, 143]. Thus, it is the delivery of oxygen (cardiac output) that is the main determinant. Cardiac output is the product of heart rate and stroke volume. As maximal heart rate does not change with exercise training and heart rates are roughly similar between subjects during maximal excursion, the ability of the heart to eject a large stroke volume of blood limits one’s VO2max [142, 143]. Thus, any intrinsic adaptations of the myocyte to increase contraction can contribute to increases one’s VO2max. In fact, it is this increases in aerobic fitness due to having a stronger heart that has a direct correlation to longevity [144].

Another stereotypical adaptation to exercise is physiological hypertrophy [145,

146]. In contrast to pathological hypertrophy (concentric growth due to pressure overload), the heart growth eccentrically (wall thickens and chamber enlarges) during the exercise-mediated increase in heart mass and is “generally” induced via volume overload

[146]. The molecular mechanisms and signaling pathways are still under investigation, but a convincing amount of data that suggest that the cardiac remodeling in response to exercise is mediated through the insulin growth factor (IGF)/phosphoinositide 3-kinase

(PI3K)/AKT pathway [147-149]. IGF is known to stimulate postnatal cardiac growth and

18 correlates with physiological hypertrophy in human athletes [150]. IGF activates the IGF- receptor and then activates PI3K, which stimulates the generation of phosphoiniositide

3,4,5 triphosphate (PIP3). In particular the PI3K(p110α) catalytic subunit has been shown to be directly involved in physiological but not pathological hypertrophy [151]. PIP3 then activates AKT that then activates further downstream targets involved in the cell growth.

AKT has also been shown to be necessary for exercised induced hypertrophy [152]. It is interesting to note that AKT is upstream of NOS1 and can phosphorylate NOS1 at

Serine1412 [88]. The transcription factor C/EBPβ also controls exercise-induced cardiac growth. Data suggest that exercise training downregulates C/EBPβ and thereby reducing

C/EBPβ’s repression on the CITED4 transcription factor, which regulates cardiac proliferation [153]. Interesting, data suggest NOS1 may regulate the expression of

C/EBPβ [154]. Although currently there is no data that directly demonstrates the role of

NOS1 in mediating exercise-induced cardiac growth, it does interact with the known keys players (AKT and C/EBPβ).

All of the adaptations induced by exercise mentioned above depend upon the intensity of the training [11, 101, 124, 155-157]. Based on evidence from animal [11] and human studies [157], a vigorous, high intense mode of training elicits a greater improvement in aerobic capacity and is more cardio-protective. Moreover, it is well established that the higher the intensity of the exercise, the greater the beneficial effects in both healthy and diseased individuals [124, 155, 157, 158]. It is the “extra push” during high intense workouts that shifts to anaerobic metabolism. Hypothetically, it has been proposed that the shift to anaerobic metabolism stimulates the mitochondria and

19 other energy metabolism pathways to greater degree and aerobic metabolism [156]. On the contrary, recent evidence suggests that prolonged high intensity exercise may be detrimental to the heart as this results in increased fibrosis, arrhythmias, and altered metabolism [159, 160]. Similar effects have been observed in long-term endurance athletes [161]. Thus, discovering the molecular mechanisms of the beneficial versus detrimental adaptations of exercise is crucial in order to design therapies (either specific exercise protocols or pharmacological targets) that maximize the beneficial aspects of HF patients.

1.6 Detrimental Adaptations - Heart Failure

HF patients have an inefficient heart that is unable to sustain sufficient blood flow to meet the demands of the body. Similar to exercise training, HF is a chronic adaptation and is attributed to systolic (contraction phase) and/or diastolic (relaxation phase) dysfunction. Alterations in ECC proteins, nitroso-redox balance, and kinase/phosphatase balance and numerous others, summate to elicit the disease.

A general characteristic of HF is the dysregulation of Ca2+ on a beat-to-beat basis.

Alterations in ion channels that result in the inability of trigger Ca2+ to sufficiently release

Ca2+ from the SR contribute to this dysregulation [162]. In addition, there is a decrease

SR Ca2+ load [22]. This is attributed to the down regulation of SERCA mRNA/protein expression or activity and decreased phosphorylation of PLB [163-166]. The slowed Ca2+ decline and decrease accumulation of SR Ca2+ contributes to both diastolic and systolic

20 dysfunction. The mechanisms responsible for the decreased PLB phosphorylation is not completely understood, but it may be in part due to nitroso-redox disequilibrium.

As mentioned previously, maintenance of the nitroso-redox balance is important for myocardial function. The loss of this equilibrium may result in pathophysiological situations. Both animal models and human HF patients have increased production of ROS

[69-71], and/or disrupted NOS localization [72, 73]. Specifically, NOS1 translocates from the SR to the plasma membrane and localizes with caveolin-3 to decrease the β-AR response [72, 73, 167]. Overall, NOS1 appears to be beneficial. Specifically, NOS1 knockout mice do worse (adverse remodeling, contractility, mortality) after myocardial infarction [168, 169] while NOS1 overexpression is protective (infarct size, recovery of left ventricular developed pressure) after ischemia/reperfusion injury [170]. However, during HF, the translocation and altered activity disrupts the nitroso-redox balance. In addition to NOS1, NOS3 expression levels decrease during cardiomyopathies and is uncoupled, resulting in elevated ROS levels [171]. The loss or altered NOS activity accompanied with abnormal ROS levels contributes to the decline of myocardial function in disease.

Oxidative stress is associated with elevated levels of ROS and is generally the result of uncoupled cellular reactions. A defense mode to combat oxidative stress

(superoxide dismutase, catalase, and glutathinone peroxidase etc [172, 173]) is present under physiological conditions, but is decreased during HF [174]. Chronic elevated ROS levels can be disadvantageous to the myocyte by stimulating apoptosis and decrease myocyte contraction via deleterious effects on ECC [175]. The negative effects on

21 myocyte contraction may be the result of destructive effects on SERCA activity and/or increasing SR Ca2+ release [176, 177]. Both alterations contribute to decreased SR Ca2+ load (hallmark of HF as previous mentioned). Finally, another ramification is to alter the kinase/phosphatase balance.

As mentioned above, the phosphorylation of key proteins (L-type Ca2+ channel,

PLB, RyR2, TnI) via kinases enhance contraction and relengthening of the myocyte.

Furthermore, altered kinase activity, phosphatase activity, and phosphorylation status contribute to the contractile anomalies observed in HF [178-184]. One might speculate that these alterations result from increased oxidative stress. In fact, both kinase and phosphatase are activated by ROS and RNS [48, 81, 185-187]. The elevated ROS levels can activate phosphatases and decreases phosphorylation levels on select protein targets.

Thus, the increased oxidative stress from the nitroso-redox disequilibrium in HF may contribute to the altered phosphorylation statues and regulation of key ECC proteins by directly altering the kinase/phosphatase balance.

1.7 Fixing Heart Failure with Exercise

Understanding the natural/innate mechanisms by which the normal heart adapts

(remodels) to chronic exercise may identify novel therapeutic targets for the management of HF. In fact, regular exercise is beneficial for HF patients [2, 5, 188-190]. Although the benefits are vastly known, cardiac patients, whether mentally unwilling or physically unable to, do not prioritize exercise enough. Once the mechanism responsible for the beneficial action of exercise has been identified, the it may be possible to develop a

“exercise pill” [191, 192]. One could term this new-age approach: Exercise-Like Induced

Medicine (ELIM). This novel therapeutic approach would attempt to mimic exercise adaptations through medicinal intervention. Mimicking exercise through possible future manipulation therapies (gene therapy, siRNA, adenovirus, etc), one could provoke these enhancements and could potentially lead to a novel therapeutic treatment (ELIM). This treatment is not limited to the heart since exercise induces adaptations on other systems.

1.7.1 Role of NO in Exercise Adaptations

Exercise was initially deemed unsafe for heart failure patients. With the help of courageous physicians and basic scientists, now it is widely excepted that not only is exercise well tolerated by HF patients/animal models but also promotes recovery by increasing in aerobic capacity (VO2max) [5, 101, 155, 190, 193] and provides protection against primary and secondary cardiac diseases of the heart [3, 194-196]. Could NO also be involved in the beneficial effects of exercise on the heart? It is well documented that

NO plays a major role for the systematic adaptive effects of exercise.

1.7.2 Systemic Adaptations

Countless cardiomyopatheis result as the consequence of bad “plumbing” (i.e. vasculature). Over the past decade, convincing evidence suggests an improved endothelial function induced by, exercise, at least in part, to enhanced NOS3 signaling

[197, 198]. As mentioned previously, NOS3 is the enzyme responsible for producing the potent endothelium-derived relaxing factor in the form of NO [90-93]. NOS3 is stimulated during exercise in several ways. First, the increased shear stress during exercise through the vast network of arteries/capillary/veins simulates NOS3 mediated 23

NO production [197, 199]. Secondly, NOS3 is triggered by the oxidative stress observed during exercise [198]. Thirdly, NOS3 is stimulated when oxygen availability is limited

(during anaerobic exercise) [200]. All of the exercise-induced increases of vascular

NOS3 expression and activation (eNOS Ser-1177 phosphorylation) are believed to protect endothelial function and/or aid in vasodilation [197-202].

Exercise-mediated NOS regulation of the vasculature has been observed in other organs, such as the lungs and the penis. For example, exercise increased NOS3 mRNA and protein levels in the lungs [203]. Additionally, exercise induced an increase in protein expressions of NOS3 and NOS1 to improve penile erection [204]. In skeletal muscle, exercise training has been shown to increase NO produced via NOS1 [205] and total NOS activity [206]. In addition, during acute mild exercise, NOS1 mediated regulation of skeletal muscle perfusion limits fatigue [207, 208]. However, perfusion is normalized during sustained exercise [209]. Thus, the beneficial effects of exercise- induced increases in NO are not limited to the heart.

Another important pool of NO is the circulating nitrites (and the other NO metabolite conjugate, nitrate). These NO metabolites are reduced to NO and nitrosothiols.

Interestingly, the act of exercising increases blood nitrite levels [210, 211]. This excess of available NO is particular beneficial during periods of ischemia or hypoxia [212].

Interestingly, the exercise-induced increases in blood nitrite levels have recently been shown to be beneficial to heart [211].

1.7.3 Cardiac Adaptations

A complete understanding of the beneficial effects of NO induced by exercise remains to be elucidated. In addition to the circulating nitrite levels induced by exercise also protect myocardial infarction (MI), but also stored nitrite levels in the heart [211].

Exercise mediated NOS3 regulation is also cardioprotective against MI [198, 213].

As far as NOS1 is concerned, limited research exists examining the effects of

NOS1 due to exercise on cardiac function. One cardiac study measured increased protein expression of NOS1 in the atria following a training period [214]. Interestingly, this study concluded that change of NOS1 played an important role in establishing bradycardia

(common phenotype) following chronic exercise. However, the role of NOS signaling in modulating the increased ventricular myocyte contraction, aerobic fitness (VO2max), and physiological hypertrophy observed following exercise have not been studied. Therefore, we investigated the contribution of changes in NOS1 to cardiac adaptive response to exercise.

1.8 Specific Aims, Objectives, and Rationales

Ventricular myocytes from Ex mice exhibit increased SR Ca2+ cycling and contraction-relaxation rates [6, 9, 11]. The molecular mechanisms behind these adaptations are not fully understood and studies examining signaling pathways are limited. An important signaling molecule modified over a training period in skeletal muscle is NO produced via NOS1. Intriguingly, NOS1 is also constitutively expressed within cardiac myocytes and its signaling is strikingly similar to the beneficial 25 adaptations to the heart following exercise. That is, NOS1 signaling modulates myocytes contraction/relaxation via enhanced SR Ca2+ cycling. However, limited studies have investigated the role of nitric oxide, especially NOS1 signaling, on the positive effects of exercise on cardiac myocyte function. Therefore, the objective of this thesis is to determine if NOS1 signaling contributes to the beneficial cardiac effects of exercise.

Overall, we hypothesize that exercise enhances NOS1 signaling to increase the strength of contraction and accelerate relaxation. To test this hypothesis, mice were assigned to either high intense aerobic treadmill training 5 days a week for 8 weeks or sedentary groups.

1.8.1 Determine if exercise enhances NOS1 protein expression and NOS1 dependent

NO bioavailibity in murine ventricular myocytes

Exercise has previously shown to induce the upregulation of NOS1 protein in the in skeletal muscle [205] and in the atria [214]. To our best knowledge, no one has investigated NOS1 protein expression in the ventricles following a high intense interval treadmill training protocol. First we want to determine via Western Blot analysis whether exercise increases NOS1 protein expression in the isoloated ventricular myocytes. Next, we would want to see if any protein differences between Ex and Sed correlates with alterations in NO bioavailability. This will be determined via DAF-2 AM fluorescence in isolated myocytes. In addition, aerobic fitness will be measured via VO2max and physiology hypertrophy will be measured via the heart weight-to-tibia length ratio. These experiments should confirm whether or not 1) our exercise protocol is sufficient to induce a training effec and 2) that exercise modulates NOS1 in the ventricular myocytes.

1.8.2 Determine if NOS1 signaling contributes to the exercise–mediated enhancement of murine myocyte contraction.

NOS1 signals to increase SR Ca2+ cycling and thereby enhances myocyte contraction. To determine if NOS1 signaling plays a role in the enhanced myocyte contraction observed following exercise, simultaneous measurements of Ca2+ transients and cell shortening in the presences and absence of acute NOS1 inhibition (SMLT) in isolated myocytes from both Ex and Sed mice was preformed. These experiments were designed to determine the role of NOS1 signaling by examining the effect of NOS1 inhibition in Ex and Sed.

1.8.3 Determine the molecular mechanisms of the NOS1-mediated increase in murine contraction.

NOS1 is localized to the SR and signals via specific compartmentalized molecular pathways that target phospholamban (PLB) to enhance SR Ca2+ uptake and RyR2 to increase SR Ca2+ release. Several methods will be used to determine if this pathway is enhanced following exercise. First, SR Ca2+ load and fractional release will be measured with application of 10 mM caffeine in Ex and Sed ± acute NOS1 inhibition. Next, the role of the exercise-induced NOS1 signaling on modulating SR Ca2+ uptake will be determined by measuring PLB phosphorylation via Western Blot analysis in Ex and Sed

± acute NOS1 inhibition. These experiments will determine if the downstream targets of

NOS1 signaling plays a significant role in the enhanced SR Ca2+ cycling observed with exercise.

1.8.4 Investigate the exercise-mediated cardiac adaptations in NOS1KO mice

If NOS1 signaling plays a major role in the enhanced myocyte contraction observed following exercise, then training the NOS1KO mice should elicit a decreased effect of exercise on myocyte contraction. Since the NOS1KO mouse is a global KO and the loss of NOS1 in the skeletal muscle and other tissue may alter training, weekly training speeds will be determined separate to that of the WT mice by measuring VO2peak biweekly. Following the 8 week training protocol, simultaneous measurements of Ca2+ transients and cell shortening in Sed-NOS1KO and Ex-NOS1KO myocytes will be used to determine the effects of exercise without NOS1. In addition, aerobic fitness will be measured via VO2max and physiology hypertrophy will be measured via the heart weight- to-tibia length ratio. These experiments could strengthen the case that NOS1 is important for the full beneficial effects of exercise on the heart.

1.8.5 Determine if NOS1 signaling contributes to the exercise-mediated enhancement of contraction in a large mammal model

It is well documented that contractility is different between rodents and larger mammals (e.g., dog, humans). Therefore, we will evaluate the contribution of the NOS1 signaling pathway to the cardiac adaptive response to exercise training in a canine model.

Following a 10 week aerobic exercise training protocol, simultaneous measurements of

Ca2+ transients and cell shortening in the presences and absence of acute NOS1 inhibition

(SMLT) in isolated myocytes from both exercise-trained and sedentary dogs will be preformed. These experiments will determine if NOS1 signaling has similar role in mediating the response to exercise in both small and large mammals. 28

Figure 1 Excitation – contraction coupling in cardiac myocytes [1]. This cartoon depicts the necessary steps to couple an electrical signal to the contraction of a myocyte. The red arrows represent the influx and upstroke of the Ca2+ transient to activate the myofilaments. The green arrows represent the decline and efflux of the Ca2+ transient to contribute to relaxation.

Figure 2 NOS1 and NOS3 localization and signaling pathways [14]. This cartoon depicts the NOS1 localized to the SR and signaling via the cGMP-independent pathway and NOS3 localized to the caveolae and signaling via the cGMP-dependent pathway.

Chapter 2: The Neuronal Nitric Oxide Synthase is Indispensable for the Cardiac Adaptive Effects of Exercise

2.1 INTRODUCTION

Regular exercise has many beneficial effects [3], including direct adaptations of the heart that result in greater aerobic fitness (VO2max), physiological hypertrophy, enhanced contraction, and accelerated relaxation. These adaptations have clinical implications as exercise has been shown to decrease the risk for the development of cardiomyopathies [2]. In addition, exercise is used as a rehabilitation tool for cardiac patients to improve the quality of life and to decrease mortality [5]. These intrinsic exercise-induced adaptations can be observed at the myocyte level [9]. For example, ventricular myocytes from exercise-trained mice exhibit increased size, greater contraction due to enhanced sarcoplasmic reticulum (SR) Ca2+ cycling, and faster contraction-relaxation rates. Previous studies have proposed that the contractile adaptations of exercise result from increases in SR Ca2+ATPase (SERCA) expression [12,

140]. However, it has been shown that the exercise-induced cardiac adaptations are still present in SERCA knockout mice [141], suggesting that mechanisms other than SERCA must also contribute to the beneficial cardiac adaptations of exercise.

In skeletal muscle, exercise training has been shown to increase nitric oxide (NO) produced via neuronal NO synthase (NOS1) [205]. NOS1 is also constitutively expressed within ventricular myocytes, and its signaling leads to enhanced contraction and accelerated relaxation via increased SR Ca2+cycling [13-16, 18]. As such, the contractile effects of NOS1 signaling are similar to the contractile adaptations of exercise. However, the contribution of NOS1 signaling to exercise-induced cardiac myocyte adaptations has not been investigated.

Therefore the purpose of the study was to assess whether NOS1 signaling contributes to the exercise-induced adaptations in the myocyte. We hypothesize that exercise training will 1) increase NOS1 expression levels and NO bioavailability, and 2) increase contraction and accelerate relaxation via enhanced SR Ca2+ cycling due to amplified NOS1 signaling in isolated ventricular myocytes.

2.2 METHODS AND MATERIALS

All the 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.

2.2.1 Murine exercise protocol

A high intensity aerobic interval treadmill training protocol was adopted [215].

C57Bl/6 mice (Jackson Laboratories, Bar Harbor, Maine), 5 months of age at sacrifice, underwent treadmill (Columbus Instruments, Columbus, OH) training 5 days a week for

8 weeks starting at 30 min/day and increased to 80 min. Mice were challenged at a high

32 intensity fast pace for 4 minutes followed by 1 minute of low intensity recovery pace

(Table 1). This interval set was repeated until the designated time was up. An adequate warm-up of 10 minutes and cool down of 4 minutes was instituted. A certified supreme mini-treat (Bio-Serv, Frenchtown, NJ) was given to all participants and age-matched

Sedentary (Sed) counterparts. Exercise effects are maintained for 2 weeks [135]. Thus to avoid detraining, we isolated ventricular myocytes from Sed and Ex mice within 1 week of completing our 8 week training protocol.

2.2.2 Canine exercise protocol

The dogs in the exercise-training group ran on a motor-driven treadmill for 10 weeks, 5 days/week at approximately 70-80% of maximum heart rate. The exercise training protocol has been previously described [216-218]. Briefly, exercise intensity and duration progressively increased as follows: 1st week, 20 min at 4.8 kph/0% grade; 2nd week, 40 min at 5.6 kph/10% grade; 3rd week, 40 min at 6.4 kph/10% grade; 4th week, 60 min at 6.4 kph/10% grade; 5th week, 60 min at 6.4 kph/12% grade; 6th week, 75 min at

6.4 kph/12% grade, 7th week, 90 min at 6.4 kph/12% grade; 8th – 10th weeks, 90 min at

6.4 kph/14% grade. Each exercise session included 5-min warm-up and 5-min cool-down periods (running at a low intensity, 0% grade and speed, 4.8 kph). The dogs in the sedentary group were placed in transport cage for equivalent time periods but without exercise. The effectiveness of the exercise-training program was evaluated by measuring the heart rate response to submaximal (comparison before and after the 10-week exercise or 10-week sedentary period - Table 2) as previously described [216-218].

2.2.3 VO2peak and VO2max testing protocol and training speeds

A metabolic chamber (Columbus Instruments) and Oxymax analyzer (Columbus

Instruments) were used for measurements. A protocol was adopted to measure peak O2 consumption (VO2peak) and maximal O2 consumption (VO2max) [219]. Briefly, mice underwent a brief warm-up with 2 min at 10 m/min at 0º incline, 2 min at 12 m/min at

10º incline, and 2 min at 15 m/min at 20º incline. Then, the treadmill speed increased 1 m/min at 20º incline every 30 sec. VO2peak was defined as a plateau in the VO2 even though intensity increased. VO2max was defined as the absolute maximal VO2 with a respiratory exchange ratio (RER) above 1 and exhaustion was reached (mice unwilling to run and neglecting shock). Weekly training speeds were determined by taking ~90% of the speed reached for the VO2peak.

2.2.4 Murine Cardiomyocyte isolation

Post-training, Ex mice were sacrificed and compared to age-matched Sed mice.

Ventricular myocytes were isolated from anesthetized mice via pentobarbital, as previously described [18, 220, 221]. Briefly, the heart was cannulated and hung on a

Langendorff apparatus. It was then perfused with Ca2+ free tyrode solution (see solutions and drugs below) for 4 min. The solution was then switched to a tyrode solution containing Liberase Blendzyme II (0.077 mg/ml) (Roche Applied Science, Indianapolis,

IN). After 3-5 min, the heart was taken down, the ventricles minced, and myocytes were dissociated by trituration. Subsequently the myocytes were filtered, centrifuged, and resuspended in tyrode solution containing 200 μmol/L Ca2+. Myocytes were used within

6 hrs of isolation. 34

2.2.5 Measurement of myocyte Ca2+ transient and shortening

Simultaneous Ca2+ transient and shortening measurements were performed at room temperature, as previously described [18, 220, 221]. Briefly, myocytes were loaded at room temperature with Fluo-4 AM (10 μmol/L, Molecular Probes, Eugene, OR) for 30 min. An additional 30 min were allowed for intracellular de-esterification. The instrumentation used for cell fluorescence measurements was a Cairn Research Limited

2+ (Faversham, UK) epifluorescence system. [Ca ]i was measured by Fluo-4 epifluorescence with excitation at 480±20 nm and emission at 535±25 nm. The illumination field was restricted to collect the emission of a single cell. Data are expressed as ΔF/F0, where F is the fluorescence intensity and F0 is the intensity at rest.

Data for cell shortening were collected using a video edge detection system (Crescent

Electronics). Myocytes were stimulated at 1 Hz for mice and 0.5 Hz for dogs via platinum electrodes connected to a Grass Telefactor S48 stimulator (West Warwick, RI).

2.2.6 Canine Cardiac Myocyte isolation

In brief, heartworm free mongrel canines were anesthetized and the heart was excised. Septum chunks were harvested and placed in cardioplegia solution (in mmol/L:

140 NaCl, 25 KCl, 1 MgCl2, 10 glucose, 5 HEPES, pH 7.4). To isolate myocytes the 5-5-

5-3-3-3-3-3… chunk method was used. Chunks were minced into smaller cube chunks with the height, length, and width of roughly 1 cm. Chucks were digested with Liberase

Blendzyme II (0.077 mg/ml) (Roche Applied Science, Indianapolis, IN) in a minimum essential medium eagle (MEM) enzyme solution that consisted of (in mmol/L): 2.4

NaHCO3, 3.4 MgCl2, 10 taurine, 0.009 BSA, 0.0008 insulin, 2 pyruvic acid, 0.005 35 trypsin, 0.05 CaCl2, aerated with 95 O2/5% CO2, pH 7.35. After gently swirling for 5 minutes at 37º, chunks were split in half and gentle swirled at 37º for an additional 5 minutes. Chunks were minced in half again and myocytes were dissociated by titration.

Solution was discarded and additional MEM-enzyme solution was added. Chunks were cut again and swirled for 5 minutes and myocytes were dissociated by titration. Myocytes were collected. Additional MEM-enzyme solution was added. Chunks were cut again and swirled for 3 minutes. Myocytes were dissociated by titration and collected. Further cutting, 3 minute swirling at 37º, titration, and myocyte collection is repeated until cells look good (usually round 5). Subsequently, the myocytes were filtered, centrifuged, and resuspended in tyrode storage solution containing 1 mmol/L Ca2+. Myocytes were used within 6 hrs of isolation.

2.2.7 Measurement of NO production with DAF-2 AM fluorescence

Isolated myocytes were loaded at room temperature with DAF-2 AM (NO- dependent fluorescent dye - 10 µM) for 20 minutes and allowed to de-esterify for an additional 20 minutes. Fluorescence was observed on an Olympus Fluoview 1000 laser scanning confocal microscope in x-y mode with the pinhole set at 400 µm. Fluorescence data was acquired for 3 seconds every 30 seconds for 15 minutes. To correct for photobleaching, unstimulated myocyte fluorescence was measured for 15 minutes and the slope of the fitted line was added back to all experimental groups. Data was normalized to F0. Myocytes were stimulated at 1 Hz.

2.2.8 Force frequency response

Ca2+ transients and shortening measurements were obtained at stimulating frequencies of 0.2, 0.5, 1, and 2 Hz until a steady state was reached.

2.2.9 Post rest potentiation

Ca2+ transients and shortening amplitudes were obtained by stopping stimulation with pauses of 5, 10, 20, 30, and 60 seconds. Steady state was reached between each pause and the first twitch following the pause was used for analysis.

2.2.10 SR Ca2+ load and SR Ca2+ fractional release

SR Ca2+ load was measured by the addition of 10 mM caffeine for 10 seconds.

The caffeine-induced Ca2+ transient amplitude was defined as the SR Ca2+ load. Whereas,

SR Ca2+ fractional release is defined as the fraction of the basal Ca2+ transient amplitude divided by the caffeine induced Ca2+ transient amplitude [18, 222].

2.2.11 Western Blot analysis

Homogenized ventricular myocytes were used to measure NOS1 protein expression (Santa Cruz, Santa Cruz, CA, 1:10,000) Total protein was normalized to

GAPDH (Cell Signaling Technology, Danvers, MA, 1:20,000). Western blot analysis, were preformed as previously described [18]. Homogenized ventricles were used to measure specific phospholamban phosphorylation at serine16 and threonine17 (Badrilla,

Leeds, UK) with phospho-specific antibodies and normalized to GAPDH and total phospholamban (Custom - Zymed, Invitrogen; Carlsbad, CA).

2.2.12 Solutions and drugs

Normal Tyrode (NT) solution consisted of (in mmol/L): 140 NaCl, 4 KCl, 1

MgCl2, 1 CaCl2 (2 CaCl2 for canine), 10 glucose, 5 HEPES, pH 7.4 adjusted with NaOH or HCl. A specific NOS1 inhibitor, S-methyl-L-thiocitrulline, which has been shown to have no effect in NOS1KO [18], (SMLT, 10 µM, Calbiochem, La Jolla, CA), was prepared each day from frozen aliquots. All chemicals were from Sigma (St. Louis, MO) except where indicated. PKA was inhibited by PKI 14-22 amide, myristoylated (5 µM,

Tocris Biosciences, Bristol, United Kingdom. Protein phosphatase 1 and 2a was inhibited by okadaic acid (OA, 5 µM, LC Laboratories, Woburn, MA). Peroxynitritie was inhibited with a peroxynitrite decomposing catalyst (FeTPPS, 5 µM, Calbiochem, La Jolla, CA).

2.2.13 Statistical Analysis

Data are presented as mean±SEM. Differences between two groups were evaluated for statistical significance (P< 0.05) by unpaired Student's t tests or by one way or two way (repeated measurements of one factor) ANOVA for multiple groups.

2.3 RESULTS

2.3.1 Exercise induced adaptations

Following an 8 week high intense, aerobic interval treadmill training, exercise- trained (Ex) mice (C57Bl/6) had elevated VO2max levels and physiological hypertrophy

(Table 3) as compared to sedentary control (Sed) mice. Therefore, the training protocol had achieved a state of fitness and produced a “physiological” ventricular hypertrophy.

2.3.2 Exercise increases myocyte NOS1 protein expression, NO bioavailability, and contraction/relaxation

Exercise training elicited a significant increase in NOS1 expression in isolated ventricular myocytes from Ex mice compared to Sed mice (Fig 3A).This was accompanied by an increase in NO production in Ex myocytes (Fig 3B), that could be normalized by acute NOS1 inhibition (SMLT, 10 µM).

In agreement with previous studies (4, 14) Ex myocytes also exhibited a greater contraction (observed as increased Ca2+transient and shortening amplitudes) and a faster

2+ relaxation (measured as time to 50% relaxation, RT50) (faster Ca transient and relengthening RT50, Fig 3C-G). Remarkably, acute NOS1 inhibition (+SMLT) normalized

Ex myocyte contraction (Ca2+ transients and cell shortening amplitude) and Ca2+ decline to Sed myocyte levels (Fig 3D-F). Furthermore, the force-frequency relationship (FFR) was enhanced via Ex and normalized with acute NOS1 inhibition (Fig 4). These data are consistent with the effects of Ex [10, 11] and NOS1 signaling [15, 18] on FFR.

2.3.3 Effects of Ex and NOS1 signaling on SR Ca2+ cycling

Since exercise enhances SR Ca2+cycling [9], SR Ca2+ load and SR Ca2+ fractional release (twitch Ca2+ amplitude / SR Ca2+ load) were examined. Ex myocytes had both an increased SR Ca2+ load (similar to a previous study) [6], and an increased SR Ca2+ fractional release compared to Sed myocytes. These differences were normalized by acute NOS1 inhibition (Fig 5A and 5B). SR Ca2+ cycling was further examined with post-rest potentiation. Post rest potentiation is the enhanced contraction observed after a period of rest. It gives insight into SR Ca2+ uptake and release [223-225]. Shown in

Figure 6, Ex myocytes reached a greater Ca2+ transient and shortening amplitude compared to Sed on the first twitch following short periods of rest. This can be contributed to the accelerated Ca2+ uptake and/or faster RYR2 recovery. To better understand the contribution of SR Ca2+ and the rate at which the SR is replenished, SR

Ca2+ loads were measured over the same time course (Fig 6C). Acute NOS1 inhibition with SMLT trended to decrease Ca2+ transient and shortening amplitudes following short periods of rest, but was similar at longer rest periods. Thus, the Ex-induced increases in myocyte SR Ca2+ cycling resulted from enhanced NOS1 signaling.

Accelerated SR Ca2+ uptake occurs via increasing phosphorylation of phospholamban (PLB). Previous studies have shown that Ex [137, 138] and NOS1 signaling increase PLB phosphorylation [17, 18, 226]. In the present study, Ex myocytes

(compared to Sed myocytes) had increased PLB serine16 phosphorylation, which is the

PKA site, but not threonine17 phosphorylation, the CaMKII site (Sed: 0.24±0.03, Ex:

0.27±0.07 A.U.). Once again, acute NOS1 inhibition normalized Ex serine16 phosphorylation to Sed levels (Fig 5C). There was no effect of NOS1 inhibition on threonine17 phosphorylation (Sed+SMLT: 0.28±0.05, Ex+SMLT: 0.28±0.05 A.U.).

Thus, these data suggest that the molecular mechanism responsible for the enhanced SR

Ca2+ cycling induced by exercise is a NOS1 mediated increase in PLB phosphorylation.

The increase in serine16 phosphorylation with Ex suggest a change in PKA and/or phosphatase activity. Hence, we investigated the roles of PKA and phosphatase 1 and 2a on Ex and Sed myocyte contraction. Interestingly, contraction in Ex myocytes was abrogated to Sed levels with either phosphatase inhibition (okadaic acid, OA) or PKA

40 inhibition (PKI) (Fig 7). Thus, these data suggest that exercise shifts the kinase/phosphatase balance in myocytes.

2.3.4 Effects of training NOS1 deficient mice

Since acute NOS1 inhibition reversed the adaptive contractile effects of exercise in WT mice, we wanted to examine the effects of Ex in a model deficient in NOS1. Thus, the cardiac effects of a similar exercise training protocol were evaluated in NOS1 knockout (KO) mice (Table 1). Surprisingly, Ex-NOS1KO mice did not exhibit any of the cardiac adaptations as observed in WT mice. That is, Ex-NOS1KO mice did not have increased VO2max or hypertrophy compared to Sed-NOS1KO mice (Table 3).

Furthermore, contraction, FFR, and post-rest potentiation were not increased in Ex-

NOS1KO myocytes (Fig 8 and 9). Surprisingly, we observed Ca2+ mismanagement in these myocytes. That is, Ex-NOS1KO myocytes had significantly decreased Ca2+

2+ 2+ transient amplitudes, SR Ca loads, and slowed Ca transient RT50 (Fig 8). Thus, exercise training did not produce beneficial cardiac adaptations in NOS1KO mice and actually caused deleterious effects on myocyte Ca2+ handling.

2.3.5 Effects of Ex and NOS1 on canines

It is well documented that contractility is different between rodents and larger mammals (e.g., dog, humans). Therefore, we evaluated the contribution of the NOS1 signaling pathway to the cardiac adaptive response to exercise training in a canine model.

Acute NOS1 inhibition (SMLT) had similar effects in isolated canine myocytes as were noted for murine myocytes (Fig 10). These data suggest a more universal role for

41 exercise induced enhancement of NOS1 signaling in both large and small mammalian species.

2.3.6 Enhanced role of NOS1 and its downstream targets with Ex

Since NOS1 inhibition normalized contractile function in Ex myocytes to Sed levels, the role of NOS1 with Ex was further investigated. NOS1 signals, in part, via peroxynitrite [18, 48]. FeTPPS, a peroxynitrite decomposition catalyst, had a similar effect on contraction as NOS1 inhibition in both Ex and Sed murine myocytes (Fig 11).

However, the functional effects of FeTPPS were much greater in Ex vs Sed myocytes

(Fig 12). This is consistent with the contractile effects of NOS1 inhibition (SMLT in murine and canine myocytes), PKA inhibition with PKI, and phosphatase inhibition with

OA (smaller effect consistent with the kinase/phosphatase shift) (Fig 12). NOS1 inhibition also had a greater effect on its downstream target PLB. Specifically, NOS1 inhibition resulted in a greater decrease in serine16 PLB phosphorylation in Ex vs Sed myocytes (-0.40±0.05 vs -0.18±0.03 A.U., P<0.05). Thus, these data suggest that the role of NOS1 and its downstream signaling targets are the major mediators in the enhanced contraction and accelerated Ca2+decline produced by exercise training.

2.4 DISCUSSION

The molecular mechanism(s) by which the heart adapts to regular exercise is/are not completely known. This study demonstrates that NOS1 signaling plays an essential role in these adaptations via: 1) increased VO2max, hypertrophy, and contraction in WT but not NOS1KO mice; 2) increased ventricular myocyte NOS1 expression and NO

42 production; and 3) normalization and greater contractile effects of inhibiting NOS1 (and its downstream targets) in Ex myocytes.

2.4.1 The role of NOS1 signaling in the exercise induced beneficial cardiac adaptations

The health benefits of exercise and the adverse consequences of inactivity are widely recognized. Regular exercise is associated with a lower risk for both cardiovascular and non-cardiovascular disease and increases the quality of life [2, 3, 5]. It may then be possible to design therapies (either specific exercise protocols or pharmacological targets) that maximize the beneficial aspects of exercise. Thus, it is imperative that the molecular mechanisms responsible for the beneficial cardiac effects of exercise are identified.

Exercise training produces physiological hypertrophy and improvements in

VO2max and myocyte contraction in both mice [11] and humans [5]. In the present study, in marked contrast to WT mice, aerobic interval training did not elicit these adaptations in NOS1KO mice (Table 3, Fig 3 & 8). Since VO2max is limited by maximal stroke volume [143], the lack of improvement in trained NOS1KO mice may be due to decreased myocyte contraction caused by NOS1 ablation. It is widely accepted that physiological hypertrophy is dependent upon the insulin-growth factor/PI3K/AKT pathway [148]. AKT is able to phosphorylate NOS1 [88], and our current data suggests that NOS1 is also essential for physiological hypertrophy and possibly downstream in the

AKT pathway. Overall, our observations strongly suggest that the NOS1 signaling pathway plays an essential role in cardiac adaptation to exercise.

Increased SR Ca2+ cycling and greater fractional shortening are also widely accepted as an adaptive response to exercise [6, 9] (Fig 3, 5, 6, 7, and 10). Previous studies focused on exercise-induced changes in excitation-contraction coupling protein profiles (i.e. SERCA2a) [12, 136-138]. The present study expands these findings, demonstrating that the NOS1 signaling pathway is also a key component in the exercise- induced contractile adaptations (Fig 3, 10 and 12). NOS1, which is localized to the SR, modulates SR Ca2+cycling by targeting PLB (Fig 2). Studies have shown that increased

PLB phosphorylation is responsible for the exercise induced increase in SR Ca2+ cycling

[137, 138]. Our results extend this observation by demonstrating that enhanced NOS1 signaling is responsible for this effect. We believe that the molecular mechanism for the increased phosphorylation is a shift in the kinase/phosphatase balance (Fig 7). These data are consistent with previous reports that demonstrated increased PKA activity with Ex [6] and NOS1 signaling activating PKA [17, 48] and inhibiting phosphatase activity [17].

While Ex has been shown to increase NOS3 expression [201], NO produced via NOS3 does not contribute to the faster relengthening or the other observed contractile effects reported in the present study since NOS3 only modulates contractile function during β-

AR stimulation and not basal function [97, 227]. Indeed, basal NO bioavailability was similar between Ex and Sed myocytes after NOS1 inhibition (Fig 3B). Taken together, these data suggest that exercise training increases contraction and accelerates Ca2+decline at baseline due to augmented NOS1 and its downstream targets (Fig 12).

2.4.2 Training NOS1 deficient mice results in Ca2+mishandling

Not only did exercise training fail to induce adaptive cardiac effects (VO2max and hypertrophy) in the NOS1KO mice, but actually provoked detrimental actions on myocyte Ca2+handling (Fig 8). As this is a global knockout, it is difficult to delineate the mechanism(s) responsible for this adverse cardiac response to exercise training. For example, the contribution of NO produced via NOS1 in the skeletal muscle cannot be overlooked. In fact, mice deficient in NOS1 have increased skeletal muscle fatigue due to an immediate decrease in vasodilation after brief, mild exercise`e even though the strength of the muscle was sustained [208]. However, NOS1 signaling may not limit flow during sustained exercise [209]. As such, an increased vulnerability to fatigue could have reduced the exercise capacity (i.e., weekly training speeds) and thereby, potentially limiting the exercise-induced cardiac adaptations. However, this seems unlikely as training intensity was matched to similar levels in both the WT and NOS1KO mice (i.e., mice were trained at ~90% of the speed of the peak oxygen consumption (VO2peak) (Fig

13). Since exercise induced cardiac adaptations are dependent upon training intensity

[11], similar intensities between groups one would expect that the exercise training effects should yield similar cardiac adaptive responses in both WT and NOS1KO mice, regardless of any potential skeletal muscle fatigue. However, only the WT mice exhibited exercise-induced enhancement on VO2 peak.

In a similar manner, NOS1 signaling also increases coronary blood flow [228].

Thus, NOS1KO mice may not have been able to increase coronary blood flow to match the increased myocardial oxygen consumption during exercise, resulting in ischemia and

45 enhanced superoxide production [229]. Hence, implementing the 8 week training protocol in NOS1KO mice may have potentially exacerbated the already high cardiac superoxide levels [85, 230, 231] culminating in deleterious effects on SR Ca2+ cycling.

These data provide further evidence that NOS1 is indispensible for the adaptive cardiac effect of exercise.

2.4.3 Beneficial effects of exercise

By understanding the natural/innate mechanisms by which a normal heart is remodeled into an athlete’s heart, one might gain the knowledge necessary to design therapies to heal the diseased heart. Alternates to exercise must be identified to treat those cardiac patients that are mentally unwilling or physically unable to exercise. This might be achieved by pharmacological approaches to target the beneficial signaling pathways that are activated by exercise training to mimic “exercise in a pill” [191, 192]. One could term this new-age approach: Exercise-Like Induced Medicine (ELIM). Our results showing that NOS1 is indispensible for the beneficial cardiac effects of exercise suggest that the manipulation of NOS1 protein expression or activation of components of the

NOS1 signaling pathway might represent one such pharmacological approach. In this regards, it is interesting that studies have shown that NOS1 knockout mice do worse

(adverse remodeling, contractility, mortality) after myocardial infarction [168, 169] while

NOS1 overexpression is protective (infarct size, recovery of left ventricular developed pressure) after ischemia/reperfusion injury [170]. These results further suggest that NOS1 overexpression may mimic the beneficial effects of exercise and could potentially be a novel therapeutic (ELIM) treatment.

In conclusion, as far as we are aware, the present study is the first to show that

NOS1 signaling contributes to the adaptive cardiac effects of exercise. Specifically, exercise increases ventricular myocyte NOS1 expression and NO bioavailability, which is essential for aerobic fitness, hypertrophy and enhanced contraction/relaxation.

Weeks Incline Total Number Fast Recover Fast Pace Recover (°) Daily of Sets Pace y Pace (m/min) y Pace Time (m/min) (m/min) NOS1KO (m/min) (min) WT WT NOS1KO 1 5 40 8 18 10 16 10 2 10 50 10 20 10 17 10 3 15 60 12 22 11 17 10 4 20 70 14 24 11 18 11 5 20 80 16 26 12 19 11 6 20 80 16 28 12 20 11 7 20 80 16 30 13 21 12 8 20 80 16 32 13 22 12

Table 1 Aerobic Interval Treadmill Training Protocol for WT and NOS1KO mice. Treadmill incline in degrees (º), time in minutes (min), and treadmill belt speed in meters/minute (m/min).

PRE POST Baseline Peak Baseline Peak Exercise Exercise HR (beats/min) Ex 125.6 ± 8.5 193.2 ± 6.8+ 114.8 ± 8.4* 179.6 ± 8.4+* + + Sed 125.0 ± 12.7 196.5 ± 10.4 130.5 ± 6.9 203.0 ± 12.8 HRV (ln ms2) Ex 7.04 ± 0.28 0.94 ± 0.42+ 8.48 ± 0.39* 3.08 ± 0.56+* Sed 7.02 ± 0.84 1.40 ± 0.37+ 6.62 ± 0.39 1.20 ± 0.50+

Table 2 The Canine Heart Rate and Heart Rate Variability Responses to Acute Exercise. HR = heart rate, HRV = heart rate variability measured as the high frequency component of the R-R interval variability (0.24 to 1.04 Hz), EX = 10 week exercise train (n = 5); Sed = 10 week sedentary period (n = 4), Pre = before 10 week exercise or sedentary period, Post = after 10 week exercise training or sedentary period. + P <0.05 Basleine vs. Peak exercise; * P < 0.05 Pre vs. Post

Sed Ex Sed- Ex- NOS1KO NOS1KO

Heart weight (mg) 121 ± 2 129 ± 3 * 112 ± 3 111 ± 4 Body weight (g) 22.2 ± 0.4 20.4 ± 0.2 * 21.8 ± 0.5 20.2 ± 0.5 * Heart to body ratio (mg/g) 5.5 ± 0.1 6.3 ± 0.1 * 5.2 ± 0.1 5.5 ± 0.2 Tibia length (mm) 22.0 ± 0.1 21.9 ± 0.1 21.0 ± 0.2 21.2 ± 0.4 Heart to tibia ratio (mg/mm) 5.8 ± 0.1 6.2 ± 0.2 * 5.4 ± 0.1 5.2 ± 0.2 -0.75 VO2max (mL/kg /min) 52.7 ± 2.9 62.0 ± 1.4 * 50.4 ± 2.4 51.1 ± 1.6

Table 3 Physiological hypertrophy and maximal oxygen consumption (VO2max). Summary data of VO2max (n=4-6 mice), heart weight to body weight ratio and heart weight to tibia length (n=12mice) in exercised (Ex) and sedentary (Sed) WT and NOS1KO mice.*P<0.05 vs corresponding control.

A NOS1 Protein Expression B NO Production 2.0 Sed 5 Sed Ex * Ex 4 NOS1 Sed + SMLT GAPDH 0 Ex + SMLT

3 1.5 *

F/F A.U. 2 

1 (DAF-2A fluorescence) 0 1.0 Sed Ex 0.0 2.5 5.0 7.5 10.0 12.5 15.0 Time (min)

2+ 2+ 2+ C Ca Transients D Ca Transient Amplitude E Ca Transients RT 50 Sed * Ex Sed 1.5 300 Ex Sed + SMLT

Sed + SMLT 0 * 1.0 Ex + SMLT 275

Ex + SMLT F/F 1.0  250

0.5 (ms) Time F/F 0 225 250 msec 0.0 0

F G Shortening Traces Shortening Amplitude Relengthening RT 50 4 * * 350 * 2% 3 325 RCL 2 300

% RCL % 275 1 (ms) Time 250 0 0

Figure 3 Exercise increases myocyte NOS1 expression, nitric oxide (NO) production and enhances myocyte contraction, which is normalized by acute NOS1 inhibition. A) Summary data of NOS1 protein expression (A.U.–arbitrary units), n=12 mice/group. B) Summary data of NO production over a 15 minute time period (±acute NOS1 inhibition, SMLT, dashed),n=19-26 cells/3 hearts. C) Representative traces of Ca2+ transients and shortening. Summary data of Ca2+ transient amplitudes (D), Ca2+decline to 50% of its peak (RT50)(E), shortening amplitudes (F), and relengthening RT50 (G)in exercise (Ex- grey) and sedentary (Sed-black)myocytes (±acute NOS1 inhibition, SMLT, striped), n=39-42 cells/7-10 hearts, *P< 0.05 vs corresponding Sed.

A Ca2+ Transient Amplitudes B Shortening Amplitudes * * Sed 3 Sed +SMLT 10 Ex

0 8 2 * Ex + SMLT

F/F * 6 *  1 * %RCL 4 * * 2 0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 Frequency (Hz) Frequency (Hz)

Figure 4 Exercise enhances the force frequency relationship, which is normalized by acute NOS1 inhibition. Summary data of Ca2+ transient amplitudes (A) and shortening amplitudes (B) in exercise (Ex-grey) and sedentary (Sed-black) myocytes (±acute NOS1 inhibition, SMLT, dashed), n=9-23 cells/4-8 hearts, *P < 0.05 vs corresponding Sed.

2+ 2+ A SR Ca Load B SR Ca Fractional Release 5.5 * *

i 0.3

[Ca]

0 Load

4.5  0.2

F/F 

0.1 Twitch 3.5 SRCa 0 0.0 PLB-PSer16 C - SMLT + SMLT Sed Ex Sed Ex Ser16 PLB total 0.6 * Sed 0.5 Ex 0.4 Sed + SMLT

0.3 Ex + SMLT A.U. 0.2 0.1 0.0

Figure 5 Exercise enhances SR Ca2+cycling and is normalized by acute NOS1 inhibition. Summary data of SR Ca2+ load (A) and SR Ca2+ fractional release (B), n=26-35 cells/11- 12 hearts. C) Summary data of PLB Serine16 phosphorylation n=6 hearts in exercise (Ex- grey) and sedentary (Sed-black) homogenates (±acute NOS1 inhibition, SMLT, striped), *P< 0.05 vs corresponding Sed.

A Ca2+ Transient Amplitude B Shortening Amplitudes 25 5 * 20

0 4 * 15 * Sed

F/F 3 * 

Ex %RCL 10 Sed +SMLT 2 Ex + SMLT 5 1 0 0 15 30 45 60 0 15 30 45 60 Time (sec) Time (sec)

C PRP (SR Ca2+ Load) 6.5 Sed Ex 6.0 Sed + SMLT Ex + SMLT 5.5 *

0 5.0

F/F  4.5

4.0

3.5 0 5 10 20 30 60 Time (sec)

Figure 6 Exercise enhances post-rest potentiation, which is normalized by acute NOS1 inhibition. Summary data of Ca2+ transient amplitudes (A), shortening amplitudes (B), and SR Ca2+ loads (C) in exercise (Ex-grey) and sedentary (Sed-black) myocytes (±acute NOS1 inhibition, SMLT), n=6-33 cells/4-8 hearts, *P < 0.05 vs corresponding Sed.

2+ 2+ A Ca Transient Amplitude B Ca Transients RT50 2.5 2.0 Sed 325

0 Ex 1.5 *

F/F 250

 1.0 * Time (ms) Time 0.5 175 0.0 0 Cont +OA +PKI Cont +OA +PKI

C Shortening Amplitude D Relengthening RT 50

6 400 5 * 4 3 300 *

% RCL % 2 Time (ms) Time 1 0 0 Cont +OA +PKI Cont +OA +PKI

Figure 7 Protein phosphatase 1 and 2a inhibition (okadaic acid, OA) and PKA inhibition (PKI) abrogated myocyte contraction. Summary data of Ca2+ transient amplitudes (A), 2+ Ca transient RT50 (B), shortening amplitudes and (C), relengthening RT50 (D) in exercise (Ex-grey) and sedentary (Sed-black) myocytes. n=10-22 cells/4 hearts, *P< 0.05 vs corresponding Sed.

NOS1KO

2+ 2+ A Ca Transient Amplitude B Ca Transient RT 50 1.00 300 * 0.75 * 290 0 280

F/F 0.50 270  260 0.25 (ms) Time 250 0.00 0 Sed-NOS1KO Ex-NOS1KO Sed-NOS1KO Ex-NOS1KO

C Shortening Amplitude D Relengthening RT 50 375 2.0 350 1.5 325 1.0 300

% RCL % 275 Time (ms) Time 0.5 250 0.0 0 Sed-NOS1KO Ex-NOS1KO Sed-NOS1KO Ex-NOS1KO

E SR Ca2+ Load F SR Ca2+ Fractional Release

4.25 0.3 i

4.00

[Ca] 0

3.75 Load 0.2

 2+

F/F 3.50 * 

3.25 0.1 Twitch 3.00 Ca SR 0 0.0 Sed-NOS1KO Ex-NOS1KO Sed-NOS1KO Ex-NOS1KO

Figure 8 Negative effects of exercising on Ca2+handling in NOS1 deficient myocytes. 2+ 2+ Summary data of Ca transient amplitudes (A), Ca transient RT50 (B), shortening 2+ 2+ amplitudes (C), relengthening RT50 (D), SR Ca load (E), and SR Ca fractional release (F) in exercise (Ex-NOS1KO-grey) and sedentary (Sed-NOS1KO-black), n=22-42 cells/5-6 hearts, *P< 0.05 vs corresponding Sed.

NOS1KO A Ca2+ Transient Amplitudes B Shortening Amplitudes

3 Sed-NOS1KO 10

Ex-NOS1KO 8 0

2 6

F/F  1 %RCL 4 * 2 0 0 0.0 0.5 1.0 1.5 2.0 0.0 0.5 1.0 1.5 2.0 Frequency (Hz) Frequency (Hz)

C Ca2+ Transient Amplitude D Shortening Amplitudes 25 5 20 4 0 15

F/F %RCL  10 2 1 5 0 0 0 10 20 30 40 50 60 0 15 30 45 60 Time (sec) Time (sec)

Figure 9 No enhancement of the force-frequency response or post-rest potentiation with exercise in NOS1 deficient myocytes. Summary data of the force frequency relationship (Ca2+ transient amplitudes (A) and shortening amplitudes (B) and post-rest potentiation (Ca2+ transient amplitudes (C), shortening amplitudes (D) in exercise (Ex-NOS1KO- grey) and sedentary (Sed-NOS1KO-black) NOS1KO myocytes, n=7-21 cells/4-5 hearts *P < 0.05 vs corresponding Sed.

Canine

Ca2+ Transient Amplitude 2+ A Sed Ex B Ca Transients RT50 Sed + SMLT * Ex + SMLT 1.5 600

* 550 * 0

1.0 500 F/F  450

0.5 (ms) Time 400 0.0 0 C D Shortening Amplitude Relengthening RT 50 600 20 * * 15 * 500

10 % RCL %

5 (ms) Time 400 0 0 E SR Ca2+ Load F SR Ca2+ Fractional Release 2.5 * * i

2.0 * 0.9

[Ca]

0 Load

1.5  2+

F/F 0.8

 1.0

0.5 Twitch SRCa 0.7 0.0 0

Figure 10 Exercise in a trained canine model elicited similar results with NOS1 inhibition on myocyte contraction. Summary data of Ca2+ transient amplitudes (A), Ca2+ transient 2+ RT50 (B), shortening amplitudes (C), relengthening RT50 (D), SR Ca load (E), and SR Ca2+ fractional release (F) in exercise (Ex-grey) and sedentary (Sed- black), (±acute NOS1 inhibition, SMLT, striped), n = 10-33 cells/3-4 hearts, *P< 0.05 vs corresponding Sed. 58

2+ 2+ A Ca Transient Amplitude B Ca Transients RT 50

2.0 Cont 275 FeTPPS * 1.5 * 0 * 250 * F/F 1.0

 225

0.5 (ms) Time 200 0.0 0 Sed Ex Sed Ex

C Shortening Amplitude D Relengthening RT 50

6 350 * 5 4 * 250 3 % RCL % * 2 (ms) Time 1 150 0 0 Sed Ex Sed Ex

Figure 11 The role of a peroxynitrite decomposing catalyst (FeTPPS) on myocyte 2+ 2+ contraction. Summary data of Ca transient amplitudes (A), Ca transient RT50 (B), shortening amplitudes (C), and relengthening RT50 (D) in exercise (Ex- grey) and sedentary (Sed- black) myocytes, n=15-18 cells/4 hearts, *P < 0.05 vs corresponding Sed.

2+ 2+ A Ca Transient Amplitude B Ca Transient RT 50 Sed 125 * ) Ex o 1.0 100

F/F 75 *  0.5 *

50 *

SMLT

SMLT PKI FeTPPS 0.0 (Canine) 25 *

0 OA OA -25 -0.5 PKI

* * (ms) CONT from SMLT

from CONT ( CONT from -50

 (Canine)  * -1.0 * -75 FeTPPS -100

Shortening Amplitude* SMLT C D Relengthening RT50 4

2 80 *

SMLT

FeTPPS SMLT PKI (Canine) * 0 60

OA 40 -2 * 20

* OA

-4 * 0 from CONT (%RCL) CONT from

-6 -20 PKI

from CONT (ms) CONT from



SMLT SMLT

-40 (Canine)

-8 FeTPPS 2+ * -60 E SR Ca Load F SR fractional release SMLT SMLT (Canine) 0.00 SMLT SMLT (Canine)

) o

-0.25 F/F

 -10 -0.50 * -20

-0.75 * from CONT (%) CONT from

from CONT ( CONT from -1.00 -30

  -1.25 * -40 *

Figure 12 NOS1 and its downstream signaling targets have a greater effect with exercise. Summary data shown as change from control of Ca2+ transient amplitudes (A), 2+ Ca decline to 50% of its peak (RT50) (B), shortening amplitudes (C), and relengthening 2+ 2+ RT50 (D), SR Ca load (E), and SR Ca fractional release (F) in exercise (Ex-grey) and sedentary (Sed-black) myocytes, *P< 0.05 vs corresponding Sed.

A VO2 peak B VO2 peak Speed 55.0 37.5 WT 52.5 NOS1KO

/min) 32.5 2

-0.75 50.0 27.5

VO m/min

47.5 22.5 (mL/kg 45.0 17.5 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Weeks Weeks

Figure 13 VO2peak was measured to determine weekly training speeds, n= 4-5 mice.

Chapter 3: Role of Neuronal Nitric Oxide Synthase in the Antioxidant Effects of Exercise

3.1 INTRODUCTION

Exercise is beneficial to one’s health, reduces the risk of cardiomyopathies, and is utilized as a therapeutic intervention after disease [2, 3, 5, 7, 232]. This is due, in part, to the heart’s enhanced ability to increase contraction, accelerate relaxation, and to augment the anti-oxidant system [8]. The previous chapter demonstrated that the neuronal nitric oxide synthase (NOS1) is indispensible for the enhanced myocyte contraction. Localized to the sarcoplasmic reticulum (SR), compartmentalized NOS1 signaling modulates basal contraction by maintaining the nitroso-redox equilibrium. Part of this regulation occurs at the level of phospholamban (PLB) [18]. That is, acute NOS1 inhibition (with S-methyl-

L-thiocitrulline (SMLT)) or NOS1 knockout (NOS1KO) decreases PLB Serine16 phosphorylation (the PKA site) [18] and myocyte-specific NOS1 overexpression increases PLB Serine16 phosphorylation [80]. Thus, NOS1 signaling increases PLB

Serine16 phosphorylation to accelerate Ca2+ decline, load the SR with Ca2+, and increase myocyte contraction. The previous chapter demonstrated that this molecular pathway is enhanced with exercise training. Specifically, an 8 week high intense treadmill training protocol unregulated NOS1 protein expression, increased nitric oxide production, and increased PLB Serine16 phosphorylation. In addition, we also demonstrated that exercise- 62 trained NOS1KO (Ex-NOS1KO) did not result in beneficial cardiac adaptations but caused deleterious effects on myocyte Ca2+ handling. The mechanism(s) governing this adaptation are currently unresolved. Thus, the objective of the current manuscript is to resolve the mechanism of the Ca2+ mishandling in Ex-NOS1KO.

Previous studies have reported elevated reactive oxygen species (ROS) levels in the NOS1KO mouse [85, 230, 231], suggesting NOS1 is involved in regulating ROS levels in the myocyte. There are three major sources of ROS found in the myocytes; xanthine oxidase (XO), NADPH oxidase, and the mitochondria complex I. Interestingly, similar to NOS1, XO is also localized to the SR [13] and it co-immunoprecipitates with

NOS1 [85]. NOS1-dervived NO has been shown to directly inhibit XO activity [86, 87] and mice deficient in NOS1 have elevated XO-dependent ROS production [85, 230].

Another study demonstrated that the elevated ROS levels in the NOS1KO are also due to

NADPH oxidase. [231] Furthermore, mice overexpressing NOS1 have a significant increase in mitochondria nitrite levels and suppressed mitochondria activity [170], suggesting NOS1 may also regulate mitochondrial ROS. During an acute bout of exercise, ROS production increases to create an imbalance between ROS production and

ROS scavenging, a situation known as oxidative stress [233-239]. Thus, we hypothesize that the Ex-NOS1KO Ca2+ mishandling is due to the additional oxidative stress of exercise training which exacerbated ROS levels to result in deleterious SR Ca2+ cycling effects.

3.2 METHODS AND MATERIALS

3.2.1 Murine exercise protocol

A high intense aerobic interval treadmill training protocol was adopted [215].

C57Bl/6 mice (Jackson Laboratories, Bar Harbor, Maine), 5 months at sacrifice, underwent treadmill (Columbus Instruments, Columbus, OH) training 5 days a week for

8 weeks starting at 30 min/day and increased to 80 min. Mice were challenged at a high intense fast pace for 4 minutes followed by 1 minute of low intensity recovery pace

(Chapter 2 - Table 1). This interval set was repeated until the designated time was up. An adequate warm-up of 10 minutes and cool down of 4 minutes was instituted. Exercise effects are maintained for 2 weeks [135]. Thus to avoid detraining, we isolated ventricular myocytes from Sed and Ex mice within 1 week of completing our 8 week training protocol.

3.2.2 Cardiomyocyte isolation

Post-training, Ex mice were sacrificed for experiments and compared to age- matched Sed mice. Ventricular myocytes were isolated from anaestized mice via isopentobarbrotal, as previously described [18, 220, 221]. Briefly, the heart was cannulated and hung on a Langendorff apparatus. It was then perfused with Ca2+ free tyrode solution (see solutions and drugs below) for 4 min. The solution was then switched to a tyrode solution containing Liberase Blendzyme II (0.077 mg/ml) (Roche

Applied Science, Indianapolis, IN). After 3-5 min, the heart was taken down, the ventricles minced, and myocytes were dissociated by trituration. Subsequently the

64 myocytes were filtered, centrifuged, and resuspended in tyrode solution containing 200

μmol/L Ca2+. Myocytes were used within 4 hrs of isolation. All the 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.

3.2.3 Measurement of myocyte Ca2+ transients and shortening

Ca2+ transient and shortening measurements were performed at room temperature, as previously described [18, 220, 221]. Briefly, myocytes were loaded at room temperature with Fluo-4 AM (10 μmol/L, Molecular Probes, Eugene, OR) for 30 min. An additional 30 min were allowed for intracellular de-esterification. The instrumentation used for cell fluorescence measurements was a Cairn Research Limited (Faversham, UK)

2+ epifluorescence system. [Ca ]i was measured by Fluo-4 epifluorescence with excitation at 480±20 nm and emission at 535±25 nm. The illumination field was restricted to collect the emission of a single cell. Data is expressed as ΔF/F0, where F is the fluorescence intensity and F0 is the intensity at rest. Data for cell shortening was collected using a video edge detection system (Crescent Electronics). Myocytes were stimulated at 1 Hz via platinum electrodes connected to a Grass Telefactor S48 stimulator (West Warwick,

RI).

3.2.4 Measurement of ROS Production with CM-H2DCFDA Fluorescence

Isolated myocytes were loaded at room temperature with CM-H2DCFDA, (ROS- sensitive fluorescent dye - 10 µM) for 20 minutes and allowed to de-esterify for an additional 20 minutes. Fluorescence was observed on an Olympus Fluoview 1000 laser

65 scanning confocal microscope by exciting at 488 nm line of an argon laser and emission was collected at 500-560 nm. Data was normalized to F0 and background subtracted.

Myocytes were stimulated at 1 Hz.

3.2.5 Western blot analysis

Western blot analysis, were preformed as previously described [18].

Homogenized ventricles were used to measure specific phospholamban phosphorylation at Serine16 (Badrilla, Leeds, UK) with phospho-specific antibodies and normalized to

GAPDH (Cell Signaling Technology, Danvers, MA, 1:20,000) and total phospholamban

(custom - Zymed, San Francisco, CA).

3.2.6 Protein Phosphatase Activity

Hearts were langendorf perfused for 5 mins with 1.8 mM Ca2+ with or without methyl-ester nitroxide (MENO). Protein phosphatase activity was examined using the

Sensolyte Protein Phosphatase Assay Kit (AnaSpec, San Jose, CA). The assay buffer consisted of (in mmol/L): Tris-HCl pH 7.0 (50), Na2EDTA (0.1), 0.01% Brij 35. The reaction was then initiated upon addition of 50 μL of the provided pNPP reaction mixture to cardiac homogenates (100 μg) each experimental well. Total well volume was 100 μL.

The protein phosphatase assay was allowed to continue for 90 min at 37°C. After which time the absorbance signal was measured at 405 nm using a BioTek PowerWave XS

(Winooski, VT). Enzyme activity (nmol/min) was calculated using the following formula: (V × vol)/(ε × l), where V is the reaction velocity (OD405/min), vol is the reaction volume in liters, ε is the extinction coefficient of pNPP (1.78 × 104 M−1cm−1) and l is the path length of light through the sample in cm (for 100 mL sample, l = 0.5

66 cm). Enzyme activity was then calculated per mg of protein by dividing the enzyme activity by 0.1 mg.

3.2.7 VO2max testing protocol and training speeds

A metabolic chamber (Columbus Instruments) and Oxymax analyzer (Columbus

Instruments) were used for measurements. A protocol was adopted to measure maximal

O2 consumption (VO2max), [219]. Briefly, mice underwent a brief warm-up with 2 min at

10 m/min at 0º incline, 2 min at 12 m/min at 10º incline, and 2 min at 15 m/min at 20º incline. Then, the treadmill speed increased 1 m/min at 20º incline every 30 sec. VO2max was defined as the absolute maximal VO2 with a respiratory exchange ratio (RER) above

1 and exhaustion was reached (mice unwilling to run and neglecting shock).

3.2.8 Solutions and drugs

Normal Tyrode (NT) solution consisted of (in mmol/L): 140 NaCl, 4 KCl, 1

MgCl2, 1 CaCl2 (2 CaCl2 for canine), 10 glucose, 5 HEPES, pH 7.4 adjusted with NaOH or HCl. Myocytes were superfused with MENO (5 µmol/L). EMEPO (1 mmol/L) was synthesized as previously described [240] and was incubated for 30 minutes. This is the only time myocytes were exposed to EMEPO. All chemicals were from Sigma (St. Louis,

MO) except where indicated.

3.2.9 Echocardiography

Echocardiography was performed on WT and AAV-9 injected mice to measure the in vivo function of the heart using the Vevo 2100 (Visualsonics, Toronto, Ontario, Canada). One mouse at a time was placed into the anesthesia chamber and anesthetized using 2.0 % Isoflurane that was added to 95% O2 / 5% CO2 at a rate of ~ 0.8 L/min. Once it was sufficiently

67 anesthetized (did not move while being touched), the mouse was removed from the chamber and placed in the supine position on the heated handling table. Care was taken to avoid over anesthetizing the mouse, which can drastically reduce the respiratory rate (< 50-90 respirations/minute), as well as the heart rate and its function. Anesthesia was maintained through the use of a nose cone that provided oxygen and 1% Isoflurane. A small amount of electrode gel was placed on the four EKG leads on the platform and the four mouse limbs were taped to the platform. The EKG was monitored while positioning the limbs to make sure there was adequate electrical conduction for data collection. A temperature probe was inserted into the rectum of the mouse to monitor the core temperature which was maintained at ~ 35oC. The heated platform temperature was set at 42oC and a heat lamp was placed over top of the mouse to regulate and maintain the temperature. Nair (Church & Dwight Co., Princeton, NJ) containing baby oil was used to remove the hair from the chest, which was vital for a strong signal during the echocardiography.

Temperature, respiration rate, and the EKG with heart rate output were collected while simultaneously performing the echocardiography. The MS-400 transducer was used to collect the long and short axis, B and M mode echocardiographs for each of the mice. Basic measurements collected from the M-mode included the internal dimensions of the left ventricle during diastole and systole of the heart. To collect the data from the long axis M-mode the probe was positioned to have the apex, the aorta, and the papillary muscle all in the same field of view and then the cursor was placed slightly inferior to the papillary muscle. For the short axis data collection the cursor was positioned superior of the apex so that two papillary muscles were in the field of view about half way up the heart. The cursor to collect the measurements was then

68 placed just to the side of the papillary muscles to measure the anterior and posterior wall movement. From these values, the computer was able to calculate diastolic and systolic internal dimensions, diastolic and systolic chamber volumes, stroke volume, ejection fraction, fractional shortening, and mass of left ventricle.

3.2.10 Statistical Analysis

Data were presented as mean±SEM. Differences between 2 groups were evaluated for statistical significance (P < 0.05) by paired or unpaired Student's t tests. To test statistical difference between multiple groups, a one-way ANOVA was used.

3.3 RESULTS

3.3.1 Exercise decreases ROS production in WT and increases in NOS1KO mice

Similar to our previous data, Ex-NOS1KO had depressed left ventricular function

(fractional shortening and ejection fraction) compared to Sed-NOS1KO (Table 4).

Since the NOS1KO mouse have elevated ROS levels [85, 230, 231] and acute bouts of exercise increases oxidative stress [233-239], we wanted to determine if NOS1 signaling is necessary to combat the oxidative stress. We measured ROS levels in sedentary (Sed) and exercise-trained (Ex) WT and NOS1KO mice with CM-H2DCFDA fluorescence. Shown in Figure 14A, Ex had decreased ROS production in WT mice compared to corresponding Sed. Consistent with previous work [85, 230, 231], Sed-

NOS1KO had elevated ROS levels compared to WT mice. Interestingly, Ex-NOS1KO

69 had further exacerbated ROS levels compared to Sed-NOS1KO. As expected, superfusing the myocytes with a cell permeable superoxide scavenger methyl-ester nitroxide (MENO) reduced ROS production in the NOS1KO mice to WT levels (Figure

14B). Thus, these data suggest that training NOS1KO mice results in exacerbated ROS levels.

3.3.2 Superoxide scavenger rescues myocyte contraction from trained NOS1KO

To determine if the elevated ROS was the main perpetrator of the Ca2+ mishandling, we measured Ca2+ transient amplitude and Ca2+ transient decline to 50% of its max (RT50). Shown in Figure 14C-D, MENO had no effect on WT but significantly increased Ca2+ transient amplitude and Ca2+ decline in the both Ex-NOS1KO and Sed-

NOS1KO as show as % change from control (i.e. without MENO). Additionally, MENO had a greater effect to increase Ca2+ transient amplitude and accelerate Ca2+ decline in

Ex-NOS1KO compared to Sed-NOS1KO. We did not observe a difference in contraction with MENO between Sed-NOS1KO and Ex-NOS1KO, consistent with Chapter 2 -

Figure 8. See Figure 15 for cell shortening data. Raw data is shown in Figure 14E-F.

Thus, these data suggest these elevated ROS levels contribute to the Ca2+ mishandling in

Ex-NOS1KO.

3.3.3 Identification of the ROS source

To determined the source of ROS, we measured Ca2+ transients and selectively inhibited each of the three major sources of ROS found in the myocyte; XO, NADPH oxidase, and the mitochondria complex I. Shown in Figure 16A-B, XO inhibition

(allopurinol) had no effect on WT, but significantly increased Ca2+ transient amplitude

70 and accelerated Ca2+ decline in the both Ex-NOS1KO and Sed-NOS1KO shown as % change from control. Interestingly, allopurinol had a significantly greater effect in the Ex-

NOS1KO compared to Sed-NOS1KO. NADPH oxidase inhibition (apocynin) and mitochondria complex I inhibition (roteone) both increased Ca2+ transient amplitude in all four groups and accelerated the Ca2+ decline in both NOS1KO groups. Moreover, inhibition with apocynin and rotenone had a greater effect in Ex-NOS1KO compared to

Sed-NOS1KO. Raw data is shown in Figure 16C-H. See Figure 17 for cell shortening data. Thus, these data suggest that all three sources contribute to the exercise-derived

ROS in Ex-NOS1KO.

3.3.4 Mechanisms responsible for the Ca2+ mishandling

Our previous chapter demonstrated that NOS1-mediated increase in PLB Serine16 phosphorylation contributed to the enhanced myocyte contraction following exercise.

16 Therefore, we wanted to determine the phosphorylation status of PLB Serine post- training in the NOS1KO. Shown in Figure 18A, Ex-NOS1KO had decreased PLB

Serine16 phosphorylation compared to Sed-NOS1KO. This was rescued with a superoxide scavenger (MENO), suggesting the elevated ROS was responsible for the decreased PLB

Serine16 phosphorylation. Since, protein phosphatase 1 (PP1) and PP2a, which are responsible for PLB dephosphorylation, are known to be activated by increased oxidative stress [81], we measured protein phosphatase activity in the absence or presence of

MENO. Interestingly, exercise significantly decreased protein phosphatase activity in

WT, consistent with ROS levels, but increased activity in NOS1KO compared to Sed controls (Figure 18B). Additionally, MENO rescued and normalized phosphatase activity

71 in Ex-NOS1KO and Sed-NOS1KO to Sed WT levels. Thus, the elevated ROS levels increases protein phosphatase activity and subsequently decrease PLB Serine16 phosphorylation.

3.3.5 Restoration of myocyte contraction with EMEPO

In order for proper myocyte contraction, the redox levels must be in balance with nitroso levels (i.e. nitroso-redox equilibrium). Since NOS1KO myocytes have increased

ROS levels and decreased NOS1-dependent NO, there is a shift in this equilibrium that contributes to the depressed myocyte contraction. We determined if the stress of exercise could be detrimental to myocytes lacking NOS1 and further contribute to this nitroso- redox disequilibrium. To test this, we used a cell permeable nitrone (EMEPO) [241], which acts as both a superoxide scavenger and NO donor, which will restore the nitroso- redox equilibrium. Shown in Figure 19A-B, EMEPO significantly increased Ca2+ transient amplitude and Ca2+ decline in the both Ex-NOS1KO and Sed-NOS1KO shown as % change from control. Interestingly, EMEPO had a greater effect in the Ex-

NOS1KO compared to Sed-NOS1KO. Raw data is shown in Figure 19C-D. See Figure

20 for cell shortening data. Thus, this is suggestive that there is an intensified nitroso- redox disequilibrium in the Ex-NOS1KO myocytes.

3.4 DISCUSSION

The molecular mechanism(s) by which training NOS1KO mice results in Ca2+ mishandling is not known. This study demonstrates that Ex-NOS1KO mice have a nitroso-redox disequilibrium partially due to elevated ROS levels from XO, NADPH

72 oxidase, and mitochondria complex I that activates phosphatase activity to decrease PLB

Serine16 phosphorylation.

3.4.1 The role of NOS1 signaling in exercise induced adaptations

NOS1 signaling regulates basal contraction and does so by playing a major role in maintaining localized nitroso-redox equilibrium at the SR. NOS1 maintains the nitroso side of the nitroso-redox equilibrium by synthesizing NO. NOS1-derived NO signals through the cGMP-independent signaling pathway (i.e – S-nitrosylation/formation of peroxynitrite). NOS1 signaling alters kinase activity by activating PKA via peroxynitrite

[17, 18, 48]. Activated PKA phosphorylates PLB Serine16 to increase SR Ca2+ uptake

[48]. Our previous chapter suggests that exercise increases NOS1 protein expression and nitric oxide/peroxynitrite production in ventricular myocytes to increase PLB Serine16 phosphorylation to accelerate Ca2+ decline, load the SR with Ca2+, and increase myocyte contraction.

NOS1-derived NO also regulates the redox side of the nitroso-redox equilibrium.

The interaction between NO and ROS is vital for adequate myocyte contraction.

Together, in equilibrium, the NO and ROS tandem maintains physiological signaling pathways [67]. One direct positive outcome of this interaction is that NO can act to

“buffer” ROS. In fact, in the absence of adequate NO (acute NOS1 inhibition or

NOS1KO), the equilibrium is shifted and ROS levels go awry [85, 230, 231]. This disequilibrium can result in pathophysiological conditions. This is partially due to the increased phosphatase activity, which is activated by increased oxidative stress [81].

Dephosphorylation of PLB by protein phophatases PP1 and PP2a [33] will increase PLB- mediated inhibition of SERCA and result in slowed Ca2+ decline and a lower SR Ca2+ load. Data from the current study suggests that the lack of NOS1-derived NO combined with the stress of exercise, elevated ROS levels (Fig 14) compared to the Sed-NOS1KO.

This in turn, activated phosphatases (Fig 18) to decrease PLB Serine16 phosphorylation

(Fig 18) and resulted in Ca2+ mishandling. [175].

3.4.2 Exercise-derived sources of ROS

An acute bout of exercise can create an imbalance between ROS production and

ROS scavenging, a situation known as oxidative stress. Oxidative stress is a key contributor to many cardiomyopathies [242]. Mild oxidative stress (as observed during exercise) can stimulant the antioxidant system and generate physiological adaptations

[236]. One of the major beneficial adaptations of exercise is to augment the anti-oxidant system (Fig 14) [8] and prevent excessive ROS damage to the myocyte. During acute exercise, XO [234-236], NADPH oxidase [233, 239], and the mitochondria [243] are significantly more active than at rest. The adaptive response and cardioprotection with exercise training is heavily reliant on manganese superoxide dismutase (MnSOD) [244,

245]. However, the elevated ROS levels in Ex-NOS1KO suggest that NOS1 also plays a major role in the antioxidant defense. Shown in Fig 14A-B, inhibition of each of the three sources had the greatest effect to increase Ca2+ transient amplitude and accelerate the

Ca2+ decline in the Ex-NOS1KO, suggesting that NOS1 plays a role in combating the increased ROS produced from each source.

Similar to NOS1, XO is also localized to the SR [13], co-immunoprecipitates with

NOS1 [85], and is responsible, in part, for the elevated ROS levels in the NOS1KO [85,

230], which is due to the absence of NOS1-dervived NO directly inhibiting XO activity

[86, 87]. Studies have shown that acute exercise increases XO-dependent ROS [234,

237]. Our results demonstrate that XO inhibition (allopurinol) was ineffective in WT mice (Sed and Ex), but significantly affected intracellular [Ca2+] effect in both Sed-

NOS1KO and Ex-NOS1KO, these data are consistent with previous studies [85, 86, 230].

Also, we observed that acute XO inhibition had the greatest effect in Ex-NOS1KO (Fig

16), suggesting that XO activity was further elevated in Ex-NOS1KO compared to Sed-

NOS1KO to depress Ca2+ transient amplitude and slow Ca2+ decline. Consistent with our

Ex-NOS1KO data, chronic treatment of L-arginine (initial substrate to synthesize NO) rescued the increase XO activity observed during acute exercise [235]. Hence, these data suggest that without NOS1 regulating XO, the exercise-derived elevated ROS levels in the Ex-NOS1KO elicited a detrimental adaptation

NADPH oxidase has been shown to be a source of ROS in the NOS1KO mouse

[231] and can modulate the SR Ca2+ release channel (RYR2) activity under normal conditions [246, 247] and in response to exercise training [239]. Acute NOS1 inhibition of NADPH oxidase (apocynin) increased the Ca2+ transient amplitude in all four groups.

However, the greatest effect was observed in the Ex-NOS1KO (Fig 16), suggesting that during exercise, NOS1 protects the myocytes from NADPH oxidase dependent ROS.

Mice overexpressing NOS1 have a significant increase in mitochondria nitrite levels and suppressed mitochondria activity [170], suggesting NOS1 regulates

75 mitochondrial ROS. The existence of a mitochondrial NOS (mtNOS) is still debatable but some, believe it to be NOS1 [248, 249]. Interestingly, exercise training increases cardiac mtNOS activity by 45% [250]. Furthermore, the idea that exercise training increases mtNOS to protect/regulate the mitochondria from oxidative stress is consistent with the role of mitochondria-derived NO (to inhibit the mitochondrial complex 1 [251]) and evidence that exercise training decreases the resting mitochondrial rate of ROS production [252]. Mitochondrial complex 1 inhibition (rotenone) had the greatest effect on Ca2+ transient amplitude and Ca2+ transient decline on Ex-NOS1KO (Fig 16), suggesting there may be an exercise-dependent NOS1 regulation of the mitochondria.

Therefore, we hypothesize an exercise induced phenomenon that mediates the modulation of NOS1 to protect the cell from oxidative stress by regulating the mitochondria ROS following chronic exercise. The cardioprotective role of mtNOS in response strenuous exercise has been previously hypothesized [248]. Our data suggests that all three sources contribute to the exercise-derived elevation of ROS in mice deficient in NOS1.

3.4.3 NOS1 plays a critical role in regulating the nitroso-redox equilibrium in response to exercise

To test the nitroso-redox equilibrium, we used our compound (EMEPO) and

2+ 2+ rescued the blunted Ca transient amplitude and accelerate the slowed [Ca ]i decline in

Sed-NOS1KO and Ex-NOS1KO myocytes (Fig 19). Interestingly, there was a greater effect in Ex-NOS1KO. This is suggestive that the Ex-NOS1KO had greater nitroso-redox disequilibrium. In fact, ROS levels were elevated in the Ex-NOS1KO (Fig 14).

Consistent with high levels of ROS causing myocyte contractile dysfunction [253, 254], a superoxide scavenger (MENO) increased Ca2+ transient amplitude and accelerate the

Ca2+ decline (Fig 14). In agreement with the increased ROS production in Ex-NOS1KO compared to Sed-NOS1KO myocytes, MENO had a greater effect on Ca2+ transient and

Ca2+ decline. EMEPO and MENO had minimal effect in Ex and Sed WT mice. Thus, these data suggests the increased ROS levels heavily contributed to the Ca2+ mishandling and nitroso-redox disequilibrium.

The negative effects of elevated ROS on Ca2+ mishandling may not solely be the result of increased phosphatase activity. Redox post translational modifications can alter the function of key players involved in regulating Ca2+ handling. Through the years, evidence of direct effects of ROS has been reported on various kinases [185, 186, 255,

256], ion channels [257-259], such as on SERCA [176] or RYR2 [260, 261], structural and contractile proteins [262]. Thus, the deleterious effects of the elevated ROS levels observed in the Ex-NOS1KO are not limited to increase phosphatase activity, and warrant further investigation.

In diseased myocardium (i.e heart failure), it has also been demonstrated that ROS levels are increased with all three major sources (XO, NADPH oxidase, mitochondria) contributing [254, 258, 263-268]. Interestingly, exercise has been shown to reduce ROS levels in heart failure [269, 270], suggesting that part of the beneficial effects of Ex may be via reducing oxidative stress. Data from this chapter suggests that NOS1 contributes to the beneficial antioxidant effect of exercise and thus, NOS1 may be utilized as a good therapeutic target in treating cardiomyopathies.

3.4.4 Conclusion

Training NOS1KO results in Ca2+ mishandling due to elevated ROS levels. This is due to XO, NADPH oxidase, and mitochondria dependent ROS mediated increase in phosphatase activity to decrease PLB Serine16 phosphorylation. These data suggest that targeting NOS1 may be beneficial against the oxidative stress.

Sed Ex Sed- NOS1-/- Ex- NOS1-/- Diastolic LV diameter, mm 3.5±0.1 3.6±0.1 3.4±0.1 3.5±0.1

Systolic LV diameter, mm 2.1±0.1 2.3±0.1 2.2±0.1 2.4±0.1

Fractional shortening, % 38.3±1.7 34.8±1.4 35.0±1.5 32.7±0.6*

Heart rate, beats/min 498±10 463±17 435±11 423±11*

Ejection Fraction, % 61±2 60±3 65±1 55±1*

Table 4 LV function as assessed by echocardiography. * P<0.05 vs Sed. n=10 mice/group.

A ROS Production B ROS Production 0.4 0.025 # Sed Sed 0.3 Ex 0.020 Sed-NOS1KO Ex F/min 0.015 Ex-NOS1KO  MENO 0.2 *

0.010 DCFDA 0.1 Fluoresence 0.005 # * Slope, * * 0.0 0.000 0 2 4 6 8 10 12 14 WT NOS1KO Time (min)

2+ 2+ Ca Transient Amplitude Ca Transients RT 50 C + MENO D + MENO Sed 110 160 Ex # Sed-NOS1KO * 140 Ex-NOS1KO 100 120

* 90 * # (-MENO)

(-MENO) 100 * % of control of % % of control of % 80 80 0 0

2+ 2+ Ca Transient Amplitude Ca Transients RT 50 E + MENO F + MENO 2.0 Control 300 # +MENO *

1.5 275 0 * 250 * F/F 1.0 # *  225 0.5 (ms) Time 200 0.0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

Figure 14 Exercise-trained NOS1KO myocytes have increased reactive oxygen species (ROS) production and a greater effect of superoxide scavenger on myocyte contraction. Summary data of ROS production over a 15 minute time period (A). Pooled data for ROS accumulation slopes in the presence or absence of a superoxide scavenger (MENO) (B), n=19-42 cells/3-4 hearts. Summary data % control of Ca2+ transient amplitudes (C) and 2+ 2+ Ca decline to 50% of its peak (RT50) (D). Summary of raw data of Ca transient 2+ amplitudes (E) and Ca transient RT50 (F), n=15-23 cells/4 hearts, *P< 0.05 vs without MENO, #P<0.05 vs corresponding Sed.

Shortening Amplitude Relengthening RT50 A + MENO B + MENO 150 * 110 125 * 100

100 90 * *

(-MENO)

(-MENO) % of control of % % of control of % 75 80 0 0

C Shortening Amplitude D Relengthening RT 50 + MENO + MENO 7 # 380 6 360 5 340 * 4 * 3 * 320 # % RCL % * 300 2 (ms) Time 1 280 0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

Figure 15 Effects of on myocyte contraction with a superoxide scavenger (MENO), on wildtype and NOS1KO sedentary (Sed) and exercise-trained (Ex) myocytes. Summary data as % control of short amplitudes (A) and relengthening to 50% of its peak (RT50) (B). Summary of raw data of shortening amplitudes (C) and relengthening RT50 (D), n=20-47 cells/3-4 hearts, *P< 0.05 vs corresponding Sed.

2+ 2+ A Ca Transient Amplitude B Ca Transients RT 50 Sed Sed-NOS1KO 150 Ex Ex-NOS1KO 140 100 130 120 90 110

100 % of control of % % of control of % 90 80 0 0 +Allopurinol +Apocyin +Roteone +Allopurinol +Apocyin +Roteone

2+ 2+ Ca Transient Amplitude Ca Transients RT 50 + Allopurinol + Allopurinol C D 2.0 Control 300

1.5 Allopurinol 275 0 250

F/F 1.0  225 0.5 (ms) Time 200 0.0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO 2+ 2+ Ca Transient Amplitude Ca Transients RT 50 E + Apocynin F + Apocynin 2.0 Control 300 Apocynin

1.5 275 0 250

F/F 1.0  225 0.5 (ms) Time 200 0.0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO 2+ 2+ Ca Transient Amplitude Ca Transients RT 50 + Rotenone + Rotenone G H 2.0 Control 300 Rotenone

1.5 275 0 250

F/F 1.0  225 0.5 (ms) Time 200 0.0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

Figure 16 Effects of acute inhibition with allopurinol (xanthine oxidase), apocynin (NADPH oxidase), and rotenone (mitochondrial complex 1) on Ca2+ handling from WT and NOS1KO sedentary (Sed) and exercise-trained (Ex) myocytes. Summary data % control of short amplitudes (A) and relengthening to 50% of its peak (RT50) (B). 2+ 2+ Summary of raw data of Ca transient amplitudes + allopurinol (C), Ca transient RT50 2+ 2+ + allopurinol (D), Ca transient amplitudes + apocynin (E), Ca transient RT50 + 2+ 2+ apocynin (F), Ca transient amplitudes + rotenone (G), and Ca transient RT50 + rotenone (H), n=8-14 cells/3-4 hearts, *P< 0.05 vs without treatment, #P<0.05 vs corresponding Sed.

Shortening Amplitude Relengthening RT A B 50

175 110 100 150 90 125 80 70 100

60 % of control of % % of control of % 75 50 0 0 +Allopurinol +Apocyin +Roteone +Allopurinol +Apocyin +Roteone

Shortening Amplitude Relengthening RT 50 C + Allopurinol D + Allopurinol 8 350 7 6 5 4 250

% RCL % 3

2 (ms) Time 1 150 0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

Shortening Amplitude Relengthening RT 50 E + Apocynin F + Apocynin 8 350 7 6 5 4 250

% RCL % 3

2 (ms) Time 1 150 0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

G Shortening Amplitude H Relengthening RT 50 + Rotenone + Rotenone

8 350 7 6 5 4 250

% RCL % 3

2 (ms) Time 1 150 0 0.0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

Figure 17 Effect of acute inhibition on myocyte contraction with allopurinol (xanthine oxidase), apocynin (NADPH oxidase), and rotenone (mitochondrial complex 1) on wildtype and NOS1KO sedentary (Sed) and exercise-trained (Ex) myocytes. Summary data % control of short amplitudes (A) and relengthening to 50% of its peak (RT50) (B). Summary of raw data of shortening amplitudes + allopurinol (C), relengthening RT50 + allopurinol (D), shortening amplitudes + apocynin (E), relengthening RT50 + apocynin (F), shortening amplitudes + rotenone (G), and relengthening RT50 + rotenone (H), n=8- 14 cells/3-4 hearts.

PLB-PSer16

- MENO + MENO Phosphatase activity Sed Ex Sed Ex Sed Ser16 Ex-NOS1KO Ex Sed-NOS1KO +Meno A PLB total B Sed-NOS1KO Ex-NOS1KO +Meno 2.5 Sed-NOS1KO 0.8 # 2.0 Ex-NOS1KO * 0.6 1.5 * * 0.4 # A.U. 1.0 # 0.2

0.5 nmol/min/mg 0.0 0.0 Control + MENO

Figure 18 Decreased PLB Serine16 phosphorylation and increased phosphatase activity was rescued with a superoxide scavenger [234] in Ex-NOS1KO. (A) Summary data of PLB Serine16 phosphorylation n=4 hearts, checkered), (B) Summary data of protein phosphatase activity, n=4 hearts, *P< 0.05 vs corresponding Sed.

2+ 2+ A Ca Transient Amplitude B Ca Transients RT50 + EMEPO + EMEPO Sed Ex 110 # 160 # Sed-NOS1KO * 100 140 Ex-NOS1KO * 90 * 120 * #

100 80

(-EMEPO)

(-EMEPO) % of control of % % of control of % 80 70 0 0

2+ 2+ Ca Transient Amplitude Ca Transients RT50 C D +EMEPO Control +EMEPO # +EMEPO 300 # 1.5 * 275

0 * 1.0 # 250 F/F * *

 # 225

0.5 (ms) Time 200 0.0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

Figure 19 A synthesized compound used as a superoxide scavenger and nitric oxide donor (EMEPO) has a greatest effect in exercise-trained NOS1KO mice (Ex-NOS1KO). Summary data shown % change from control of Ca2+ transient amplitudes (A) and Ca2+ 2+ decline to 50% of its peak (RT50) (B). Summary raw data of Ca transient amplitudes (C) 2+ and Ca transient RT50 (D), n=20-47 cells/3-4 hearts, *P< 0.05 vs without EMEPO, #P<0.05 vs corresponding Sed.

Relengthening RT A Shortening Amplitude B 50 + EMEPO + EMEPO 400 * # 100 300 * 80 *

200 60 *

(-EMEPO) (-EMEPO) 100 control of % * * % of control of % 40 0 0.0

Shortening Amplitude Relengthening RT50 C + EMEPO D + EMEPO 7 # 350 6 * * # 5 300 4 250 3 * % RCL % 200 2 # (ms) Time * 1 150 * * 0 0 Sed Ex Sed- Ex- Sed Ex Sed- Ex- NOS1KO NOS1KO NOS1KO NOS1KO

Figure 20 Effects of a compound that acts both as a superoxide scavenger and nitric oxide donor (EMEPO) on wildtype and NOS1KO sedentary (Sed) and exercise-trained (Ex) myocytes. Summary data as % control of short amplitudes (A) and relengthening to 50% of its peak (RT50) (B). Summary of raw data of shortening amplitudes (C) and relengthening RT50 (D), n=20-47 cells/3-4 hearts, *P< 0.05 vs corresponding Sed.

Chapter 4: Future Directions

This dissertation would not be complete without tying up loose ends, providing instructions for future experiments, and delivering dream worthy experiments with endless possibilities. While most of the experiments performed during the completion of the thesis supported the overall hypothesis and the current literature, some experiments did not. One question that remains to be answered is; how does exercise training affect local PKA activity at the SR? We experimentally tested global PKA activity and found that there was no difference in the Ex group compared to Sed. This finding is not surprising since others have shown that chronic exercise can modulate the autonomic nervous system by increasing parasympathetic and decreasing sympathetic tone at rest

[271, 272]. A decrease in circulating catecholamines would decrease PKA activity

(downstream of the β-AR signaling pathway). Thus, specifically measuring PKA activity at the SR would determine if the NOS1 mediated increase in PLB phosphorylation is due to increased PKA activity or decreased phosphatase activity. To best attack this problem, four experiments could be developed. First, the experiment could be repeated but measured in isolated myocytes instead of whole heart preparation. Secondly, local PKA activity could be measured with live cell imaging using the FRET based biosensor SR-

AKAR3 [273]. Thirdly, PKA activity could be measured in isolated SR vesicles homogenates. Lastly, the use of two different common PKA phosphorylation sites as 87 readout could work. In this experiment, one could use PLB phosphorylation for the SR readout and TnI Serine23/24 as a common PKA phosphorylation site outside the SR.

However, exercise could also alter TnI phosphorylation via other pathways (see below).

Thus, the first approach may provide a more accurate assessment of PKA activity at the

SR.

A loose end that was never resolved was that in the presence of NOS1 inhibition,

Ex still had a significantly faster myocyte relengthening compared to Sed. Thus, a mechanism independent of NOS1 inhibition also contributes to the faster relengthening in

Ex compared to Sed. This could be due to increased TnI phosphorylation at basal levels, that would desensitizes the myofilaments to Ca2+ and thereby, contribute to a faster relaxation. In fact, TnI phosphorylation has been previously shown to be altered over a training period [125]. Additional work would be needed, as this was not the focus of this thesis.

One of the most unclear phenomenon was that the Ex-NOS1KO did not obtain physiological hypertrophy (as measured by the heart-to-tibia ratio). As mentioned earlier, the cardiac remodeling in response to exercise is mediated through the IGF/ PI3K/ AKT pathway [147-149] and the transcription factor C/EBPβ [153]. In fact, AKT is upstream of NOS1 [88] and NOS1 may regulate the expression of C/EBPβ [154]. Thus, it’s quite possible that NOS1 may be involved in physiological hypertrophy. It is also interesting to note that, although not statistically significant, Sed-NOS1KO hearts tread to be smaller

(Chapter 2 – Table 3 & [169]). A good way to determine if NOS1 signaling is needed physiological hypertrophy is to train the WT and NOS1KO with 4 weeks of swimming.

Compared to the high intense treadmill training, which only elicits a ~7% change, swimming can result in 20% increase in hypertrophy compared to Sed [153, 274]. This greater difference would give a clearer conclusion whether or not NOS1 is involved.

The activity of NOS1 was not compared between Ex and Sed post training. These data would help understand the whole picture. However, we did observe increased

NOS1-dependent NO production, but it’s unclear whether this was due to the increased

NOS1 expression and/or activity. We know that NOS1 protein expression levels are up, but that might not be enough to elicit the NO production levels that we observed. To determine NOS1 activity levels one has 2 main approaches. First, NOS activity could be measured in Sed vs Ex myocyte homogenates with a commercially available NOS activity assay kit (Sigma). This kit measures the enzymatic conversion to radiolabeled

3H-arginine to 3H-citrulline. Radioactivity could be quantified with a liquid scintillation counter. To control for background, a reaction mixture could be boiled or omit one of the cofactors. The second approach is to measure NOS1 phosphorylation levels. NOS1 can be activated by phosphorylation by AKT at Serine1412 [88]. Deactivation of NOS1 can be, in part, regulated by its phosphorylation status at Serine847 by CaMKII [89]. We preformed these Western blot analyses, but due to the lack of quality antibodies, no firm conclusions were able to be made. If more select antibodies are developed, then the experiments could be repeated in order to give a clearer conclusion whether or not NOS1 activity is altered by AKT or CaMKII.

Finally, if endless resources, time, man power, and possibilities are allowed, what experiments would I do to fully understand if the exercise induced NOS1 signaling could

89 have medicinal purposes? To answer the question, I would need Jango Fett (yes, that’s right, Jango Fett). The father of the infamous bounty hunter named Boba Fett. He is the one whose genetic template was used to clone an army for the Republic in Star Wars II: The

Clone Wars. This army was comprised of countless Imperial Storm Troopers. Therefore, by using

Jango Fett, I could create my own army made specifically for scientific purposes. Not only are they human, but these elite soldiers obey every command. Thus, the variability based on motivation would be nonexistent. In a mad scientist’s perfect world (ignoring morals and ethics), studying science on humans would probably elicit great results to cure human disease. Thus, if I could train storm troopers like mice, it would be the best way to determine if ELIM could become reality. Here are the proposed experiments.

Investigation 1: Confirm NOS1 signaling is responsible for the exercise induced increases in the intrinsic capacity of the heart to contract. This will be done by testing NOS1 protein levels and activity in exercise-trained storm troopers versus Sed. Sed storm troopers will maintain daily activities around the Death Star and may not engage in any form of strenuous exercise or combat in battle. To elicit a trained state, storm troopers will undergo an 8 week high intense interval treadmill or cycling training protocol. Speeds for the fast pace will be determined by taking 90% of the speed at which they reached their VO2peak. Cardiac function will be monitored using a variety of techniques including MRI and echo. Upon sacrificing, ex vivo pressure development and isolated myocytes will be assessed. We hypothesize that exercise will increase NOS1 protein expression and activity. If NOS1 protein or activity is indeed upregulated with exercise, we will continue on with Investigation 2.

Investigation 2: Determine if NOS1 overexpression via gene therapy can mimic exercise adaptations and promote longevity in senescent storm troopers. Storm troopers will be split into 5 groups: 1) Sed, 2) Ex-old, 3) NOS1-AAV-old, 4) Ex-young, and 5) NOS1-AAV-young. Again, 90

Sed storm troopers will maintain daily activities around the Death Star and may not engage in any form of strenuous exercise or combat in battle. Ex-old and NOS1-AAV-old will begin treatment during senescent years when decreasing cardiac function has been diagnosed. Cardiac function will be monitored using MRI, Echo and VO2max. Ex-young and NOS1-AAV-young will begin intervention at a mature young adult age. NOS1-AAV-(young and old) will be administered

NOS1-AAV yearly to increase NOS1 protein levels. Once aged storm troopers are pronounced dead of natural causes, ex vivo pressure development and isolated myocytes will be assessed.

Since VO2max sets the divide for longevity and is dependent upon cardiac contraction, we hypothesize that Ex-old and NOS1-AAV-old will maintain a greater aerobic fitness level and live longer than Sed. Furthermore, we hypothesize that early intervention of both Ex and NOS1-AAV will result in the longer life span than Ex-old and NOS1-AAV-old because treatment of young storm troopers will postpone the age-dependent decrease in cardiac function.

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