Effects of Neuronal Nitric Oxide Synthase Signaling on Myocyte
Contraction during β- Adrenergic Stimulation
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate School of
The Ohio State University
By
Lifei Tang
Biophysics Graduate Program
The Ohio State University
2013
Dissertation Committee:
Dr. Mark T. Ziolo, PhD Advisor
Dr. Brandon Biesiedecki, PhD
Dr. Jonathan Davis, PhD
Dr. Sandor Gyorke, PhD
a
Copyright by
Lifei Tang
2013
i
ABSTRACT
Nitric oxide (NO) is known to be a key regulator of cardiac contraction.
Within ventricular myocytes, NO is produced by two constitutively expressed NO synthase (NOS) isozymes, NOS1 and NOS3. It is well defined that NOS1 signaling results in positive inotropic and lusitropic effects under baseline conditions. This effect is largely due to the phosphorylation of phospholamban
(PLB) at Ser16 by the cAMP-dependent protein kinase (PKA) up-regulating sarcoplasmic reticulum (SR) Ca2+ uptake. In addition, our lab also demonstrated that NOS1 increases ryanodine receptor (RyR) activity via S-nitrosylation up- regulating SR Ca2+ release. Physiologically, heart function is largely regulated by the β-adrenergic (β-AR) pathway leading to positive inotropy and lusitropy.
Alterations in the β-AR pathway contribute to the contractile dysfunction, adverse remodeling, and arrhythmias in many cardiac diseases (i.e. heart failure (HF)).
The purpose of this dissertation is to investigate the role of NOS1 signaling during β-AR stimulation.
Previous studies have shown that NOS1 signaling contributes to the positive inotropy, but not lusitropy, during β-AR stimulation. Interestingly, unlike under baseline conditions, PLB phosphorylation is not altered in the condition of
NOS1 deficiency (acute NOS1 inhibition or NOS1 knockout) during β-AR stimulation. Thus, it is clear that there are other end targets of NOS1 other than
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PLB during stimulation of the β-AR pathway, possibly RyR. Hence the molecular mechanisms of NOS1 signaling during -AR stimulation are not known.
Phosphorylation of RyR by the Ca2+/Calmodulin-dependent protein kinase II
(CaMKII) has been reported to be the main cause of the enhanced SR Ca2+ release during β-AR stimulation. CaMKII is activated in response to β-AR stimulation and also has positive inotropic effects. Unfortunately, increased
CaMKII activity, which occurs in many heart diseases (i.e. hypertrophy and HF), leads to Ca2+ mishandling. Given that RyR is reported to be co-localized with
NOS1 and CaMKII, we are interested in whether the CaMKII pathway crosstalks with the NOS1 pathway to regulate RyR activity during β-AR stimulation. In addition, the upstream activator of NOS1 during β-AR stimulation is not known.
There is an Akt dependent phosphorylation site (Ser1416) on NOS1 which is reported to stimulate NOS1 activity, and it is known that Akt activity is increased during β-AR stimulation. Therefore, we will also investigate the possible activation of NOS1 by Akt during -AR stimulation.
We first investigated if NOS1 signaling was necessary for the enhanced
SR Ca2+ leak during β-AR stimulation. Rabbit ventricular myocytes displayed decreased -AR stimulated (isoproterenol, ISO) SR Ca2+ leak in the presence of acute, non-specific NOS inhibition by L-NG-Nitroarginine methyl ester (L-NAME).
Additional experiments indicated that NOS1 but not NOS3 was the NOS isozyme involved. Specifically, β-AR stimulated SR Ca2+ leak was only decreased by
NOS1 inhibition (S-Methyl-L-thiocitrulline (SMLT)), but not NOS3 inhibition (L-N5-
(1-Iminoethyl) ornithine (L-NIO)). We next determined if the NOS1 mediated
iii increase in SR Ca2+ leak during β-AR stimulation is via CaMKII. In isolated wildtype (WT) mouse myocytes, β-AR stimulated SR Ca2+ leak was decreased withCaMKII inhibition by KN93. However, KN93 had no effect on the already blunted -AR stimulated SR Ca2+ leak in NOS1 knockout myocytes (NOS1-/-).
Simply resupplying NO with S-Nitroso-N-acetyl-DL-penicillamine (SNAP) increased SR Ca2+ leak; and this effect was blocked by CaMKII inhibition, suggesting that CaMKII is downstream of NOS1. We then determined if the
NOS1/CaMKII-mediated increase in SR Ca2+ leak during β-AR stimulation also involves RyR modulation. RyR Ser2814 phosphorylation (CaMKII site) was increased in WT hearts during β-AR stimulation and was significantly blunted in
NOS1-/- hearts. We performed further experiments using RyR knockin mice
(CaMKII phosphorylation constitutively active (S2814D) or ablated (S2814A)) to determine if RyR is the end target of NOS1/CaMKII signaling. During -AR stimulation, NOS1 inhibition decreased SR Ca2+leak in WT myocytes but showed no effect in the RyR knockin myocytes. These data suggest that RyR is the end target of NOS1/CaMKII signaling.
We then investigated if the NOS1/CaMKII/RyR axis contributes to the positive inotropic effect of β-AR stimulation. In WT myocytes, CaMKII inhibition decreased -AR stimulated myocyte contraction (simultaneous measurement of
Ca2+ transients with Fluo-4 and cell shortening by edge detection). In NOS1-/- myocytes, CaMKII inhibition had no effect on the already blunted -AR stimulated contraction. Interestingly, resupply of NO via SNAP increased -AR stimulated contraction in NOS1-/- myocytes, and this effect was completely blocked by
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CaMKII inhibition. Consistent with these data, -AR stimulated fractional release
(an indicator of RyR activity) was decreased with CaMKII inhibiton in WT myocytes but not in NOS1-/- myocytes. We investigated the molecular mechanisms of the NOS1-mediated CaMKII activation. Western blot revealed that CaMKII autophosphorylation (Thr287) was increased in WT but not altered in
NOS1-/-myocytes during -AR stimulation. This effect was associated with an increase in CaMKII S-nitrosylation levels (western blot). Further, we found that
NO (SNAP) can directly activate CaMKII in vitro (CaMKII activity kit). These data indicate that NOS1 signaling directly increases CaMKII activity via S-nitrosylation to increase autophosphorylation levels. We performed additional experiments and excluded other potential mediators of CaMKII activation (Epac and oxidation) and RyR activity (PKA phosphorylation).
We designed experiments to determine if NOS1 signaling is activated by
Akt during β-AR stimulation. Our data demonstrate that both SR Ca2+ leak and
NOS1 phosphorylation at Ser1416 (Akt site) were decreased by Akt inhibition
(Akt inhibitor X) in isolated rabbit myocytes. Further, Akt inhibition (Akt inhibitor V) decrease β-AR-stimulated myocyte contraction in WT myocytes but had no effect in NOS1-/- myocytes.
Lastly, we determined if NO-dependent increase in RyR activity is involved in arrhythmogenesis during β-AR stimulation. NOS inhibition (L-NAME) attenuated ISO-stimulated spontaneous Ca2+ waves (SCaW) in rabbit myocytes and Ca2+aftertransients in failing human trabeculae.These data suggest that
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NOS-mediated CaMKII activitation in the pathological heart may be of significance.
In conclusion, these data suggest that during β-AR stimulation, NOS1 is activated by Akt and contributes to the positive inotropy via CaMKII-mediated
RyR activation. This pathway may also be involved in the arrhythmogenesis of the heart.
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DEDICATED TO MY FAMILY – SHANJIN TANG, GUIXIU LI, AND HAIYING XING.
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ACKNOWLEDGEMENT
Foremost, I would like to express my deepest appreciation to my advisor,
Dr. Mark Ziolo, for his continuous support of my Ph.D study. He was always patient in teaching me experimental design, experimental techniques, data analysis, and helped me improve my writing/speaking skills. He can promptly unravel the positive information from my research results and point out the right direction with his immense knowledge and sharp observation. His motivation and enthusiasm on science encourage me to bravely face the frustration and distress in research. He is generous and always fully supportive of my applications to grants and conferences. In the past five years, his continuous guidance helped me in my research and writing of this thesis. Dr. Ziolo is not only a good advisor, but also a good friend. He has a great sense of humor, and curiosity to foreign cultures. His lab has a very comforting environment. I could not have imagined having a better advisor and mentor for my time as a Ph.D. student.
Besides my advisor, I would like to thank the rest of my thesis committee:
Dr. Sandor Gyorke, Dr. Jonathan Davis, and Dr. Brandon Biesiadecki, for their encouragement, fair and sharp questions, insightful suggestions, and collaboration.
This thesis was born from a discussion between Drs. Tom Shannon, Jerry
Curran and Mark Ziolo. Thus, I would like to thank Drs. Shannon (and his group) viii and Curran for their indispensable and key contributions to this thesis. Further, I would like to thank the support and help given by the other collaborators and their group members: Drs. Peter Mohler, Thomas Hund and Paul Janssen.
Next I want to thank my current and previous labmates: Dr. Honglan Wang,
Dr. Bo Zhang, Dr. Steve Roof, Xin Huang, and Dr. Christopher Traynham for their help and guidance during my time in the lab.
Last but not least, I would like to thank my family: my parents Shanjin
Tang and Guixiu Li for giving birth to me and trusting me throughout my life; my husband Haiying Xing, the magic man I met in the US, for waiting and supporting me for the past 4 years.
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VITA
April 6th, 1984...... Born, Hunan, China
2001-2005...... B.S. Biotechnology, Wuhan University, China
2005-2008...... M.S. Biochemistry and Molecular Biology, Wuhan
University, China
2008-2013...... Graduate Research Associate, Department of
Physiology and Cell Biology, The Ohio State University
PUBLICATIONS
1. Tang L, Wang H, Ziolo MT. Targeting NOS as a Therapeutic Approach in Heart Failure. Pharmacology & Therapeutics. ACCEPTED
2. Curran J*, Tang L*, Roof SR, Velmurugan S, Millard A, Shonts S, Wang H, Ahmad DSU, Perryman M, Bers DM, Mohler PJ, Ziolo MT*, Shannon TR*. Nitric Oxide-dependent Activation of CaMKII Increases Diastolic Sarcoplasmic Reticulum Calcium Release in Cardiac Myocytes in Response to Adrenergic Stimulation. PLOS ONE. SUBMITTED *Authors contributed equally
3. Tang L, Roof SR, Huang X, Mohler PJ, Wehrens XH, Ziolo MT. Contribution of the NOS1/CaMKII/RyR2 Axis in the Positive Inotropic Response to β- adrenergic Stimulation in MouseVentricular Myocytes. IN PREPARATION
4. Wang H, Tang L, Zhang Bo, Roof SR, Huang X, Janssen PM, Davis JP, Kirschner LS, Villamena F, Biesiadecki BJ*; Ziolo MT*. NOS1 Orchestrates a Localized Nitroso-Redox Signaling Network in Ventricular Myocytes. IN PREPARATION x
*Authors contributed equally
5. Roof SR, Ho HT, 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
6. Roof SR, Tang L, Ostler JE, Periasamy M, Györke S, Ziolo MT. Neuronal Nitric Oxide Synthase is Indispensable for the Adaptative Contractile Effects of Exercise. Basic Res Cardiol. 2013 Mar;108(2):332.
7. Traynham CJ, Roof SR, Wang H, Prosak RA, Tang L, Viatchenko-Karpinski S, Ho H, Racoma IO, Catalano DJ, Huang X, Han Y, Kim S, Gyorke S, Billman GE, Villamena FA, Ziolo MT. Di-esterified Nitrone Restores Nitroso- Redox Balance and Increases Myocyte Contraction via Increased SR Ca2+ handling. PLoS One. 2012, 7(12):e52005.
8. He Y, Wang Y, Tang L, Liu H, Chen W, Zheng Z and Zou G. Binding of Puerarin to Human Serum Albumin: A Spectroscopic Analysis and Molecular Docking. Journal of Fluorescence, 2008, 18(2):433-42
9. Yang Y, Zhu S, Tang L, Liu D, Huang J, Zou G. Chemical Composition and Antimicrobial Activity of the Essential Oil of Cacalia tangutica (Maxim.) Hand.- Mazz.. Wuhan University Journal of Natural Sciences (in Chinese), 2007, 53(2): 198-203
10. Wang J, Tang L, Fu Y, Zhong J. Affects of E.coli K12 Culture Liquids and Sauces on Its Growth. Amino Acids and Biotic Resources (in Chinese), 2005, 27(1): 71-73
11. Fu Y, Tang L, Wang J, Zhong J. Communication among Bacteria. Amino Acids and Biotic Resources(in Chinese), 2004,26(4):62~64
FIELDS OF STUDY
Major Field: Biophysics Graduate Program
1. Cellular and Integrative Biophysics Track
2. Cardiac Physiology
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TABLE OF CONTENTS
ABSTRACT ...... ii
ACKNOWLEDGEMENT ...... viii
VITA ...... x
LIST OF FIGURES ...... xvii
Chapter 1: Introduction ...... 1
1.1 General Introduction ...... 1
1.2 Excitation-Contraction Coupling ...... 2
1.2.1 Action Potential ...... 2
2+ 2+ 1.2.2 Ca Transient, [Ca ]i ...... 3
1.2.3 Myocyte Contraction ...... 5
1.3 Regulation of ECC ...... 6
1.3.1 FFR ...... 7
1.3.2 The β-adrenergic receptor (β-AR) signaling ...... 8
1.3.3 NO signaling ...... 9
1.3.4 Role of CaMKII in ECC ...... 17
1.4 Heart Failure ...... 19
1.5 NOS signaling in heart failure ...... 20
1.5.1 NOS1 signaling in heart failure ...... 21
1.5.2 NOS3 signaling in heart failure ...... 23
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1.5.3 NOS2 signaling in heart failure ...... 23
1.6 Specific Aims, Objectives, and Rationales ...... 24
Chapter 2: CaMKII mediated SR Ca2+ leak is involved in the positive inotropy of
NOS1 signaling during β-AR stimulation ...... 38
2.1 Introduction ...... 38
2.2 Materials and methods ...... 40
2.2.1 Rabbit ventricular myocyte isolation ...... 40
2.2.2 Mouse ventricular myocyte isolation ...... 40
2.2.3 SR Ca2+ Leak Measurement ...... 41
2.2.4 SR Ca2+ load measurement ...... 41
2.2.5 Measurement of NO Production ...... 41
2.2.6 Simultaneous measurement of Ca2+ transients and myocyte shortening.
...... 42
2.2.7 SR Ca2+ load and SR Ca2+ fractional release ...... 43
2.2.8 Western blot analysis ...... 43
2.2.9 Solution and drugs ...... 43
2.2.9 Statistical analysis ...... 44
2.3 Results ...... 45
2.3.1 Enhanced diastolic SR Ca2+ leak during β-Adrenergic stimulation is
NOS dependent ...... 45
2.3.2 -AR stimulation in ventricular myocytes increases NO production .... 45
2.3.3 Increased diastolic SR Ca2+ leak during -AR stimulation is NOS1
dependent ...... 46
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2.3.4 Enhanced diastolic SR Ca2+ leak via NOS1 is CaMKII dependent...... 46
2.3.5 CaMKII regulates RyR activity via Phosphorylation at Ser2814 ...... 48
2.3.6 CaMKII phosphorylation of RyR is sufficient for the NOS1 mediated
increase in diasolic SR Ca2+ leak during β-AR stimulation ...... 48
2.3.7 CaMKII is involved in the positive inotropy of NOS1 signaling during β-
AR stimulation ...... 49
2.3.8 Resupply of NO in NOS1-/- myocytes rescues the blunted inotropic
response which is prevented by CaMKII inhibition ...... 50
2.3.9 The NOS1-mediated postivie inotropic effect during -AR stimulation is
via CaMKII modulation of RyR activity ...... 51
2.4 Discussion ...... 52
2.4.1 Effects of NOS1 during β-AR stimulation...... 52
2.4.2 Involvement of CaMKII in the NOS1 signaling pathway during β-AR
stimulation ...... 53
2.4.3 End targets of NOS1 signaling pathway during β-AR stimulation ...... 54
Chapter 3: Direct activation of CaMKII by NO and the upstream activator of
NOS1 during β-AR signaling ...... 71
3.1 Introduction ...... 71
3.2 Materials and Methods ...... 73
3.2.1 Rabbit ventricular myocyte isolation ...... 73
3.2.2 Mouse ventricular myocyte isolation ...... 73
3.2.3 Ca2+ Transient Measurement in Human Trabeculae ...... 74
3.2.4 Western Blot analysis ...... 75
xiv
3.2.5 Measurement of CaMKII Activity ...... 75
3.2.6 S-NO Western Blotting ...... 75
3.2.7 SR Ca2+ leak measurement ...... 76
3.2.8 SR Ca2+ load measurement ...... 77
3.2.9 Simultaneous measurement of Ca2+ transients and myocyte shortening.
...... 77
3.2.10 Spontaneous Ca2+ wave measurement ...... 77
3.2.11 Solution and drugs ...... 78
3.2.12 Statistical analysis ...... 78
3.3 Results ...... 79
3.3.1 Adrenergic stimulation Leads to NO-dependent, Ca2+ independent
CaMKII Activation ...... 79
3.3.2 CaMKII-dependent diastolic SR Ca2+ leak is not mediated through
oxidation ...... 80
3.3.3 CaMKII-dependent increase in diastolic SR Ca2+ leak is not mediated
through Epac ...... 81
3.3.4 The increase in RyR activity with ISO is Akt-dependent ...... 82
3.3.5 Inhibition of NOS attenuates arrhythmogenic spontaneous Ca2+ Waves
...... 83
3.3.6 Inhibition of NOS attenuates aftertransients in human heart failure .... 83
3.4 Discussion ...... 85
3.4.1 Effects of NOS1 on CaMKII activation...... 85
3.4.2 Akt is the upstream activator of NOS1 during β-AR stimulation ...... 87
xv
3.4.3 Relevance to cardiac diseases ...... 87
Chapter 4: Discussion ...... 99
4.1 Principle findings ...... 99
4.2 Future directions ...... 100
4.2.1 Physiological implication of NOS1 signaling cascade during β-AR
stimulation ...... 100
4.2.2 Pathological implications of the NOS1 signaling cascade ...... 103
4.3 Therapeutic targets of NOS1 dysfunction in heart disease ...... 105
4.4 Final Remarks ...... 106
LIST OF REFERENCES ...... 107
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LIST OF FIGURES
Figure 1. Excitation-contraction coupling in the cardiac myocyte...... 31
Figure 2. The cardiac action potential...... 32
Figure 3. β-adrenergic receptor signaling pathway in the cardiac myocyte...... 33
Figure 4. NOS structure ...... 34
Figure 5. Constitutively expressed NOS isoforms exert differential effects on myocyte contraction...... 35
Figure 6 NOS isozymes in HF myocardium...... 36
Figure 7 CaMKII activity ...... 37
Figure 8. Schematic of the diastolic SR Ca2+ leak protocol...... 57
Figure 9. ISO-dependent leak is attenuated by the non-specific NOS inhibitor L-
NAME...... 58
Figure 10 : ISO increases myocyte NO production in isolated rabbit myocytes.. 59
Figure 11 : Inhibition of NOS1 but not NOS3 reverses the ISO-dependent increase in SR Ca2+ leak...... 60
Figure 12: NOS1-/- myocytes show attenuated CaMKII-dependent leak...... 61
Figure 13: SNAP-dependent diastolic SR Ca2+ leak...... 62
Figure 14: Blunted RyR phosphorylation by CaMKII in NOS1-/- heart...... 63
Figure 15. No effect of acute NOS1 inhibition in RyR knockin myocytes...... 64
Figure 16: PKA affect on RyR is similar between WT and NOS1-/- hearts...... 65
xvii
Figure 17: Effects of CaMKII inhibition on cell contraction in WT and NOS1-/- myocytes...... 66
Figure 18: Resupply of NO to NOS1-/- myocytes rescues the functional response to β-AR stimulation, which is prevented by CaMKII inhibition...... 67
Figure 19: During β-AR stimulation, CaMKII inhibition decreases fractional SR
Ca2+ release in WT myocytes, but has no effect in NOS1-/- myocytes...... 68
Figure 20. KN92 has no effect on myocyte contraction...... 69
Figure 21. CaMKII inhibition does not change Ca2+ transient decline but slows the time to peak in WT compared to NOS1-/- myocytes during β-AR stimulation. 70
Figure 22. NOS1-/- hearts show attenuated CaMKII-autophosphorylation...... 89
Figure 23. NO increases CaMKII activity via S-nitrosylation...... 90
Figure 24. NADPH oxidase inhibitor is unable to decrease diastolic SR Ca2+ leak.
...... 91
Figure 25. No difference in CaMKII oxidation between WT and NOS1-/- hearts. 92
Figure 26. Epac activator 8-CPT has the same effects on diastolic SR Ca2+ leak in WT and NOS1-/- myocytes...... 93
Figure 27. Akt activates NOS1...... 94
Figure 28. Effects of Akt inhibition on cell contraction in WT and NOS1-/- myocytes...... 95
Figure 29. Inhibition of NOS attenuates SCaW formation in ISO treated myocytes.
...... 96
Figure 30: Inhibition of NOS attenuates Ca2+ aftertransients in ISO treated failing human ventricular trabeculae...... 97
xviii
Figure 31: Summary of NOS1 signaling pathway during β-AR stimulation ...... 98
xix
Chapter 1: Introduction
1.1 General Introduction
The cardiovascular system consists of the heart, blood, and blood vessels.
It is responsible for transport of oxygen, carbon dioxide, hormones, nutrients (i.e. amino acids, glucose, and electrolytes), blood cells, etc. to and from every organ; thereby maintaining homeostasis of the body. It is also responsible for waste removal of the body. The heart pumps de-oxygenated blood to the lungs and oxygenated blood to the periphery. Once the blood pumped to the body fails to reach the body demands, a person is at high risk. Therefore, a healthy, strong, electrically stable heart is crucial to survival. In fact, in the US, cardiovascular diseases caused 24.2% of all deaths in 2010[1], and there are 5,700,000 patients who have heart failure[2]. To better treat cardiovascular diseases, it is indispensable to bring in basic studies. The knowledge of mechanisms of heart functions could help us develop efficient treatments to tackle heart problems. The process that regulates heart contraction is termed “Excitation-Contraction coupling” (ECC), and a large field of research on ECC is focused at the level of ventricular myocytes.
1
1.2 Excitation-Contraction Coupling
Heart contraction is based on a process termed Excitation-Contraction
Coupling (ECC)[3]. ECC is initiated by an action potential (AP), causing ion movements across the plasma membrane, leading to secondary signaling inside the cell and generation of contractile force (Figure 1). More details of this process are explained below to provide sufficient background of this dissertation study.
1.2.1 Action Potential
The cardiac action potential (AP) is initiated at the sinoatrial (SA) node.
The pacemaker cells at the SA node spontaneously depolarize, and this waveform is transmitted to the atria via gap junctions causing atrial depolarization.
Next, this AP is carried to the atrioventricular (AV) node; where the propagation is slowed down to allow enough time for the atria to eject blood into the ventricles.
Then the AP is transmitted via the Bundle of His and the purkinje fibers to the left and right ventricles, leading to ventricular depolarization.
The AP presents different waveforms throughout the heart due to different ion channel expression and gating properties (Figure 2). In the SA node, the AP waveform is divided into 4 phases: A slow depolarization (Phase 4) is caused by inward Na+ current (the funny current, If)[4] and decreased permeability of K+ efflux. Subsequently an upstroke of the AP termed as Phase 0, is governed by inward Ca2+ current via the L-type (LTCC) and T-type (TTCC) Ca2+ channels[5].
To repolarize the cell, permeability of K+ channels (Ikr, Iks) is increased at Phase
3. However, SA nodal cells cannot reach a resting membrane potential like
2 ventricular cells at Phase 4, allowing these cells to spontaneously excite.
Therefore, the SA node regulates heart rate and the APs are termed as
“pacemaker potentials”[4]. In the ventricular myocytes, the AP is described with 5 phases. At Phase 0, the voltage-gated Na+ channels open due to the depolarization of the sarcolemmal membrane potential, leading to an inward Na+ current and upstroke of the AP. Right after the peak of the AP upstroke, there is a notch caused by an efflux of K+ ions, termed as Phase 1. The depolarization of the cell membrane triggers the opening of both the voltage-gated Ca2+ (L-type,
LTCC) and K+ channels. The inward Ca2+ and outward K+ current maintain a plateau phase (Phase 2) of the AP. Last, further K+ efflux contributes to the repolarization of the myocytes (Phase 3). The cell membrane potential returns to resting levels at Phase 4, and the Na+, K+, Ca2+ are returned to diastolic levels to await the next AP propagation via the Na+/ K+ -ATPase, Na+/ Ca2+ exchanger
(NCX), and sarcoplasmic reticulum Ca2+ ATPase (SERCA). During the action potential of ventricular myocytes, there is a triggered intracellular Ca2+ flux referred to as the “Ca2+ transient”, leading to a cascade of cell signaling and resulting in myocytes contraction.
2+ 2+ 1.2.2 Ca Transient, [Ca ]i
The Ca2+ transient begins when LTCC opens during an AP and causes an influx of Ca2+ into the cytosol of myocytes. This influx of Ca2+ triggers more Ca2+ release via ryanodine receptors (RyRs) from the sarcoplasmic reticulum (SR), the main source of Ca2+ storage within myocytes, resulting in increased cytosolic
[Ca2+][3, 6]. This process is termed calcium-induced-calcium-release (CICR).
3
Increased cytosolic [Ca2+] is a key regulator of myofilament contractile function in myocytes. Further, the open probability of RyR is also influenced by the amount of Ca2+ within the SR (SR Ca2+ load)[7]. Data shows that increased trigger Ca2+ from LTCC and SR Ca2+ load both lead to increases of fractional release from the
SR (Twitch SR leak/ SR load)[8]. Therefore maintenance of SR Ca2+ load is very important for proper myocyte contraction.
The decline of the Ca2+ transient and refilling of the SR with Ca2+ is necessary to maintain SR Ca2+ load for the consequent contraction. The decline of the Ca2+ transient is largely regulated by the sarcoplasmic reticulum Ca2+
ATPase (SERCA) and sodium-calcium exchanger (NCX). SERCA is a Ca2+
ATPase which pumps 2 Ca2+ ions back into the SR with 1 adenosine tri- phosphate (ATP) utilization[9, 10]. SERCA is responsible for ~ 70% of the Ca2+ transient decline in large mammals (i.e. rabbits and humans) while ~ 92% of the
Ca2+ transient decline in small rodents (i.e. mice and rats) is due to SERCA[6, 11].
The remaining Ca2+ transient decline is mainly regulated by NCX. NCX is an antiporter located on the sarcolemmal membrane with 2 pumping modes: in the forward mode, it pumps 1 Ca2+ ion out of the cell for 3 Na+ ions into the cell.
Worth to point out, these Na+ ions will eventually be pumped out of the cell via the Na+/ K+ ATPase and Na+/H+ exchanger to keep intracellular electrolyte balance. In the case of high [Na+] within myocytes, NCX functions in a reverse mode to pump one Ca2+ ion into the cell with 3 Na+ ions out of the cell[12]. In addition, it is reported that the mitochondria plays a role in Ca2+ decline[13], and
2+ 2+ this mitochondrial Ca uptake accounts for ~1-2 % of total [Ca ]i decline in small
4 and large mammals[14]. Therefore, mitochondrial Ca2+ uptake is a minor regulator of Ca2+ transient decline within myocytes.
Phospholamban (PLB), a pentameric protein associated with SERCA, also plays an important role in regulating SERCA function. PLB can be phosphorylated at Ser16 by the cAMP-dependent protein kinase A (PKA) or
Thr17 by the Ca2+/calmodulin-dependent protein kinase II (CaMKII). Our lab has shown that the Ca2+ transient decline of myocytes is predominantly regulated by
PLB phosphorylation at Ser16 [15]. In its dephosphorylated state, PLB is closely associated with SERCA, decreasing the affinity of SERCA for Ca2+, thereby blocking its pump function [16-18]. This “brake” effect on SERCA is released once PLB gets phosphorylated, resulting in increased SR Ca2+ load[19].
Furthermore, the Ser16 phosphorylation of PLB has been shown to precede the
Thr17 phosphorylation during β-AR stimulation[20]. In addition, PLB is dephosphorylated by protein phosphatases (PPs). Specifically, PP1 has been shown to account for ~ 70% of Ser16 dephosphorylation while PP2A is responsible for the rest (~ 30%)[21]. PP1 is also reported to account for ~ 74% of
Thr17 dephosphorylation[21]. In summary, the SERCA/PLB complex, NCX and mitochondria are responsible for the Ca2+ transient decline which is a key regulator of myocyte relaxation.
1.2.3 Myocyte Contraction
The contractile force is generated through a coordination of a protein complex termed “myofilaments”. The myofilaments consist of thick and thin filaments, which slide over one another to generate contraction. The thin filament 5 is comprised of troponin I (TnI), troponin C (TnC), troponin T (TnT), tropomyosin and actin, while the thick filament is mainly comprised of myosin[22]. At low cytosolic [Ca2+], actin is blocked by troponin and tropomyosin, and disassociated from the thick filament. Once cytosolic [Ca2+] increases during ECC, Ca2+ binds to TnC, resulting in the dissociation of TnI from actin and forming a tight association of TnC/TnI. This conformational change leads to the movement of tropomyosin, exposing the myosin binding site on actin. Next, with the hydrolysis of ATP, myosin interacts with actin and forms a cross-bridge of myosin and actin.
A power stroke is generated to shorten myocytes and produce force when inorganic phosphate and ADP are released from myosin. As [Ca2+] declines, the reverse occurs with ATP binding to myosin and causing dissociation of myosin from the thin filament resulting in myocyte relaxation.
1.3 Regulation of ECC
Since every myocyte contracts with every heartbeat, and external cues such as exercise, emotion, and disease conditions highly influence the demand of body, myocyte contraction needs to be highly regulated. There are numerous mechanisms regulating ECC to match the beat-to-beat changes or long term adaptations (i.e. chronic exercise, aging, and HF), such as the force frequency response (FFR), β adrenergic receptor (β-AR) stimulation, and nitric oxide signaling.
6
1.3.1 FFR
Myocyte contraction is modulated by frequency (i.e., heart rate), which is defined as the force-frequency-response (FFR). Specifically, with increasing stimulation rate, Ca2+ transient amplitude and myocyte contraction are increased, which is termed as a positive FFR[23] [24]. Positive FFR is correlated with increased SR Ca2+ load and increased SERCA Ca2+ uptake [25]. A suggested explanation is that increased heart rate leads to a shortened time for Ca2+ efflux via NCX, thereby leading to elevated cytosolic [Ca2+] and enhanced SERCA Ca2+ uptake. Subsequently, the SR Ca2+ load is increased and more Ca2+ can be released in the next beat. To match the increase in heart rate, the rate of relaxation has to be increased to ensure proper filling, known as force-dependent acceleration of relaxation (FDAR). The mechanism of FDAR is not fully understood yet. It has been suggested that activation of the SERCA/PLB complex contributes to the accelerated Ca2+ decline thereby increasing SR Ca2+ load and accelerating myocytes relaxation[26]. CaMKII activity is found to be increased in response to elevating diastolic [Ca2+] during FFR [27], and CaMKII regulates the FFR and FDAR via a PLB independent mechanism[28]. In addition, myofilament Ca2+ sensitivity has been shown to be decreased with the accelerated stimulation rate via TnI phosphorylation[28, 29],which is also involved in the FDAR. In vivo, the heart rate is mainly regulated by the parasympathetic (vagal) and sympathetic nervous system. The vagal system decreases heart rate via the neurotransmitter acetylcholine, while the sympathetic system increases heart rate and is responsible for the fight/ flight
7 response via the neurotransmitters of epinephrine and norepinephrine (i.e., catecholamines). The β-adrerergic receptor is the key receptor of the sympathetic nervous system in the heart. Increased β-AR signaling is usually accompanied with increased heart rate and stronger contractile force generation.
1.3.2 The β-adrenergic receptor (β-AR) signaling
The β-AR signaling pathway is a key regulator of ECC (Figure 3.).
Activation of the β-AR signaling leads to positive inotropy (contraction) and lusitropy (relaxation) in the heart [30]. The primary β-adrenergic receptors are β1 and β2. β1 and β2 receptors account for 75-80% and ~20% of all β-AR receptors, respectively[31]. The β-AR signaling pathway begins when an agonist (i.e. catecholamine) binds to the β-AR, which activates the associated G-protein. The primary G-proteins are the stimulatory G-proteins (Gs). Gs will then activate adenylate cyclase （AC） to increase cyclic AMP (cAMP) levels. cAMP binds to
PKA, leading to activation of PKA and phosphorylation of a variety of ECC proteins including LTCC, PLB, RyR, and TnI. PKA dependent phosphorylation of the LTCC increases Ca2+ influx, providing more trigger Ca2+ to the SR, therefore leading to increased CICR from the SR[32, 33]. PKA dependent PLB phosphorylation at Ser16 also releases SERCA from the “brake” status, and accelerates the Ca2+ uptake into the SR. Increased SR Ca2+ uptake leads to an increase in the SR Ca2+ load to enhance the SR Ca2+ release. The net outcome of the phosphorylation of LTCC and PLB is increased Ca2+ transient and cell contraction. The positive lusitropy is mainly regulated by two mechanisms, one is the acceleration of Ca2+ uptake via PLB phosphorylation, the other is the PKA
8 phosphorylation on TnI at Ser23/24 which decreases the Ca2+ sensitivity of myofilaments therefore promoting faster relaxation[34]. In addition, it is reported that PKA can phosphorylate RyR at Ser2808[35]. However, the significance of this phosphorylation is still under debate during β-AR stimulation[36].
Furthermore, the activation of CaMKII is also reported to be involved in the positive inotropy during β-AR stimulation which will be discussed later.
1.3.3 NO signaling
Nitric oxide (NO) is a soluble and highly diffusible gas that functions as a signaling molecule in the human body. Besides its well-recognized roles in endothelium-derived smooth muscle relaxation[37], synaptic transmission and immunological responses[38], NO is also a key regulator of heart function via modulation of excitation-contraction coupling (ECC) [39] and myocardial growth
[40].
NO signals via either the cyclic GMP (cGMP)-dependent or cGMP- independent pathways [41]. In the cGMP-dependent signaling pathway, NO activates soluble guanylate cyclase (GC) to increase cGMP levels, which modulates cGMP-regulated phosphodiesterases (PDE; cGMP-stimulated: PDE2; cGMP-inhibited: PDE3) or the activation of cGMP-dependent protein kinase G
(PKG) to modulate a variety of protein targets [42-45]. In the cGMP independent pathway, NO regulates protein function via the post-translational modification of
S-nitrosylation, which is the addition of a nitrosyl group to a free thiol on a cysteine residue of the target protein[46]. Furthermore, NO can rapidly react with
.- - - the superoxide anion radical (O2 ) to form peroxynitrite (ONOO )[47]. ONOO acts 9 as a buffer to ROS levels [48], as well as a signaling molecule[49]. Low levels of
ONOO- are reported to mediate beneficial protein glutathionylation and nitrosylation, while high levels of ONOO- can lead to irreversible and harmful nitration or oxidation of proteins [50, 51].
1.3.3.1. NO Synthases
NO is endogenously produced by enzymes termed Nitric Oxide Synthase
(NOS) (Figure 4). In mammals, three isoforms of NOS are encoded by different genes [52]. Within ventricular myocytes, neuronal NOS (NOS1, nNOS) and endothelial NOS (NOS3, eNOS) are constitutively expressed [53, 54], while inducible NOS (NOS2, iNOS) is expressed due to environmental cues (i.e. cytokine production during immune responses) [55]. Production of NO via NOS1 and NOS3 is calcium-dependent [56, 57], while NOS2 is a calcium-independent enzyme [58]. Furthermore, within cardiac myocytes, NOS2 produces much higher levels of NO compared to NOS1 and NOS3 [59].
NOS functions as a dimer composed of two identical monomers. The C- terminus (reductase domain) of the monomer consists of nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) binding sites, while the N-terminus (oxygenase domain) consists of tetrahydrobiopterin (BH4), oxygen (O2), and L-arginine binding sites along with a heme group for NOS dimerization[60, 61]. There is also a calmodulin binding domain in between the reductase and oxygenase domains.
Flavin serves to transfer an electron from NADPH in the reductase domain of one
10 monomer to the oxygenase domain of the other monomer resulting in the production of NO via oxidation of L-arginine to L-citrulline [62, 63]
A state of oxidative stress (i.e., increased levels of reactive oxygen species- ROS) can interrupt NO production by NOS leading to the generation of
- superoxide anion (O2∙ ) instead of NO, termed NOS uncoupling [64, 65]. BH4 is known to bind to NOS at the interface of two monomers to stabilize the dimer structure. Depletion or oxidation (to BH2) of BH4 causes the electrons to flow from the reductase domain to oxygen at the oxidase domain within the monomer,
.- producing O2 [66]. Additionally, glutathionylation of NOS3 at Cys689 and Cys908
- blocks the reduction of NADPH to NADP, leading to O2∙ generation at the reductase domain [67]. ONOO- toxicity is also reported to uncouple NOS3 via oxidation of the central zinc bound in the heme domain [68]. Thus, NOS serves as the primary source of NO within ventricular myocytes. However, under
- - disease conditions, it can also result in O2∙ production and increased ONOO levels.
1.3.3.2. NOS1 signaling
NOS1 was first identified in the brain [69], and was termed neuronal (or brain) NOS. However, expression of this isozyme has been found in numerous cell types (including ventricular myocytes) and has since been renamed NOS1.
In the myocyte, NOS1 is known to be predominantly localized to the SR [53, 70], but it also found at the mitochondria [71]and sarcolemmal membrane [72]. At the
SR, NOS1 has been shown to co-immunoprecipitate with RyR and xanthine oxidoreductase (XOR) [53, 70, 73]. NOS1 signals through the cGMP- 11 independent signaling pathway (i.e, S-nitrosylation via generation of low ONOO-)
[74], and acts as a major regulator of ECC (Figure 5).
It appears that the chief source of NO to modulate myocyte Ca2+ handling is by NOS1. Contractile studies performed on myocytes with genetic deletion of
NOS1 (NOS1-/-) demonstrated that these myocytes had depressed basal contraction along with prolonged relaxation [74]. Our lab believes that these observed functional effects in NOS1-/- myocytes were due to loss of NOS1 signaling (and not some compensatory adaptation) since these results could be recapitulated with acute NOS1 inhibition (S-methyl-L-thiocitrulline- SMLT) in wildtype (WT) myocytes [70, 74-76]. NOS1 signaling is also imperative for the positive force-frequency response (FFR) and the β-adrenegic (β-AR)-induced positive inotropy [70, 74, 76]. That is, with NOS1 knockout or acute inhibition of
NOS1, myocytes have a blunted FFR and reduced contractile response to β-AR stimulation. The major factor for the positive FFR and inotropy with β-AR stimulation is an increase in the Ca2 +transient amplitude, which is not observed with NOS1 knockout or inhibition. As we mentioned above, a major determinant of Ca2+ transient amplitude is the SR Ca2+load [7], we and others have reported that there is a significant reduction in the SR Ca2+ load with NOS1 knockout or inhibition [74, 76]. In NOS1-/- myocytes, basal PLB serine16 phosphorylation is decreased [74, 75]. Thus, the decrease in PLB phosphorylation is the main cause of the slowed Ca2+transient decline and reduced SR Ca2+ load[77]. Our lab believes the decreased phosphorylation is due to a decrease in basal PKA activity. Specifically, NOS1 signals via ONOO- formation, and ONOO- can directly
12
(in the absence of cAMP) activate PKA [78], most likely via S-nitrosylation[79].
Consistent with these findings, myocyte-specific NOS1 over-expression mice showed increased basal PLB Serine16 phosphorylation [80].However, it still remains to be directly shown that NOS1 signaling regulates PKA activity. There is also evidence that NOS1-/- myocytes have increased phosphatase (both PP1 and PP2a) activity [75].This is most likely due to the increased oxidative stress
- that is present in these myocytes [73, 81-83], as O2∙ can activate both PP1 and
PP2a [84, 85]. In fact, in canine and murine exercise models, we have found upregulated NOS1 expression, and increased PLB Serine16 phosphorylation due to increased PKA activity and decreased PP activity [86].
Interestingly, there was no difference between WT and NOS1-/- myocytes in PLB Ser16 phosphorylation and Ca2+transient decline during β-AR stimulation
[74, 77], suggesting that the cAMP-induced activation of PKA is normal in NOS1-
/- myocytes. Thus, it appears NOS1 signaling mediates PLB phosphorylation at
Ser16 via regulation of PKA and PP activity under basal conditions, leading to positive inotropy. However, during β-AR stimulation, although PKA activation is intact, the functional response to β-AR stimulation is blunted in NOS1-/- myocytes
[70, 74]. These data indicate that NOS1 signaling has another target, which is the ryanodine receptor (RyR).
Provocatively, RyR protein levels are increased in NOS1-/- myocytes [87-
89], which the lab believes to be a compensatory mechanism for the decreased
SR Ca2+ release and Ca2+ cycling in the cell. The lab has previously showed that
NOS1 signaling activates RyR via S-nitrosylation under baseline conditions [89].
13
Therefore, the decrease in RyR activity is due to a lack of S-nitrosylation that limits Ca2+release from the SR reducing basal contraction. XOR, which is
- involved in purine degradation and is one of the major sources of O2∙ [90], also
- co-localizes with NOS1 and RyR at the SR [73]. O2∙ production by XOR is inhibited by NO [91]. The loss of XOR inhibition in the NOS1 knockout is the foremost reason for the oxidative stress in NOS1-/- myocytes [73]. Hence, in addition to S-nitrosylation, NOS1 signaling also helps maintain normal RyR activity by preventing RyR oxidation. However, whether S-nitrosylation of RyR can explain the positive inotropy of NOS1 during β-AR stimulation is still unclear.
Further studies are still needed to validate if there are other mechanisms to regulate RyR activity by NOS1 signaling during β-AR stimulation. This is the purpose of my thesis.
NOS1 signaling occurs at the subcellular SR domain to directly (S- nitrosylation) and indirectly (phosphorylation via possibly regulating PKA and PP activity) regulate protein function and contraction. Loss of this compartmentalized signaling results in decreased contraction and prolonged relaxation through alterations in PLB phosphorylation, RyR activity, and increased XOR function.
Interestingly, these phenomenon are broadly observed in heart failure as well [92]
Therefore, dysfunction of NOS1 is very likely involved in the process of heart failure, and studies on NOS1 deficiency models can help to reveal, in part, the molecular mechanisms of heart failure.
14
1.3.3.3 NOS3 signaling
NOS3 was first identified in the coronary endothelium and was termed endothelial NOS [93]. However, expression of this isozyme has been found in numerous cell types (including ventricular myocytes) and has since been renamed NOS3. In the myocyte, NOS3 is known to be localized to caveolae by
2+ binding with Caveolin-3 (Cav-3) [70, 94]. In addition to increased [Ca ]i, NOS3 is also activated via phosphorylation at Ser1179 by Akt (protein kinase B)[95], which is important for cardioprotection in end-stage heart failure[96].
Although NO is a highly diffusible gas, NOS signaling is temporally and spatially localized (i.e., compartmentalized signaling) [97]. Since NOS1 and
NOS3 are localized to different domains (caveolae vs SR, respectively) within the ventricular myocyte, each has unique signaling pathways, protein end targets, and functional effects on the heart (Figure 5) [39, 70, 94]. Unlike NOS1, NOS3 signaling does not regulate basal myocyte contraction but only β-AR stimulated contraction [70, 76, 98]. In the caveolar microdomain, NOS3 closely localizes with L-type Ca2+ channels (LTCC) and β-adrenergic receptors [99].Superoxide
.- dismutase (SOD), which degrades O2 , is also localized with NOS3 [100]. The
.- buffering of O2 by SOD drives NOS3 to signal via the cGMP/sGC/PKG pathway to phosphorylate various protein targets (i.e. LTCC). In contrast to NOS1-/- myocytes, NOS3-/- myocytes showed an increased functional response to β-AR receptor stimuli [70, 98, 101, 102], whereas mice with NOS3 overexpression showed a decreased functional response to β-AR stimulated contraction [103,
104].
15
Due to the same subcellular localization, NOS3 modulates LTCC.
Specifically, acute NOS3 inhibition in WT myocytes or NOS3 knockout (NOS3-/-)
2+ myocytes had a greater β-AR stimulated Ca current (ICa) [98]. In addition, our data demonstrated that PKG is involved in this pathway and likely phosphorylates LTCC at Ser496 on the β2a subunit [42]. Furthermore, we determined that the effect of NOS3 on LTCC is restricted to caveolar microdomain via phosphodiesterases type 5 (PDE5)[105]. PDE5 is specific for cGMP and thus will prevent cGMP from freely diffusing. There also appears to be gender differences with NOS3 signaling. Sun et al. observed greater effects of
NOS3 on LTCC in female myocytes compared to male myocytes [106]. I believe it is this NOS3/PDE5/PKG/LTCC pathway that blunts the contractile response to
β-AR stimulation.
In addition to limiting β-AR stimulated Ca2+ influx, NOS3 signaling also shortens the action potential waveform. Specifically, NOS3-/- mice exhibit prolonged action potential (AP) duration with β-AR stimulation[98]. A key determinant of AP duration is the various K+ channels [107]. However, our lab
+ observed no differences in K channels (transient outward (Ito), sustained (IKsus),
-/- or inward rectifier (IK1)) between WT and NOS3 myocytes [108]. Thus, I deduce that the increased Ca2+ influx in NOS3-/-myocytes results in greater
Na+/Ca2+exchange (NCX) current that prolongs AP duration [109].
While NOS3 signaling does blunt the contractile effects of β-AR stimulation, this effect is minor and not always observed [110]. Therefore, NO produced via NOS3 has a different role compared to NOS1. The lab proposes
16 that NOS3 signaling is protective against adrenergic toxicity. In summary, NOS3- derived NO is a negative regulator of β-AR stimulated contraction via inhibition of
LTCC and limiting Ca2+influx.
1.3.4 Role of CaMKII in ECC
CaMKII is a serine/threonine protein kinase. There are 4 CaMKII isoforms,
CaMKIIα, CaMKIIβ, CaMKIIγ, and CaMKIIδ in the body. CaMKIIδ is the predominant isoform in the heart. CaMKIIδc is the cytosolic isoform and
CaMKIIδb is the isoform located in the nucleus. CaMKII activation is initiated in response to elevated [Ca2+] via binding of the Ca2+/Calmodulin complex and is then autophosphorylated at Thr287. The autophosphorylation sustains its activity independent of Ca2+, therefore keeping CaMKII active at low levels of [Ca2+] such as observed in diastole. In addition, CaMKII is reported to be activated via oxidation at Met281/282 by NADPH oxidase after the initial Ca2+ dependent activation[111, 112]. Further, elevated CaMKII activity has been shown to occur during β-AR stimulation [113]. Previous studies have suggested that the activation of CaMKII during β-AR stimulation is due to either the elevated [Ca2+] cycling[114] or Epac signaling pathway [115]. However, further studies are still required to illustrate the mechanism of CaMKII activation during β-AR stimulation, and this is also the purpose of my thesis.
CaMKIIδc involvement in regulation of Ca2+ handling during ECC is via phosphorylation of a variety of downstream targets including LTCC[116], PLB
(Thr17) [117] and RyR (Ser2814 or 2815, species dependent)[118] (Figure 7).
Previous studies have shown that specific CaMKII phosphorylation of RyR leads
17 to increased RyR open probability[119], resulting in an increased fractional SR
Ca2+ release (twitch Ca2+ transient/SR Ca2+ load)[120]. This will result in increased Ca2+ transient and shortening amplitudes. The phosphorylation of RyR via CaMKII is reported to be more dominant at increasing RyR activity than phosphorylation via PKA during β-AR stimulation [121-123]. The importance of
CaMKII specific phosphorylation of PLB remains controversial. It has been demonstrated that phosphorylation of PLB at Thr17 helps increase SR Ca2+ reuptake in more stressed conditions such as prolonged isoproterenol perfusion but not acute -AR stimulation[19, 124]. CaMKII phosphorylation at the β2a subunit (Thr498) of LTCC is reported to increase the channel open probability, therefore increase the influx of ICa. Furthermore, a recent study showed that
CaMKII can direct phoshoprylate titin, indicating an effect on myofilament function [125]. Hence, additional studies are required to fully comprehend the role of CaMKII signaling, especially during -AR stimulation.
Although CaMKII is an essential regulator of myocyte contraction, under certain diseased conditions (i.e. hypertrophy, heart failure), CaMKII has been shown to have a negative influence on heart function. Both CaMKII expression and activity have been found to be upregulated in human heart failure and failing animal models[126, 127]. It is reported that the hyperphosphorylation of RyR by
CaMKII is the major cause of increased SR Ca2+ leak, which decreases the SR
Ca2+ load and elevates diastolic cytosolic [Ca2+], thereby impairing the contractility of the heart[120-122, 128]. In addition, a hyperactive RyR also causes SR Ca2+ waves and cytosolic Ca2+ overload, which contribute to the
18 arrhythmogenesis and sudden death in failing heart[129]. CaMKII is found to phosphorylate cardiac Na+ channels [130] and K+ channels [131]. CaMKII phoshprylation on Nav 1.5 triggers the late INa current, thereby inducing arrhythmias in heart failure [132]. CaMKII phosphorylation on KV4.3 inhibits Ito, which interrupts the AP repolarization during heart failure[133].
CaMKII activity is also involved in the calcineurin/NFAT signaling pathway.
Phosphorylation of calcineurin by CaMKII leads to the nuclear translocation of
NFAT, thereby facilitating the process of hypertrophy and the transition from hypertrophy to heart failure. Data shows that CaMKII knock-out mice had cardioprotective effects after pressure overload treatment [134, 135]. Taken together, CaMKII signaling is crucial to maintain normal myocyte contraction in the healthy heart, but elevated CaMKII activity is involved in a variety pathological process such as myocardial contractile dysfunction, cardiac remodeling and arrhythmogenesis in diseased hearts.
1.4 Heart Failure
Heart failure is defined as the inability of the heart to pump enough blood to meet the metabolic demands of the body. Various experimental animal models are generated to produce heart failure, such as myocardial infraction, pressure overload, toxic drug treatment, and mutations of sarcomeric and cytoskeletal proteins [136]. Severe heart failure displays ventricular dilation and limited contractile reserve.
19
At the myocyte level, a final common pathway of HF, regardless of the causative event, is a lower SR Ca2+ load resulting in depressed cell contraction
[137]. An element of the lower SR Ca2+ load is a reduction in SR Ca2+ uptake.
This is due to decreased SERCA expression and/or reduced PLB Ser16 phosphorylation. Also, the expression and activity of NCX is increased in HF leading to a loss of Ca2+ from the myocyte. Another factor is an increase in diastolic SR Ca2+ leak due to increased RyR activity. Associated with the
Ca2+mishandling, Na+ handling is also altered (increased NCX and Na+/H+ exchanger activity, late Na+ current, etc.) leading to Na+ overload and increasing the propensity of arrhythmias.
Much of the altered Ca2+ and Na+ handling in HF can be attributed to oxidative stress [92]. Oxidative stress is due to increased ROS production (via mitochondria, NADPH oxidase, XOR, NOS uncoupling, monoamine oxidase, etc) and decreased degradation (downregulated SOD, glutathione, NO buffering etc.).
This leads to an increase in ONOO- levels[138] with a reduction in NO bioavailability. The dis-equilibrium of ROS and NO levels is termed “nitroso-redox imbalance”.
1.5 NOS signaling in heart failure
- While there is decreased [NO]i due to buffering with O2∙ in HF, there also exists vicissitudes in NOS activity resulting in lowered NO production. Thus, we will discuss how signaling via each NOS isoform is disrupted during HF (Figure
6).
20
1.5.1 NOS1 signaling in heart failure
There is ample evidence that NOS1 is involved in the contractile dysfunction in HF. Particularly, studies have shown that myocardial infarction in
NOS1-/- mice display worse ventricular remodeling, contractile dysfunction, and mortality compared to WT mice [139, 140]. Whereas, mice with cardiac myocyte- specific NOS1 overexpression developed less dilation, maintained contractile function, and prevented the development of HF in a TAC (transverse aortic constriction) model. The protein end targets and components of the NOS1 signaling pathway exhibit altered function in HF (i.e., decreased PLB Ser16 phosphorylation [141, 142], increased diastolic SR Ca2+leak [143], and increased
XOR activity[144]). The increased diastolic SR Ca2+leak is due to enhanced RyR activity and has been explained by increased RyR phosphorylation via CaMKII
[123, 145, 146], or thiol oxidation of RyR [144, 147, 148]. ROS production from
XOR is increased, which contributes to the increased RyR oxidation[144] and possibly CaMKII activation, which can occur in the absence of Ca2+/calmodulin
[111]. In addition, NOS1 is found to be uncoupled under oxidative stress in vitro
[149], but whether this process exists in HF and contributes to the oxidative stress is still not known.
Interestingly, expression and activity of NOS1 were found to be increased in human HF and a rat myocardial infarction HF model. However, NOS1 translocates from the SR to caveolae in the sarcolemmal membrane by binding to caveolin-3 [150-152]. We believe that this is analogous to knockout of NOS1 arising to similar functional effects we and others observe in NOS1-/- mice. These
21 data suggest that the loss of NOS1 signaling at the SR microdomain contributes to the dysregulation of Ca2+ handling in HF.
NOS1 signaling has also been reported to be protective against arrhythmias. The translocation of NOS1 has been shown to decrease β-AR stimulated contraction via reducing LTCC by PKG phosphorylation [151, 153] acting similar to NOS3 in healthy hearts. This occurs since NOS3 expression is decreased in HF and NOS1 recoups the loss of NO in this microdomain.
Consistent with that assertion, NOS1-/- mice have increased arrhythmias after myocardial infarction [154]. Furthermore, CAPON (carboxy-terminal PDZ ligand of NOS1), a NOS1 adaptor protein, is reported to be beneficial in preventing prolonged QT interval and sudden cardiac death [155, 156], by mediating NOS1 and ICa reduction. With the decrease in NOS3 expression [157] and/or uncoupling of NOS3 [158] in HF, we believe the translocation of NOS1 plays a compensatory mechanism for the loss of NOS3 to prevent arrhythmias. This will also limit detrimental Ca2+ influx to prevent pathological hypertrophy. Thus, a beneficial therapeutic approach is to increase NOS1 or NO. In fact, we have
- shown that simultaneously restoring NO levels and decreasing O2∙ levels was beneficial for contraction in NOS1-/- [83]. However, care must be taken to assure that this occurs at the SR microdomain. NOS1 is also shown to form a protein complex with PMCA4b, sodium channel (Nav1.5), and β-syntrophin located at a different subcellular domain- the sarcolemma membrane [72]. S-nitrosylation of
Nav 1.5 by NOS1 increases late Na+ current and derangement of this complex can lead to increased late Na+ current causing long QT syndrome [159].
22
1.5.2 NOS3 signaling in heart failure
NOS3 expression levels have been shown to be decreased in HF [157].
There is also a reduction in Caveolin-3 expression [160], which may further mediate the loss of NOS3 signaling in the correct subcellular location. The decrease in NOS3 activity decreases NO production, which is an enormous detriment to the failing heart.
The negative effect of losing NOS3 signaling is prominently illustrated in
NOS3-/-mice. After a myocardial infarction, NOS3-/- mice display worse ventricular remodeling, hypertrophy, and contractile dysfunction [161], whereas cardiomyocyte targeted NOS3 expression rescued the LV remodeling after pressure overload [162]. As previously discussed NOS3 acts as an antihypertrophic factor in a cGMP dependent manner by inhibition of the calcineurin-NFAT pathway and protects the heart from arrhythmias. Thus, in the setting of HF, increasing expression of NOS3 is an attractive therapeutic approach. However, caution must be taken with this approach since NOS3 can
. become uncoupled in HF to produce O2 and increase ROS levels in the heart
[158]. This effect may be further exacerbated by a decrease in SOD expression with HF [163]. Thus, a more advisable approach would be to target the beneficial downstream targets of NOS3 (e.g., decrease PDE5 to increase cGMP levels).
1.5.3 NOS2 signaling in heart failure
NOS2 was first identified in activated (but not quiescent) macrophages and was termed inducible NOS [164] and renamed to NOS2 to be consistent with the new nomenclature. During an inflammatory response, expression of NOS2
23 can also be induced within ventricular myocytes where it is a cytosolic protein
[165]. Since it’s observation in cardiac myocytes [55], NOS2 is widely considered to be involved in various pathophysiological conditions of the myocardium, such as ischemia-reperfusion injury [166], septicemia[167-169], aging [170], infarction
[171], and heart failure[172]. NOS2 signals via the cGMP-dependent and independent pathways leading to targeting of different end proteins with distinctive post-translational modifications (phosphorylation, S-nitrosylation, nitration, oxidation).
Expression of NOS2 has been found to contribute to the contractile
dysfunction in many diseases. In addition, in HF O2∙ production via mitochondria,
NADPH oxidase, and XOR are increased. With its extremely high rate constant,
- NO will react with O2∙ to favor the formation of high levels of ONOO [47]. It has been shown that high levels of ONOO- results in contractile dysfunction with
NOS2 expression [138]. Studies using NOS2 knockout (NOS2-/-) mice clearly show that NOS2 expression is detrimental in HF [171, 173, 174]. The mechanism by which NOS2 results in contractile dysfunction in HF is by blunting basal contraction and the positive inotropic response during β-AR stimulation by reducing Ca2+ transient amplitude[172, 175]. This is accomplished by altering
RyR activity and reducing PLB phosphorylation [167, 176, 177].
1.6 Specific Aims, Objectives, and Rationales
Nitric Oxide (NO) has been known as a key regulator of cardiac contraction for the past several decades [39]. It is well defined that signaling via the two constitutive expressed NOS isozymes, NOS1 and NOS3, are
24 compartmentalized to regulate myocyte Ca2+ cycling and cell contraction [41].
Since NOS1 appears to be the main NOS which regulates Ca2+ handling and is involved in mediating the -AR pathway[70, 74-76], my project will focus on
NOS1. NOS1 contributes to positive inotropy and lusitropy during basal myocyte contraction, largely due to the phosphorylation of PLB at Ser16 (PKA site), thereby up-regulating the SR Ca2+ uptake. In response to β-AR stimulation,
NOS1 signaling also has positive inotropic effects, but without lusitropic effects[74, 77]. It is clear that there are downstream targets of NOS1 other than
PLB for regulation of SR Ca2+ handling, but the mechanism(s) are still not understood. Given that phosphorylation of RyR by CaMKII has been reported to be the main cause of the enhanced SR Ca2+ release during β-AR stimulation[121-123], and RyR co-immunoprecipitates with NOS1[178], I am specifically interested in the role of CaMKII mediated RyR function in the NOS1 signaling pathway during β-AR stimulation. In addition, the upstream activator of
NOS1 during β-AR stimulation is not well defined. There are several post- translational modifications of NOS1 including a stimulatory phosphorylation of
NOS1 at Ser1416 by Akt [179]. In this dissertation, I also investigated the role of
Akt in activating NOS1 during β-AR stimulation. Furthermore, I am interested in
NOS1 signaling in disease conditions, and measured the effects of NOS1 inhibition on arrhythmogenesis in healthy rabbit and failing human hearts. To summarize, I hypothesize that NOS1, activated by Akt, regulates CaMKII activation, thereby mediating positive inotropy via RyR phosphorylation during β-AR stimulation.
25
To test this hypothesis, we propose the following aims:
Specific Aim 1: Determine if NOS1 signaling is necessary for diastolic SR
Ca2+ leak during β-AR stimulation.
Previous work has demonstrated that diastolic SR Ca2+ leak is increased during -AR stimulation [180]. However, the molecular mechanisms responsible for the increased diasolic SR leak are unresolved. The lab has previously shown that NOS1-/- myocytes have a decreased basal diastolic SR Ca2+ leak[89]. The purpose of this aim is to investigate if NOS1 signaling plays a role in regulation of diastolic SR Ca2+ leak during -AR stimulation. SR Ca2+ leak-load measurement will be performed in -AR stimulated isolated ventricular myocytes from rabbit (± acute inhibition of NOS1 and NOS3) and WT and NOS1-/- myocytes. I hypothesize that NOS1 deficiency via acute inhibition or genetic knockout will lead to a blunted diastolic SR Ca2+ leak-load relationship compared to WT and
NOS3 inhibition during -AR stimulation.
Specific Aim 2: Determine if NOS1 dependent SR Ca2+ leak is mediated by
CaMKII during β-AR stimulation.
The enhanced diastolic SR Ca2+ leak during -AR stimulation has been reported to be dominantly regulated by CaMKII[121-123].The purpose of this aim is to determine if CaMKII activation is NOS1 dependent. SR Ca2+ leak-load measurement will be performed in the presence of CaMKII inhibition in WT and
NOS1-/- myocytes (±-AR stimulation). Measurement of SR Ca2+ leak with exogenous supply of NO (±CaMKII inhibition) will be done to determine if CaMKII
26 is downstream of NOS1. I hypothesize that CaMKII inhibition will block the SR
Ca2+ leak in WT myocytes but have no effect in NOS1-/- myocytes. Further, exogenous NO will be enough to enhance SR Ca2+ leak and this effect will be completely blocked by CaMKII inhibition.
Specific Aim 3: Determine if RyR is the end target of the NOS1/CaMKII mediated diastolic SR Ca2+ leak during β-AR stimulation.
It is known that phosphorylation of RyR at Ser2814 by CaMKII is the prominent modification of RyR during β-AR stimulation[121]. The purpose of this aim is to determine if CaMKII–mediated RyR phosphorylation is an end target of the NOS1 signaling pathway. Western blotting will be used to measure specific
Ser2814 (CaMKII site) and Ser2808 (PKA site) phosphorylations in WT and
NOS1-/- ventricular homogenates (±-AR stimulation). In addition, RyR knockin mice with serine 2814 replaced with alanine (S2814A, phospho site ablated) or aspartic acid (S2814D, phosphorylation mimetic) will be used to measure the SR
Ca2+ leak-load relationship with NOS1 inhibition. I hypothesize that Ser2814 phosphorylation will be elevated in response to β-AR stimulation in WT but unaltered in NOS1-/- hearts; PKA phosphorylation will show no difference between WT and NOS1-/- hearts; and NOS1 inhibition will have no effects on the already depressed (S2814A) or hyperactive (S2814D) SR Ca2+ leak-load relationship.
Specific Aim 4: Determine if the NOS1/CaMKII/ RyR axis contributes to the positive inotropy during β-AR stimulation.
27
The prupose of this aim is to determine if the NOS1/CaMKII/RyR axis contributes to the positive inotropy of NOS1 signaling during β-AR stimulation.
We will simultaneously measureme cell shortening and Ca2+ transients (±CaMKII inhibition) in WT and NOS1-/- myocytes. Supply of exogenous NO (±CaMKII inhibition) will also be performed in NOS1-/- to further explore if CaMKII is downstream of NOS1. I hypothesize that blunted myocyte contraction via
CaMKII inhibition will only be observed in WT myocytes but not NOS1-/- myocytes; resupply of NO will rescue the blunted myocyte contraction in NOS1-/- myocytes and will be completely prevented by CaMKII inhibition.
Specific Aim 5: Define the mechanism of NOS1-mediatedCaMKII activation
Regulation of protein function directly by NO occurs via the post- translational modification termed S-nitrosylation [46]. The purpose of this aim is to determine if NOS1 can directly activate CaMKII via S-nitrosylation. CaMKII S- nitrosylation and activity (autophosphorylation at Thr286) will be measured via western blotting in WT and NOS1-/- hearts (± β-AR stimulation). An in vitro
CaMKII activity kit will also be used to investigate the effect of a NO donor on purified CaMKII protein. I will also examine other possible mechanisms of
CaMKII activation, such as ROS and EPAC. I hypothesize that CaMKII activity is increased in WT myocytes during β-AR stimulation, while this effect will not be observed in NOS1-/- myocytes. Furhtermore, NO will directly activate CaMKII and that S-nitrosylation (and not Epac or oxidation) of CaMKII is increased in myocytes in response to β-AR stimulation.
28
Specific Aim 6: Determine if NOS1 signaling is activated by Akt during β-
AR stimulation.
The activation of NOS1 during β-AR stimulation is not well understood.
There is an Akt dependent phosphorylation site (Ser1416) on NOS1 which is reported to stimulate NOS1 activity[179]. Akt is known to be activated during β-
AR stimulation. The purpose of this aim is to determine if Akt can activate NOS1 to stimulate the downstream CaMKII/RyR mediated SR Ca2+ release.
Phosphorylation of NOS1 at Ser1416 will be detected by western blotting (±β-AR stimulation). We will measure SR Ca2+ leak-load relationship and myocyte contraction during β-AR stimulation (±Akt inhibition). I hypothesize that NOS1
Ser1416 phosphorylation will be increased with β-AR stimulation; and Akt inhibition will decrease SR Ca2+ leak and myocyte contraction in WT myocytes, and have no effect in NOS1-/- myocytes.
Specific Aim 7: Determine if NOS1 dependent SR Ca2+ leak is involved in arrythmogenesis during β-AR stimulation.
The β-AR-dependent increase in diastolic SR Ca2+ leak is predominantly
CaMKII dependent and this increased leak is potentially arrhythmogenic[122,
123]. The purpose of this aim is to test if NOS1 mediated CaMKII activation and
SR Ca2+ leak contribute to arrhythmogenesis in healthy and diseased hearts.
Spontaneous SR Ca2+ waves (SCaW) are thought to be the precursor of arrhythmias. Therefore, the effects of NOS inhibiton on SCaW will be detected during β-AR stimulation in rabbit ventricular myocytes. Further, we will also investigate the effect of NOS inhibition on Ca2+ waves in trabeculae isolated from
29 human failing hearts. I hypothesize that NOS inhibition will block the generation of arrhythmias in both healthy myocytes and diseased human trabeculae.
30
Figure 1. Excitation-contraction coupling in the cardiac myocyte[3].
The diagram illustrates the movement of Ca2+ within the cardiac myocyte during excitation-contraction coupling. The solid arrows represent Ca2+ influx SR Ca2+
2+ release leading to the [Ca ]i transient, while the dashed arrows represent the
2+ 2+ decline of the [Ca ]i transient via Ca efflux and SR reuptake.
31
Figure 2. The cardiac action potential[4].
Ventricular and SA nodal cells exhibit significantly different AP waveforms and therefore different ion channel conductances. Ventricular AP waveforms are distinguished by four phases: Phase 0: Rapid Depolarization; Phase 1:Transient
Repolarization; Phase 2: The Plateau Phase; Phase 3: Rapid Repolarization;
Phase 4: Resting Membrane Potential. SA nodal cell AP waveforms are divided into the following phases: Phase 0: AP Upstroke; Phase 3: Repolarization; Phase
4: Slow Depolarization.
32
Figure 3. β-adrenergic receptor signaling pathway in the cardiac myocyte [3].
The following diagram illustrates the downstream effects of the β-AR signaling pathway in the cardiac myocyte.
33
Figure 4. NOS structure [63].
NOS functions as a dimer composed of two identical monomers. Each monomer consists of a reductase and oxygenase domain. The reductase domain consists of binding sites for NADPH, FAD, and FMN while the oxygenase consists of binding sites for oxygen, L- arginine, BH4 and heme. NOS also has a calmodulin binding site that lies between the oxygenase and reductase domains.
34
Figure 5. Constitutively expressed NOS isoforms exert differential effects on myocyte contraction.
NOS1 regulates both basal and β-AR receptor stimulated contraction at the SR while NOS3 only regulates β-AR receptor stimulated contraction and arrhythmogenesis at the caveolae. (Solid line: stimulatory effects; Dashed line: inhibitory effects.)
35
Figure 6 NOS isozymes in HF myocardium.
NOS1 expression is increased and it translocates to the caveolae; NOS3 is
.- down-regulated and uncouples to produce O2 ; NOS2 expression is induced and increases both NO and ROS production.
36
Figure 7 CaMKII activity[181].
CaMKII phosphorylation increases SR Ca2+ leak from RyR, increases SR Ca2+ uptake via SERCA (controversial effects), and increases Ica via LTCC.
37
Chapter 2: CaMKII mediated SR Ca2+ leak is involved in the positive inotropy of NOS1 signaling during β-AR stimulation
2.1 Introduction
β-adrenergic (β-AR) stimulation leads to positive inotropic and lusitropic effects in the heart[30]. That is, an increase of systolic [Ca2+] levels with faster time to peak leads to stronger myocyte contraction, and a shorter time of [Ca2+] decline results in faster relaxation. Various Ca2+ handling proteins, especially those located on the SR, are modified during β-AR stimulation, such as RyR and the SERCA/PLB complex. Phosphorylation of PLB at Ser16 by PKA and phosphorylation of RyR at Ser2814 by CaMKII are found to increase SR Ca2+ reuptake [16-18] and SR Ca2+ release[119] [120], respectively. These modifications are thought to be the dominant regulators of Ca2+ handling during
β-AR stimulation [20, 121-123].
Nitric oxide (NO) is also a key regulator of cardiac contractility and is synthesized by nitric oxide synthase (NOS)[39]. Neuronal nitric oxide synthase
(NOS1) is constitutively expressed within cardiac myocytes and co-localizes with
RyR on the SR[178]. NOS1 signaling modulates RyR activity by S-nitrosylation
[89] and increases PLB phosphorylation at Ser16 during basal contraction[74, 75].
During β-AR stimulation, the inotropic response is depressed in NOS1 knockout
(NOS1-/-) myocytes compared to wild type (WT). Interestingly, there is no
38 difference in PLB phosphorylation during -AR stimulation[74, 77], suggesting that there are additional protein targets (i.e. RyR) of the NOS1 signaling pathway.
Here we investigate whether NOS1 signaling affects RyR activity during β-
AR stimulation. We found that 1) NOS1 is the key regulator of SR Ca2+ release via RyR during β-AR stimulation, 2) the modulation of RyR activity by NOS1 is mediated via CaMKII phosphorylation of RyR at Ser2814, 3) the NOS1/CaMKII axis contributes to the positive inotropy during β-AR stimulation. Our data suggest that CaMKII and RyR are downstream in the NOS1 signaling cascade contributing to the positive inotropic effect of β-AR stimulation.
39
2.2 Materials and methods
2.2.1 Rabbit ventricular myocyte isolation
Rabbit ventricular myocytes were obtained as described previously [182].
Briefly, hearts excised from adult male New Zealand rabbits under pentobarbitone sodium anaesthesia (70 mg/kg) were mounted on a Langendorff perfusion apparatus. After perfusion with nominally Ca2+ -free Tyrode solution for
6 min, the hearts were perfused for 30 min with a solution containing 1 mg/ml collagenase Type B (Boehringer Mannheim) and 0.16 mg/ml protease (Type XIV,
Sigma) at 370 C. The enzymatic digestion was followed by dispersion of the ventricular tissue and repeated rinsing of the cell suspension, during which the
Ca2+ concentration was gradually raised to 2 mm.
2.2.2 Mouse ventricular myocyte isolation
Ventricular myocytes were isolated from NOS1-/-, their corresponding WT, and RyR S2814 knockin mice (S2814D, S2814A) as previously described [183].
Briefly, the heart was mounted on a Lagendorff apparatus and perfused with nominally Ca2+ -free Tyrode solution for 4 min. Blendzyme Type IV (0.077 mg/ml)
(Roche Applied Science, Indianapolis, IN) was then added to the perfusate. After
3-5 min, the heart was removed, the ventricles minced, and myocytes dissociated by trituration. Subsequently, the myocytes were filtered, centrifuged, and resuspended in normal Tyrode solution containing 200 μM Ca2+. Myocytes were used within 6 h after isolation.
40
2.2.3 SR Ca2+ Leak Measurement
SR Ca2+ leak was measured with the tetracaine (RyR inhibitor)-induced shift in diastolic [Ca2+][184, 185](Figure 8). Myocytes were loaded at 22 °C with
Fluo-4 AM (10μM, Molecular Probes, Eugene, OR, USA) for 30 min, washed out, and then an additional 30 min was allowed for intracellular de-esterification. The cells were then stimulated for 3 min in normal Tyrode solution. After reaching steady state, the solution was rapidly switched to 0 Na+, 0 Ca2+ Tyrode solution
(Na+ was replaced by Li+) plus 1mM tetracaine (Sigma) for 30 s. Afterwards, the solution was rapidly switched to 0 Na+,0 Ca2+Tyrode solution for 20 s. Tetracaine, by blocking RyR and therefore SR Ca2+ leak, caused a decrease in diastolic
2+ + [Ca ]i. The shift in diastolic [Ca2 ]i upon the removal of tetracaine was used as a measure of RyR-dependent SR Ca2+ leak. Since SR Ca2+ leak is also dependent upon the SR Ca2+ load, SR Ca2+ load of each cell was measured. Measurements were performed at room temperature.
2.2.4 SR Ca2+ load measurement
SR Ca2+ load was measured by rapid application of 10 mM caffeine for 10 s (Figure 8). The amplitude of the caffeine-induced Ca2+ transient was used as an index of SR Ca2+ load [186].
2.2.5 Measurement of NO Production
Myocytes were loaded with the cell permeable NO-dependent fluorescent dye DAF-2 AM (10 ìM) for 25 minutes, and allowed to de-esterify for an additional
25 minutes. Cells were paced at 1.0 Hz ±isoproterenol (a β-AR agonist, ISO).
41
Fluorescence was observed on an Olympus Fluoview confocal microscope in x-y mode with the pinhole set at 400 ìm. Fluorescence data was acquired for 5 seconds at 1 minute intervals over the duration of 20 minutes. The dye was sensitive to photobleaching and the signal was corrected. Unstimulated myocytes without ISO present were subjected to the same experimental conditions to assess photobleaching. A line was fit to this data, and the slope of this line was added back to all experimental groups to correct for photobleaching. SNAP was used as a positive control for DAF-2.
2.2.6 Simultaneous measurement of Ca2+ transients and myocyte shortening.
Myocytes were loaded at 22° C with Fluo-4 AM (10 μM, Molecular Probes,
Eugene, OR) for 30 min and washed out, and then an additional 30 min were allowed for intracellular de-esterification. The instrumentation used for cell fluorescence measurement was a Cairn Research (Faversham, UK) epifluorescence system. Myocytes were stimulated via platinum electrodes
2+ connected to a Grass S48 stimulator at a frequency of 1 Hz. [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 were expressed as F/F0, where F was the fluorescence intensity and F0 was the intensity at rest. Simultaneous measurement of shortening was also performed by use of an edge-detection system (Crescent Electronics, Sandy,
UT). Data were expressed as percentage of resting cell length (%RCL).
Measurements were performed at room temperature.
42
2.2.7 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. SR Ca2+ fractional release was calculated as the Ca2+ transient amplitude divided by the caffeine induced Ca2+ transient amplitude [18, 222].
2.2.8 Western blot analysis
Western blot analysis was performed as previously described [18].
Homogenized ventricles were used to measure specific RyR phosphorylation at
Ser2814 (Badrilla, Leeds, UK; 1:50.000) or Ser2808 (Badrilla, Leeds, UK;
1:5,000) Phosphorylated protein was normalized to GAPDH (Cell Signaling
Technology, Danvers, MA, 1:20,000) and total protein (Affinity BioReagents,
Golden, CO, USA; 1:50,000.).
2.2.9 Solution and drugs
Normal tyrode solution consisted of (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 1
CaCl2, 10 glucose, 5 HEPES, 1 L-arginine, pH 7.4 adjusted with HCl.
Isoproterenol (ISO), a nonselective β-AR agonist, was obtained from Sigma. The
CaMKII inhibitor KN-93, its inactive analog KN-92, cell-permeable CaMKII inhibitor Autocamtide-2 Related Inhibitory Peptide II (AIP), NOS inhibitor L-NG-
Nitroarginine methyl ester (L-NAME), NOS1 and NOS3 specific subtype inhibitors S-Methyl-L-thiocitrulline (SMLT) and L-N5-(1-Iminoethyl) ornithine (L-
NIO), PKG inhibitor peptide DT-2, and S-Nitroso-N-acetyl-DL-penicillamine,
(SNAP) were obtained from Calbiochem.
43
2.2.9 Statistical analysis
Data are reported as mean ± SEM. Student t test or ANOVA was used when appropriate. P < 0.05 was considered statistically significant. To compare
DAF-2 dependent fluorescence a non-parametric Spearman correlation test was conducted. The Spearman r-values are reported as an index of correlation of NO production with time.
44
2.3 Results
2.3.1 Enhanced diastolic SR Ca2+ leak during β-Adrenergic stimulation is
NOS dependent
We measured SR Ca2+ leak as the shift of Ca2+ from the cytosol to the SR in response to the RyR inhibitor tetracaine in isolated rabbit ventricular myocytes.
Figure 9 shows that treatment by 250 nM ISO alone left shifts the leak/load
2+ relationship away from control such that more SR Ca leak (Δ[Ca]SRT) is
2+ observed at a given SR Ca load ([Ca]SRT) consistent with previous data [122].
Conversly, those myocytes stimulated with ISO and L-NAME (non-specific NOS inhibitor) showed a leak/load relationship that was similar to control. When data was matched such that [Ca]SRT was the same for both groups (127 μM, Figure
9B), myocytes stimulated with ISO had significantly higher leak compared to control and this increase was prevented with L-NAME (10.0 ± 1.2, 3.2 ± 1.1 ,4.2 ±
1.5 μM Δ[Ca]SRT, respectively). Similarly, when selecting for myocytes such that
2+ SR Ca leak was the same for all groups (5 μM, Figure 9C), the [Ca]SRT needed to induce that leak was significantly lower in myocytes stimulated by ISO versus control and, again, this change was ablated in the presence of L-NAME.
2.3.2 -AR stimulation in ventricular myocytes increases NO production
We tracked cellular NO by using the NO-dependent fluorescent dye DAF-
2 in isolated rabbit myocytes. Both the NO donor SNAP (positive control) and
ISO increase NO compared with control (Figure 10, Spearman r = 1.0, 0.9 and -
0.05, respectively). These data are in line with previous findings[187], suggesting
45 that increased NO production during -AR stimulation is responsible for the observed NO-dependent effect on diastolic SR Ca2+ leak.
2.3.3 Increased diastolic SR Ca2+ leak during -AR stimulation is NOS1 dependent
Two NOS subtypes are constitutively expressed in healthy ventricular myocytes, NOS1 and NOS3[188]. We specifically inhibited each isoform in the presence of ISO (Figure 11) and measured SR Ca2+ leak. The leak/load relationship was right-shifted away from ISO alone and towards control while in the presence of ISO plus the NOS1 inhibitor SMLT (3 μM). Inhibition of NOS3 by
L-NIO (5 μM) resulted in a similar leak as ISO alone. Statistically, myocytes stimulated with ISO and ISO plus L-NIO had significantly higher leaks (8.3 ± 1.6;
6.8 ± 1.2 μM, respectively) compared with ISO plus SMLT or control (3.5 ± 1.7;
3.7 ± 1.0 μM, respectively) at the same [Ca]SRT. Similarly, cells stimulated with
ISO or ISO plus L-NIO required significantly lower [Ca]SRT (113 ± 14; 113 ± 6.6
μM respectively) compared with ISO plus SMLT or control (159 ± 14; 159 ± 10
μM, respectively) to induce the same SR Ca2+ leak.
2.3.4 Enhanced diastolic SR Ca2+ leak via NOS1 is CaMKII dependent
Since our pharmacological inhibitors of NOS may have non-specific effects (i.e., inhibition of the other NOS isoform), we further investigated the molecular mechanisms of the NOS1-dependent regulation of diastolic SR Ca2+ leak in NOS1-/- mice. SR Ca2+ leak-load relationship was measured in isolated ventricular myocytes from NOS1 knockout mice (NOS1-/-) and wildtype mice (WT)
46 in the presence of a specific CaMKII inhibitor, KN93. The ISO-dependent
2+ increase in SR Ca leak at the same [Ca]SRT was shifted back towards control levels by 1 μM KN93 in WT mice (Figure 12; 3.0 ± 0.4, 7.5 ± 0.8, 4.9 ± 0.7 μM for control (data not graphed), ISO, ISO+KN93, respectively). ISO treatment in
NOS1-/- myocytes was unable to increase SR Ca2+ leak above control levels (2.6
± 0.4 μM), and inhibition of CaMKII had no further effect on leak (2.1 ± 0.4 μM).
As the lab previously published[89], NO from NOS1 regulates RyR activity via S-nitrosylation under baseline conditions. Consistent with this observation,
KN93 does not shift SR Ca2+ leak back to control (no ISO) levels (Figure 12).
Possible reasons for this observation are that there was incomplete kinase inhibition or contributing effects of increased RyR S-nitrosylation. We further investigated if NO mediated S-nitrosylation of RyR affects ISO stimulated SR
Ca2+ leak by assessing the effects of the NO donor SNAP (100 μM) in the absence of ISO. Myocytes stimulated with SNAP had a significantly higher leak at the same load compared with SNAP plus KN93, SNAP plus the CaMKII inhibitor AIP, or control (Figure 13; 6.8 ± 0.5, 3.9 ± 0.8; 3.6 ± 0.7, 3.0 ± 1.3 μM, respectively). The [Ca]SRT needed to induce the same leak was significantly lower with the SNAP treatment versus SNAP plus KN93, SNAP plus AIP, or control.
There is still a minor but not significant elevation of SR Ca2+ leak with CaMKII inhibition (SNAP plus KN93/AIP vs control.), suggesting that there is still some minor effects of S-nitrosylation. Thus, these data indicate that the vast majority of the NO effect to enhance diastolic SR Ca2+ leak is via activation of CaMKII.
47
2.3.5 CaMKII regulates RyR activity via Phosphorylation at Ser2814
To further investigate the the role of CaMKII in the NOS1-mediated increase in RyR activity, immunoblotting for RyR Ser2814 phosphorylation was performed in NOS1-/- and WT hearts. The phosphorylation levels of the CaMKII- dependent site, S2814, of RyR increased with ISO in WT hearts, but this increase was not present in NOS-/- hearts (Figure 14). These data further support the concept that the ISO-dependent increase in SR Ca2+ leak is CaMKII- dependent and that NOS1 activity is essential.
2.3.6 CaMKII phosphorylation of RyR is sufficient for the NOS1 mediated increase in diasolic SR Ca2+ leak during β-AR stimulation
We further tested whether phosphorylation of RyR at Ser 2814 by CaMKII is the molecular mechanim for the NOS1-meidated increase in RyR activity during β-AR stimulation by using isolated myocytes from knockin mice with
CaMKII phosphorylation site of RyR constitutively active (S2814D) or genetically ablated (S2814A). RyR activity was measured via the SR Ca2+ leak/load relationship in the presence of NOS1 inhibitor SMLT (10 μm) during ISO stimulation. As seen in Fig 15, S2814D myocytes showed increased RyR activity
(0.012 ± 0.004) whereas S2814A myocytes showed decreased RyR activity
(0.003 ± 0.001) compared to WT myocytes (0.006 ± 0.001). SMLT significantly shifted the SR Ca2+ leak-load relationship downward in WT myocytes (0.002 ±
0.001), but had no effect in either S2814D (0.011 ± 0.004) or S2814A myocytes
(0.004 ± 0.001). These data suggest that CaMKII phosphorylation of RyR at Ser
48
2814 is the mechism responsible for the NOS1-dependent increase in diastolic
SR Ca2+ leak.
Since PKA is also able to phosphorylate RyR at S2808 and increase the
SR Ca2+ leak[35], I also measured PKA phosphorylation of RyR at Ser2808 in the presence of ISO. Our data demonstrated that there was no difference in
Ser2808 phosphorylation between WT and NOS1-/- hearts (Figure 16). These data indicate that PKA phosphorylation on RyR is not involved in the regulation of
SR Ca2+ leak during β-AR stimulation. Previous results showed that PLB Ser16 phosphorylation by PKA was also not different between WT and NOS1-/- hearts during -AR stimulation. Thus, I conclude that PKA is not involved in NOS1 signaling pathway during β-AR stimulation.
2.3.7 CaMKII is involved in the positive inotropy of NOS1 signaling during
β-AR stimulation
In this study, I have demonstrated that the enhanced diastolic SR Ca2+ leak during -AR stimulation is dependent upon NOS1 via CaMKII phosphorylation of RyR at Ser2814. Since NOS1 signaling is also involved in the positive inotropic effect of -AR stimulation, we further investigate this pathway. I hypothesize that the contribution of NOS1 signaling to the positive inotropic effect of -AR stimulation is CaMKII mediated. We tested this hypothesis by measuring contraction in WT and NOS1-/- myocytes by applying the specific CaMKII inhibitor
KN93 (1 μM) while in the presence of ISO (1 μM). Shown in Figure 17A are representative shortening and Ca2+ transient traces in the presence of -AR
49 stimulation (±KN93). As summarized in Figure 17B, WT myocytes incubated with
KN93 had significantly decreased shortening (7.6 ± 0.8 %RCL) and Ca2+ transient (4.7 ± 0.1 F/F0) amplitudes compared to control WT myocytes (12.4 ±
1.1 %RCL and 5.3 ± 0.1 F/F0). These data suggest that inhibition of CaMKII decreased cell contraction in WT myocytes during -AR stimulation. Consistent with previous data[89, 183], NOS1-/- myocytes showed blunted shortening (9.1 ±
2+ 0.7 %RCL) and Ca transient amplitude (4.3 ± 0.2 F/F0) compared to WT.
Interestingly, KN93 had no effects on shortening (8.0 ± 1.0 %RCL) or Ca2+
-/- transient (4.1 ± 0.3 F/F0) amplitudes in NOS1 myocytes (Fig. 17B). These data suggest activation of CaMKII contributes to the positive inotropic effect of -AR stimulation and that NOS1 is essential for this function.
2.3.8 Resupply of NO in NOS1-/- myocytes rescues the blunted inotropic response which is prevented by CaMKII inhibition
We further tested the NO/CaMKII pathway on myocyte contraction by perfusing NOS1-/- myocytes with the NO donor SNAP (1μM) ± KN93 during β-AR stimulation. Shown in Figure 18 are myocytes incubated with SNAP had
2+ significantly increased Ca transient (4.1 ± 0.2 F/F0) amplitudes compared to control myocytes (3.2 ± 0.2 F/F0). However, CaMKII inhibition with KN93
2+ completely prevented this effect (Ca transient amplitude: 3.3 ± 0.2 F/F0). Similar results were found with shortening amplitudes (data not shown). Thus, re-supply of NO successfully rescued the blunted inotropic effect of NOS1-/- myocytes during β-AR stimulation, and CaMKII inhibition blocked this effect. These data
50 confirm that CaMKII is the downstream effector of the NOS1 signaling pathway during β-AR stimulation.
2.3.9 The NOS1-mediated postivie inotropic effect during -AR stimulation is via CaMKII modulation of RyR activity
We have shown above that during β-AR stimulation the NOS1-dependent increase in diastolic SR Ca2+ leak occurs via CaMKII activation and phosphorylation of RyR at Ser 2814. We also observed similar results investigating myocyte contraction (i.e., NOS1-dependent positive inotropic effect during -AR stimulation is via CaMKII activation). We wanted to further extend our data and investigated if the NOS1 effect on contraction was also via RyR modulation. We measured fractional SR Ca2+ release in WT and NOS1-/- myocytes in the presence of ISO (±KN93). As shown in Figure 19, I observed a decreased SR Ca2+fractional release (twitch Ca2+transient amplitude/SR
Ca2+load) with KN93 (0.66 ± 0.02) compared to control (0.73 ± 0.03) in WT myocytes. In addition, similar to our contractile findings in Figure 17 and the SR
Ca2+ leak results in Figure 13, NOS1-/- myocytes showed depressed fractional SR
Ca2+ release (0.58 ± 0.05) compared to WT myocytes. In addition, KN93 had no effect on the fractional SR Ca2+ release in NOS1-/- myocytes (0.59 ± 0.03). Since fractional SR Ca2+release is dependent upon RyR activty, these data suggest that NOS1 signaling contributes to the positive inotropic effect of β-AR stimulation by regulating RyR activity via CaMKII.
51
2.4 Discussion
In this study, ISO-dependent increases in diastolic SR Ca2+ leak were observed in rabbit and mouse ventricular myocytes. This increase is dependent upon NOS1 activity but not NOS3. Further, we find that NO was sufficient to induce increased leak in the absence of ISO and that this NO-dependent effect requires CaMKII phosphorylation of RyR at Ser2814. We also find that this
NOS1/CaMKII/RyR signaling cascade contributes to the positive inotropic effect during β-AR stimulation. These data suggest that during β-AR stimulation,
CaMKII is a downstream target of NOS1 signaling to increase SR Ca2+ release and myocyte contraction via RyR modulation.
2.4.1 Effects of NOS1 during β-AR stimulation.
Our data lead us to conclude that the ISO-dependent increase in SR Ca2+ leak is mediated by a new and unique adrenergic second messenger pathway involving NO. We show directly that simply treating cardiac myocytes with ISO leads to increases in NO production (Figure 10). In these experiments the response of DAF-2 during ISO stimulation is significantly lower than that invoked by SNAP. I would propose that ISO stimulation leads to an activation of NOS1 in a highly compartmentalized NO signaling domain. It is known that NOS1, CaMKII, and RyR are spatially coupled at the SR[178, 189], whereas NOS3 is co- localized with Caveolin 3 to the caveolae[70, 94]. These two isozymes also function differently during β-AR stimulation. That is, NOS1 enhances the contractile response to β-AR stimulation via post translational modification of its target proteins, such as S-nitrosylation of RyR[39, 89]. NOS3 blunts the
52 contractile response to β-AR stimulation via phosphorylation of the L-type
Ca2+channel by the cGMP/ PKG pathway to decrease Ca2+influx[42] [39]
Therefore, only the NO produced from NOS1 leads to increased CaMKII- dependent phosphorylation of RyR as indicated by our data (Figure 11). The increased phosphorylation levels will increase RyR activity which will contribute to the positive inotropic effects of β-AR stimulation.
Data from our laboratory and others have shown that PLB expression is decreased and RyR expression is increased in NOS1-/-mice[89, 190, 191].
Furthermore, I found that the expression of CaMKII is increased in NOS1-/-mice
(Figure 22 in Chapter 3). I believe that these changes are a compensatory mechanism for the lack of NOS1. However, these genetic compensatory changes in our NOS1-/- mice should not interfere with the interpretation of our data in this study since we and others have also observed a similar blunted ISO stimulated contraction via acute NOS1 inhibition with SMLT in WT myocytes [70,
74-76].
2.4.2 Involvement of CaMKII in the NOS1 signaling pathway during β-AR stimulation
In response to β-AR stimulation, CaMKII can phosphorylate RyR at
Ser2814 and PLB at Thr17[113]. In this study, we found that CaMKII inhibition
(KN93 or AIP) significantly decreased SR Ca2+ leak and myocyte contraction (cell shortening and Ca2+ transient amplitudes) in WT myocytes, but had no effects in
NOS1-/- myocytes (Figure 17). Since CaMKII inhibition can block the effects of our NO donor SNAP on NOS1-/- myocyte contraction (Figur 18) and diastolic SR 53
Ca2+ leak (Figure 13), I believe that CaMKII is a key downstream effecter in the
NOS1 signaling pathway.
It is found in other tissues that the CaMKII inhibitor KN93 can nonspecifically affect other protein targets such as LTCC [192] or K+ channels
[193]. Thus, to test the specificity of our findings, I repeated the myocyte contraction experiments in WT and NOS1-/- myocytes using the non-active KN93 analog, KN92. The data demonstrate that KN92 did not change β-AR stimulated shortening or Ca2+ transient amplitudes in WT or NOS1-/- myocytes (Figure 20).
Thus the decreased RyR activity, diastolic SR Ca2+ leak, and contraction that we observed with KN93 are indeed due to the inhibition of CaMKII.
2.4.3 End targets of NOS1 signaling pathway during β-AR stimulation
Previous studies have shown that NOS1 can regulate PLB phosphorylation by PKA under baseline conditions[74, 75]. Interestingly, PKA phosphorylation of PLB at Ser16 or CaMKII phosphorylation of PLB at Thr17 were not different in NOS1-/- vs. WT myocytes during β-AR stimulation [74, 77].
This indicates that PLB is not a target of NOS1 signaling during β-AR stimulation.
Here, I observed that CaMKII inhibition leads to a similar increase of Ca2
+transient RT50 in WT and NOS1-/- myocytes, whereas a slower Ca2+ transient time to peak (which is related to RyR activity) was only observed in WT but not
NOS1-/- myocytes (Figure 21). These results further confirm that the
NOS1/CaMKII signaling pathway is targeting RyR and not PLB during β-AR stimulation.
54
Since studies have found that PKA does phosphorylate RyR at Ser2808 during -AR stimulation[118], we investigated if PKA was also involved in the
NOS1 signaling pathway with -AR stimulatin. I found no difference in PKA phosphorylation of RyR at Ser2808 in WT and NOS1-/- hearts (Figure 16). These data, combined with my findings using the RyR knockin mice (S2814D/A) (which showed no change in RyR activity in response to acute NOS1 inhibition-SMLT
(Figure 15)), makes me conclude that CaMKII/RyR is the end target of NOS1 signaling during β-AR stimulation. Furthermore, the NOS1-mediated increase in
CaMKII resulting in RyR Ser2814 phosphorylation is the molecular mechanism that contributes to the positive inotropic effect of -AR stimulation. Thus, during
-AR stimulation, PKA is not involved in the NOS1 signaling pathway.
Our previous study showed under basal contraction that NO will S- nitrosylate RyR and increase open probability[89]. In Figure 10, NO production is elevated during β-AR stimulation. Therefore it’s necessary to determine if RyR S- nitrosylation also plays a role with -AR stimulation. However, currently no data exists if RyR S-nitrosylation levels change during β-AR stimulation. In this study,
NOS1-/- myocytes showed blunted fractional SR Ca2+ release and SR Ca2+ leak compared to WT myocytes. While CaMKII inhibition decreased fractional release and SR Ca2+ leak in WT myocytes, these levels were still higher compared to
NOS1-/- myocytes (Figure 12 and Figure 19). Thus, these data suggest that increased RyR S-nitrosylation may also be involved in our pathway. However, with acute resupply of NO in NOS1-/-(Figure 18) or rabbit myocytes (Figure 13),
CaMKII inhibition blocked the increase in myocyte contraction and SR Ca2+ leak
55 back to control levels. These data suggest that although S-nitrosylation of RyR can affect activity; phosphorylation of RyR by CaMKII is the dominant regulator of
RyR with NOS1 signaling during β-AR stimulation to increase diastolic SR Ca2+ leak and contribute to the positive inotropic effect.
56
Figure 8. Schematic of the diastolic SR Ca2+ leak protocol.
The cartoon demonstrates how the fluo-4 dependent signal tracks changes in
2+ 2+ 2+ [Ca ]i. The SR Ca leak is proportional to the fall in [Ca ]i and the resultant rise
2+ in [Ca ]SRT in the presence of the RyR blocker, tetracaine. The steady-state shift of Ca2+ from the cytosol to the SR with tetracaine is proportional to the SR Ca2+ leak. [Ca2+] in solution was 2 mM in rabbit and 1 mM in mouse.
57
Figure 9. ISO-dependent leak is attenuated by the non-specific NOS inhibitor L-
NAME.
A) Leak/load relationship for all treatments in rabbit myocytes. Each data point
2+ represents [Ca]SRT and the shift of Ca from the cytosol to the SR in response to inhibition of RyR-dependent SR Ca leak under a given loading protocol (from low to high [Ca]SRT; resting, 1 field stimulation, 0.25 Hz, 0.5 Hz and 1 Hz stimulation, respectively). B) Matched data such that the mean [Ca]SRT was the same for all treatments (left) and the resultant SR Ca leaks (right, n=10-14). C) Data were matched such that the average SR Ca2+ leak was the same for all treatments (left) and the [Ca]SRT needed to induced that leak (right, n=11-17). *Statistically different from control, # different from ISO (p<0.05). (Data collected by Shannon lab.)
58
D A F -2 (N O -d e p e n d e n t) F lu o r e s c e n c e
1 .4 5 S N A P
0 F
/ 1 .3 5
F
e s
a 1 .2 5
e
r
c n
I IS O
1 .1 5 %
1 .0 5
0 .9 5 C trl
0 .0 2 .5 5 .0 7 .5 1 0 .0 1 2 .5 1 5 .0 1 7 .5 2 0 .0 T im e (m in )
Figure 10 : ISO increases myocyte NO production in isolated rabbit myocytes.
Shown is the NO-dependent DAF-2 fluorescence while stimulating at 1.0 Hz
(n=6). For graphical representation only, data was fit to a second order polynomial. Spearman correlation = 1.0 for SNAP, 0.9 for ISO, and -0.05 for control. (Data collected by Shannon lab.)
59
A B Matched [Ca]SRT 150 10.0 * 125
M) 7.5
100 SRT 17.5 Control ( # 75 5.0
ISO SRT 15.0 50 [Ca] ISO + NOS1 Inhib. 2.5
12.5 [Ca] 25
Control ISO ISO + eNOS inhib. eNOS + ISO ISO +NOS3 Inhib. inhib. nNOS + ISO SRT 10.0 0 0.0
7.5 [Ca]
5.0 C Matched Leak 2.5 # 0.0 8 175 150
0 50 100 150 200 6 M)
125 * * SRT ( 100 [Ca]SRT (M) 4
SRT 75 [Ca] 2 50
[Ca] 25
Control ISO ISO + nNOS inhib. nNOS + ISO 0 eNOSinhib. + ISO 0 Figure 11 : Inhibition of NOS1 but not NOS3 reverses the ISO-dependent increase in SR Ca2+ leak.
A) Leak/load relationship for all treatments in isolated rabbit myocytes. B)
Matched data such that the average [Ca]SRT was the same for all treatments (left) and resultant leaks (right, n=13-17). C) Data matched such that the average SR
2+ Ca leak was the same for all treatments (left) and the [Ca]SRT needed to induce that leak (right, n=11-19). * Statistically different from control, # different from ISO
(p<0.05). (Data collected by Shannon lab.)
60
M a tc h e d [C a ] S R T
2 5 0 9
)
)
M
+ O
2 0 0
M
S
(
O
I
+
( 6
O
*
S
1 5 0 T
I
T
S
+
O
R
I
R
-
+
S
/
S
S
-
-
]
I ]
1 0 0 /
+
-
1
a
a
3 3 *
+
1
3
S
C *
9
C
T
[
S
9 [
5 0 T
O
N
O N
W
N
W K K 0 N 0
Figure 12: NOS1-/- myocytes show attenuated CaMKII-dependent leak.
Matched data such that [Ca]SRT was the same for all treatments (left) and resultant SR Ca2+ leaks (right, n=10-22). * Statistically different from WT + ISO
(p<0.05).
61
M a tc h e d [C a ] S R T L e a k
1 2 0 7 .5
) * )
1 0 0 M
P
M
(
A
( 8 0 5 .0
T
N
T
R
l
S
R +
6 0 +
S
o
S
]
r
]
P P
3
+ a
4 0 t 2 .5
a
9
l
A A
n
C
r
C
P
[
N
o
t
I
N N
[ 2 0
C C S K S A 0 0 .0
M a tc h e d L e a k [C a ] S R T
9 1 7 5
) )
M 1 5 0
M
P (
1 2 5
(
6 A *
T T
N 1 0 0
+
R
+
l
R
S
S
o
S
] 7 5
r
]
P
P
3
+ t
a 3
a
9 l
A 5 0
A
n
C
r
P
C
[
N
o
I
t
N N
[ 2 5
S A
C S K C 0 0
Figure 13: SNAP-dependent diastolic SR Ca2+ leak.
A) Data was matched such that [Ca]SRT was the same for all treatments (left) with the resultant leaks on the right (n=9-13).B) Ca2+ leak was the same (left) with the
[Ca]SRT needed to induced that leak shown on the right (n=12-17). * Statistically different than control (p<0.05). (Data collected by Shannon lab.)
62
WT NOS1-/- ISO - - + + - - + +
P-RyR Ser 2814 Total RyR
GAPDH
Figure 14: Blunted RyR phosphorylation by CaMKII in NOS1-/- heart.
Immunoblots showing RyR Ser2814 phosphorylation by CaMKII normalized to total RyR in WT and NOS1-/- heart (±ISO, n=4-6 mice). * Statistically different from WT, # different from WT + ISO (p<0.05).
63
(A.U.)
SRT SRT
/ / [Ca]
SRT SRT Δ[Ca]
Figure 15. No effect of acute NOS1 inhibition in RyR knockin myocytes.
SR Ca2+ leak-load relationship in WT, S2814D (S14D) and S2814A (S14A) myocytes (ISO±SMLT). n=13-23/ 9-10 hearts). * Statistically different from corresponding control, (p<0.05).
64
P -R y R S e r2 8 0 8 (N o rm a liz e d to T o ta l R y R )
3 0 C O N T
2 5 IS O
2 0
. U
. 1 5 A
1 0
5
0 -/- W T N O S 1
Figure 16: PKA effect on RyR is similar between WT and NOS1-/- hearts.
Immunoblots showing that RyR Ser2808 phosphorylation by PKA normalized to total RyR (n=4-6). (P=NS, n=3-4 mice).
65
A . -/- B . W T N O S 1 C e ll S h o rte n in g A m p litu d e 1 4 IS O 1 2 IS O + K N 9 3 # 1 0
L 8 *
C R
% 6 C e ll S h o r te n in g 4
2 IS O 0 IS O + K N 9 3 -/- W T N O S 1
4 % R C L 2 + 2 5 0 m s C a T ra n s ie n t A m p litu d e
2 + 6 C a T r a n s ie n t A m p litu d e
5 * #
4
0
F /
F 3 2
1
0 F /F 0 0 .5 W T N O S 1 -/-
Figure 17: Effects of CaMKII inhibition on cell contraction in WT and NOS1-/- myocytes.
A) Representative traces of cell shortening and Ca2+ transient from myocytes with ISO (± KN93). Left: WT, Right: NOS1-/-. B) Pooled data (mean±SEM) of cell shortening and Ca2+ transient amplitudes. Open bar: ISO, Dark bar: ISO+KN93 (*
P<0.05 vs. ISO, # P<0.05 vs. WT + ISO; n=22-27 myocytes/8-12 hearts).
66
C a 2 + T ra n s ie n t A m p litu d e
6 C O N T IS O
4 *
0
F
/ F
2
0 -/- N O S 1 S N A P S N A P + K N 9 3
Figure 18: Resupply of NO to NOS1-/- myocytes rescues the functional response to β-AR stimulation, which is prevented by CaMKII inhibition.
Pooled data (mean±SEM) of Ca2+ transient amplitude with ISO. Open bar: ISO,
Dark bar: CONT. (*P<0.05 vs. other ISO groups; n=12-20 myocytes/5 hearts).
67
2 + F r a c tio n a l S R C a R e le a s e
1 .0 IS O IS O + K N -9 3
i 0 .8
d
] a
a *
o
C
[ L
0 .6
+
2
h
a
c
t
C i
0 .4
w
R
T S 0 .2
0 .0 W T N O S 1 -/-
Figure 19: During β-AR stimulation, CaMKII inhibition decreases fractional SR
Ca2+ release in WT myocytes, but has no effect in NOS1-/- myocytes.
Pooled data (mean ± SEM) of fractional SR Ca2+ release with ISO (±KN93).
(*P<0.05 vs other groups; n=9-14 myocytes/3-5 hearts.)
68
C e ll S h o rte n in g A m p litu d e (+ K N 9 2 ) C a 2 + T ra n s ie n t A m p litu d e (+ K N 9 2 )
1 3 0 1 1 0
1 2 0
l
o r
) 1 0 0
t
2 n
9 1 1 0
o
N
C
K
f
- o
( 1 0 0
9 0 % 9 0
8 0 8 0 -/- -/- W T + IS O N O S 1 + IS O W T + IS O N O S 1 + IS O
Figure 20. KN92 has no effect on myocyte contraction.
Left: Percent changes of cell shortening amplitude (ISO+ KN92 vs. ISO-KN92),
Right: Percentage changes of Ca2+ transient amplitude (ISO+ KN92 vs. ISO-
KN92). (P=ns; n=10-21 myocytes/4-5 hearts.)
69
2 + 2 + A . C a T r a n s ie n t R T 5 0 (+ K N 9 3 ) B . C a T r a n s ie n t T im e T o P e a k (+ K N 9 3 )
1 1 5 1 1 5
P = 0 .0 6
l 1 1 0 1 1 0
o
r
)
t
3
n
9
o N
C 1 0 5 1 0 5
K
f
-
o
(
% 1 0 0 1 0 0
9 5 9 5
O O O O IS IS IS IS + /- + + /- + T - T - W 1 W 1 S S O O N N
Figure 21. CaMKII inhibition does not change Ca2+ transient decline but slows the time to peak in WT compared to NOS1-/- myocytes during β-AR stimulation.
Left: Percent change of Ca2+ transient RT50 (ISO+ KN93 vs. ISO-KN93), Right:
Percent change of Ca2+ transient time to peak (ISO+ KN93 vs. ISO-KN93).
(n=20-22 myocytes/8-12 hearts)
70
Chapter 3: Direct activation of CaMKII by NO and the upstream activator of NOS1 during β-AR signaling 3.1 Introduction
Activation of the β-AR pathway leads to a large increase of CaMKII activity[113]. However, the molecular mechanisms responsible for the activation of CaMKII in response to β-AR signaling is not well understood [113]. Classically,
CaMKII is thought to rely upon increases in [Ca2+] to initiate and maintain enzyme activity. However, recent evidence has emerged supporting novel activation mechanisms of CaMKII that are independent of increases in Ca2+ [111,
194-196]. In the last chapter, I have already illustrated a novel pathway where
CaMKII is a downstream target of the NOS1 signaling pathway during β-AR stimulation. In this chapter I will further investigate how NOS1 signaling activates
CaMKII.
We found that the positive inotropic effect of NOS1 signaling is due to a
CaMKII-dependent increase in RyR activity. However, the upstream activator of
NOS1 during β-AR stimulation is also not known. Here I will investigate the possible upstream activators of NOS1. I will specifically examine the effects of
Akt inhibition on NOS1 activity, RyR activity, and myocyte contraction.
Furthermore, NOS1 has been reported to be altered (increased expression and translocation) in heart failure, which is believed to contribute to the imbalance of nitroso-redox levels within the myocytes. CaMKII is also found
71 to have elevated expression and activity during heart failure, which is thought to be the main cause of the hyperactivity of RyR and arrhythmogenesis in HF. Thus,
I will investigate the role of the NOS1/CaMKII pathway on arrhythmogenesis.
These data may result in novel therapeutic treatment strategies for heart failure.
In this chapter, I find that 1) NO directly affects CaMKII via S-nitrosylation to sustain activity and increase RyR activity, 2) Akt is the upstream activator of
NOS1 signaling to increase RyR activity and myocyte contraction during -AR stimulation, 3) inhibition of nitric oxide synthase (NOS) attenuates SCaW formation as a result of β-AR stimulation in isolated rabbit myocytes and failing human trabeculae. Collectively, these data provide evidence that
Akt/NOS1/CaMKII/RyR is a signaling cascade in the β-AR response that can lead to arrhythmogenic SCaW formation which might play an important role in cardiac myopathies.
72
3.2 Materials and Methods
3.2.1 Rabbit ventricular myocyte isolation
Rabbit ventricular myocytes were obtained as described previously [182].
Briefly, hearts excised from adult male New Zealand rabbits under pentobarbitone sodium anaesthesia (70 mg/kg) were mounted on a Langendorff perfusion apparatus. After perfusion with nominally Ca2+ -free Tyrode solution for
6 min, the hearts were perfused for 30 min with a solution containing 1 mg/ml collagenase Type B (Boehringer Mannheim) and 0.16 mg/ml protease (Type XIV,
Sigma), at 37⁰ C. The enzymatic digestion was followed by dispersion of the ventricular tissue and repeated rinsing of the cell suspension, during which the
Ca2+ concentration was gradually raised to 2 mM.
3.2.2 Mouse ventricular myocyte isolation
Ventricular myocytes were isolated from NOS1-/-, their corresponding WT, and RyR S2814 knockin mice (S2814D, S2814A) as previously described [183].
Briefly, the heart was mounted on a Lagendorff apparatus and perfused with nominally Ca2+ -free Tyrode solution for 4 min. Blendzyme Type IV (0.077 mg/ml)
(Roche Applied Science, Indianapolis, IN) was then added to the perfusate. After
3-5 min, the heart was removed, the ventricles minced, and myocytes dissociated by trituration. Subsequently, the myocytes were filtered, centrifuged, and resuspended in normal Tyrode solution containing 200 μM Ca2+. Myocytes were used within 6 h after isolation.
73
3.2.3 Ca2+ Transient Measurement in Human Trabeculae
Human multicellular trabeculae were dissected from failing donor hearts obtained via collaboration with the Lifeline of Ohio Organ Procurement program.
Briefly, immediately after cross-clamp and excision, the coronary arteries were flushed with ice-cold oxygenated standard cardioplegic solution. After transport to the laboratory (<30 minutes), multicellular trabeculae were dissected under a stereo microscope in a Krebs-Henseleit (KH) solution containing 20 mmol/L 2,3- butanedione monoxime (BDM) to minimize cutting injury[197]. Care was taken to only select thin and linear preparations from the left ventricle and these were mounted between a micromanipulator and a force transducer as described previously[198]. Thereafter, KH buffer was used to perfuse the muscles, with the omission of BDM. Muscles were stretched to an optimal length and allowed to stabilize at a baseline pacing frequency of 1 Hz before starting the experimental protocol as previously described for rabbit and dog muscles[199, 200]. All experiments were performed at room temperature. The recording chamber was situated on the stage of an Olympus FluoView 1000 laser scanning confocal microscope, equipped with a UPLSAPO 60X water objective. Muscles were loaded with the cytosolic Ca2+ indicator Rhod-2 AM (40 μM) for 45 min as previously described[201]. The muscles were then washed with fresh KH buffer containing 5 mmol/L BDM and 10-20 μmol/L blebbistatin to stop cell contraction and allow for Ca2+ imaging by reducing or halting motion artifacts. Line scan images were acquired at the rate of 8 μs/pixel. The fluorescence emitted was expressed as F/F0. After obtaining baseline calcium transients, 1 μM
74 isoproterenol was added, and calcium transients recorded. Thereafter, 300 μM L-
NAME was added and calcium transients were again recorded.
3.2.4 Western Blot analysis
Western blot analysis was performed as previously described [183].
Homogenized ventricles were used to measure specific CaMKII phosphorylation at Thr286 (Thermo Fischer Scientific, Waltham, MA, 1:5000), Akt phosphorylation at Ser437, NOS1 phosphorylation at Ser1416 and, CaMKII oxidation at Met281/282 (Cell Signaling Technology, Danvers, MA, 1:5,000).
Phosphorylated or oxidized proteins were normalized to GAPDH and total protein
(total CaMKII, Cell Signaling Technology, Danvers, MA, 1:5,000).
3.2.5 Measurement of CaMKII Activity
Purified CaMKII was incubated with 200 μM Ca and CaM for 10 min. to pre-activate the molecule. H2O2 (1 μM) or 500 μM SNAP was added and allowed to incu-bate for 30 min. EGTA (10 mM) was then added and allowed to incubate for 10 min. Radio labeled ATP (32P) was added along with 5 μL of purified β2a
L-type Ca channel subunit on nickel beads. Incorporation of 32P into β2a was allowed to proceed for 10 minutes. Phosphorylated β2a is the reporter of this assay.
3.2.6 S-NO Western Blotting
CaMKII was immunoprecipitated using the Classic Immunoprecipitation Kit
(Pierce/Thermo Scientific). Briefly, cell lysates were pelleted with a microcentrifuge for 10 minutes and the pelleted debris was discarded. Lysates
75 were then added to a spin column with agarose resin and incubated for 1 hour at
4°C. After incubation, CaMKII antibody was added to the flow through and incubated overnight at 4°C. The incubation was applied to a spin column with protein A/G agarose and incubated 1 hour at 4°C. CaMKII was eluted with elution buffer and Western blotted with 1:1000 anti-S-NO anti-body.
3.2.7 SR Ca2+ leak measurement
SR Ca2+ leak was measured with the tetracaine (RyR inhibitor)-induced shift in diastolic [Ca2+][184, 185](Figure 8). Myocytes were loaded at 22 °C with
Fluo-4 AM (10μM, Molecular Probes, Eugene, OR, USA) for 30 min, washed out, and then an additional 30 min was allowed for intracellular de-esterification. The cells were then stimulated for 3 min in normal Tyrode solution. After reaching steady state, the solution was rapidly switched to 0 Na+, 0 Ca2+ Tyrode solution
(Na+ was replaced by Li+) plus 1mM tetracaine (Sigma) for 30 s. Afterwards, the solution was rapidly switched to 0 Na+,0 Ca2+Tyrode solution for 20 s. Tetracaine, by blocking RyR and therefore SR Ca2+ leak, caused a decrease in diastolic
2+ + [Ca ]i. The shift in diastolic [Ca2 ]i upon the removal of tetracaine was used as a measure of RyR-dependent SR Ca2+ leak. Since SR Ca2+ leak is also dependent upon the SR Ca2+ load, SR Ca2+ load of each cell was measured. Measurements were performed at room temperature.
76
3.2.8 SR Ca2+ load measurement
SR Ca2+ load was measured by rapid application of 10 mM caffeine for 10 s (Figure 8). The amplitude of the caffeine-induced Ca2+ transient was used as an index of SR Ca2+ load[186].
3.2.9 Simultaneous measurement of Ca2+ transients and myocyte shortening.
Myocytes were loaded at 22 °C with Fluo-4 AM (10 μM, Molecular Probes,
Eugene, OR) for 30 min and washed out, and then an additional 30 min were allowed for intracellular de-esterification. The instrumentation used for cell fluorescence measurement was a Cairn Research (Faversham, UK) epifluorescence system. Myocytes were stimulated via platinum electrodes
2+ connected to a Grass S48 stimulator at a frequency of 1 Hz. [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 were expressed as F/F0, where F was the fluorescence intensity and F0 was the intensity at rest. Simultaneous measurement of shortening was also performed by use of an edge-detection system (Crescent Electronics, Sandy,
UT). Data were expressed as percentage of resting cell length (%RCL).
Measurements were performed at room temperature.
3.2.10 Spontaneous Ca2+ wave measurement
SCaWs were assessed as previously described[123]. Fluo-4 AM (10 μM) loaded myocytes were electrically field stimulated for at least 5 minutes before
77 data acquisition. Grading [Ca]SRT was achieved by stimulating at frequencies from 0.25 Hz to 1.0 Hz in 2 Ca NT solution. After 20 beats a rapid switch to 0
Na/0 Ca NT solution + 10 mM caffeine was applied for 2 seconds to empty the
SR of calcium. The difference between basal and peak total cytosolic [Ca2+] in
2+ the presence of caffeine is therefore total SR [Ca ]. After assessing [Ca]SRT the myocyte was loaded under the same conditions. After loading, field stimulation
2+ was terminated and [Ca ]i was continuously monitored for 90 seconds.
Spontaneous calcium release was determined by visual inspection, and confirmed if the peak signal was greater than two standard deviations above the average signal for the preceding 50 ms.
3.2.11 Solution and drugs
Normal tyrode solution consists of (in mM) 140 NaCl, 4 KCl, 1 MgCl2, 1
CaCl2, 10 glucose, 5 HEPES, 1 L-arginine, pH 7.4 adjusted with HCl.
Isoproterenol (ISO), a nonselective β-AR agonist and the activator of Epac, 8-
CPT, were obtained from Sigma. The specific Akt inhibitor V and X, were obtained from Calbiochem.
3.2.12 Statistical analysis
Results were expressed as means ± SE. Statistical significance (P < 0.05) was determined by ANOVA (followed by Newman-Keuls test) for multiple groups.
Paired or unpaired t-tests were used for comparison between two groups.
78
3.3 Results
3.3.1 Adrenergic stimulation Leads to NO-dependent, Ca2+ independent
CaMKII Activation
We measured CaMKII Thr286 autophosphorylation by western blotting as a surrogate for CaMKII activity. Though ISO increased CaMKII phosphorylation in WT myocytes, this effect was absent in NOS1-/- myocytes (Figure 22). Total
CaMKII was increased in NOS-/- myocytes compared to control (bottom left), possibly in order to compensate for a decrease in CaMKII activity (bottom right).
It has long been known that PKG activation is NO-dependent [39, 188].
However, PKG inhibition with DT-2 did not alter the diastolic SR Ca2+ leak (Figure
5) suggesting that the effect of NOS1 on RyR is independent of PKG activation.
Physiologically, NO often acts on target proteins by direct S- nitrosylation[39]. Work by Erickson, et al[111] indicated that CaMKII activity can be sustained by oxidation (and independent of Ca2+), which prompted us to investigate the possibility that NO can replicate this effect and sustain CaMKII activity via S-nitrosylation. Purified CaMKII was incubated with Ca2+ and CaM to pre-activate the molecule. This was followed by oxidation by H2O2 or 500 μM
SNAP. EGTA (10 mM) was then added to stop Ca-CaM mediated activity. Finally,
ATP32 was added along with purified L-type Ca channel β2a subunit on nickel beads. Incorporation of P32 into β2a was therefore a measure of the sustained,
Ca-CaM independent activity. Ca-CaM independent kinase activity (Figure 23A) was sustained in the presence of H2O2 (as in Erickson, et al; Lane 2) and in the
79 presence of SNAP (Lane 3) indicating that, like oxidation, NO can sustain
CaMKII activity in the absence of Ca2+, likely by direct S-nitrosylation. Consistent with these experiments, cellular homogenates from rabbit cardiac myocytes stimulated at 0.5 Hz showed an increase in S-nitrosylation by Western blotting with an antibody to S-NO (Figure 23B). This increase was reversed in the presence of the β1 receptor blocker CGP 20712A. These data suggest that during -AR stimulation NO activates CaMKII directly via S-nitrosylation.
3.3.2 CaMKII-dependent diastolic SR Ca2+ leak is not mediated through oxidation
It has been reported that β-AR stimulation elevates cytosolic reactive oxygen species (ROS) levels[202], and CaMKII can be directly activated via oxidation[111]. Therefore, we set out to determine if ROS plays a role in the
NOS1-dependent activation of CaMKII to increase diastolic SR Ca2+ leak. We stimulated rabbit myocytes with ISO in the presence of the NADPH oxidase inhibitor, DPI (250 nM). Figure 24 shows that DPI is unable to shift the leak/load away from that observed with ISO. When selecting myocytes for similar [Ca]SRT
(112 ìM, Figure 24B) the leak at that load was significantly higher in ISO and ISO plus DPI when compared with control (12.2 ± 0.9, 11.8 ± 2.6, 4.1 ± 0.9 ìM, respectively). Similarly, when matching myocytes by similar leak (7.5 ìM, Figure
24C) the [Ca]SRT needed to induce that leak was significantly lower in ISO and
ISO plus DPI versus control (104 ± 9, 115 ± 14, 178 ± 6 ìM, respectively). From these data, I conclude that NADPH oxidase-generated ROS does not mediate the acute of effects of CaMKII on RyR activity during -AR stimulation.
80
.- -/- The lab previously reported that there are higher O2 levels in NOS1 hearts[83]. However, when I measured CaMKII oxidation by immunoblotting, I found no difference in CaMKII oxidation at Met281 between WT and NOS1-/- hearts (±β-AR stimulation) (Figure 25). Thus, these data further validate that oxidation of CaMKII is not involved in the NOS1 dependent increase in RyR activity during -AR stimulation.
3.3.3 CaMKII-dependent increase in diastolic SR Ca2+ leak is not mediated through Epac
Since Epac has been found to increase CaMKII and RyR activity, we also tested if Epac was involved in the NOS1/CaMKII pathway. I evaluated the SR
Ca2+ leak-load relationship in the presence of the specific EPAC activator 8-CPT
(2 ìM). Shown in Figure 26A, WT and NOS1-/- myocytes incubated with 8-CPT exhibited increased diastolic SR Ca2+ leak. When selecting myocytes for similar
[Ca]SRT (5 F/F0, Figure 26B) the leak at that load was significantly higher in 8-
-/- CPT (WT: 0.040 ± 0.003 F/F0, NOS1 : 0.050 ± 0.002 F/F0) when compared with
-/- control (WT: 0.026 ± 0.001 F/F0, NOS1 : 0.015 ± 0.001 F/F0) in both WT and
-/- NOS1 myocytes. Similarly, when matching myocytes by similar leak (0.03 F/F0,
Figure 26C) the [Ca]SRT needed to induce that leak was significantly lower in 8-
-/- CPT (WT: 4.64 ± 0.02 F/F0, NOS1 : 4.01 ± 0.02 F/F0) versus control (WT: 5.20 ±
-/- -/- 0.03 F/F0, NOS1 : 6.38 ± 0.03 F/F0) in both WT and NOS1 myocytes. Thus,
Epac activates CaMKII to increase diastolic SR Ca2+ leak similarly in WT and
NOS1-/- myocytes. Thus, these data suggest that Epac is not involved in the regulation of CaMKII and RyR via NOS1 during -AR stimulation.
81
3.3.4 The increase in RyR activity with ISO is Akt-dependent
We set out to investigate potential upstream effectors that were responsible to increase NOS1 activation during -AR stimulation. Since Akt activity is increased with -AR stimulation and can increase NOS1 activity via phosphorylation, our studies focused on Akt. Myocyte Akt activity (measured as
Ser473 phosphorylation) showed an increase in response to ISO, which was decreased by the addition of the Akt Inhibitor X (Figure 27A). Akt inhibitor X also prevented the ISO-dependent increase in diastolic SR Ca2+ leak (Figure 27B &
C). Figure 27D shows that phosphorylation of NOS1 at the Akt site (Ser1416) is increased with ISO and prevented by the Akt Inhibitor X.
Unfortunately, the Akt-inhibitor X also severely decreased contraction in control cells. Thus, further experimentation to rule out non-specific effects was needed. I therefore used another Akt inhibitor, Akt inhibitor V (5 μm) to measure the myocyte contraction. Shown in Figure 28 are WT myocytes incubated with
Akti-V had significantly decreased shortening (9.5 ± 0.7 %RCL) and Ca2+ transient (3.2 ± 0.3 F/F0) amplitudes compared to control WT myocytes (13.5 ±
1.7 %RCL and 4.3 ± 0.5 F/F0). However, Akti-V did not affect (6.7 ± 1.0 %RCL,
2+ 2.8 ± 0.2 F/F0) the already blunted shortening (6.4 ± 1.1 %RCL) and Ca
-/- transient (3.2 ± 0.3 F/F0) amplitudes in NOS1 myocyte. Taken together, these data indicate that Akt is necessary for NOS1 activation to increase CaMKII and
RyR activity to contribute to the positive inotropic effect of -AR stimulation.
82
3.3.5 Inhibition of NOS attenuates arrhythmogenic spontaneous Ca2+
Waves
It has been recently demonstrated that the CaMKII-dependent increase in diastolic SR Ca2+ leak contributes to an increase in the incidence of arrhythmogenic spontaneous SR Ca2+ waves (SCaW) in both healthy myocytes and those isolated from failing hearts [123]. We therefore hypothesized that NO and its influence to increase CaMKII activity will increase SCaW generation in response to β-AR stimulation. To test this hypothesis, we applied the general
NOS inhibitor Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME, 100 μM) to healthy rabbit myocytes in the presence of ISO. Figure 29A shows the average
[Ca]SRT from all cells examined with the percentage of those myocytes showing
SCaW activity in Figure 29B. Untreated myocytes did not show any SCaWs but
47% of all myocytes exhibited SCaW with ISO treatment. This activity was suppressed when treated with ISO plus L-NAME (29%). After normalizing to
[Ca]SRT, ISO treated cells were significantly more active (0.49 ± 0.04) than those treated with ISO plus L-NAME (0.25 ± 0.02) (Figure 29C). ISO-treated myocytes had a significantly higher number of SCaWs per cell (1.1 ± 0.3, Figure 29D) compared to ISO plus L-NAME (0.22 ± 0.16) at the same [Ca]SRT.
3.3.6 Inhibition of NOS attenuates aftertransients in human heart failure
We also took a translational approach and examined the effect NO and arrhythmias in failing human hearts. We used trabeculae taken from failing human hearts and measured ISO stimulated Ca2+ waves (±L-NAME). Shown in
Figure 30, ISO induced Ca2+ aftertransients in the failing trabeculae which were
83 attenuated by L-NAME (n=2). These data suggest that NO signaling plays a role in the arrhythmogenesis in heart failure.
84
3.4 Discussion
In this study we show evidence for a novel NO-dependent activation scheme for CaMKII leading to increases in RyR activity in response to -AR stimulation (Figure 31). These data suggest that increased ISO-dependent Akt activity leads to NOS1 activation and increased [NO]i activates CaMKII by direct
S-nitrosylation. This novel pathway also effects the generation of arrhythmogenic
SCaWs in healthy (rabbit) and diseased hearts (human HF). We conclude that
NOS1 may be a potentially important therapeutic target for the treatment of arrhythmogenic heart disease.
3.4.1 Effects of NOS1 on CaMKII activation.
Our data lead us to conclude that the ISO-dependent increase in SR Ca2+ leak is mediated by a new and unique adrenergic second messenger pathway involving NO. I found no change in CaMKII oxidation at Met281 between WT and
NOS1-/- mice (Figure 25), even though it has been reported that β-AR stimulation elevates ROS levels [202] and NOS1-/- myocytes have higher ROS levels due to imbalance of the nitroso-redox equilibrium[83].This is in contrast to other studies demonstrating activation of CaMKII via oxidation through NADPH oxidase[111].
The local ROS levels at the SR are known to be regulated by the interaction of
- XOR with NOS1. That is, NOS1 inhibits XOR O2∙ production and NO buffers the
.- O2 produced from XOR. Based on these data we collected, it seems that ROS produced from XOR fails to oxidize CaMKII at Met281/282. However, I cannot exclude oxidation of CaMKII at other residues. Another possible explanation for
85 our results is that the amount of ROS produced from XOR (compared to that from NADPH oxidase) is not adequate to oxidize CaMKII.
Here we found that NO sustained CaMKII activity is independent of Ca2+
(Figure 23A) and via S-nitrosylation of residues within the regulatory domain
(Figure 23B), thus allowing for increased kinase activity. Though the activation of
CaMKII by SNAP makes nitrosylation more likely, an effect due to oxidation by other RNS cannot be completely ruled out [183]. Regardless, the extent to which this mechanism is involved in mediating other CaMKII-dependent effects upon the cell warrants future studies.
In addition to ROS, previous studies have shown other activating mechanisms of CaMKII independent of Ca2+, such as exchange protein activated by cAMP (Epac). However, I showed that in the presence of 8-CPT, an analog of c-AMP which will specifically activate Epac, the increase in diastolic SR Ca2+ leak was similar between WT and NOS1-/- myocytes (Figure 26). These data indicates that Epac is not a downstream effector of NOS1 signaling. Given that NOS1 is located at the SR, whereas Epac is reported to be in a complex with CaMKII, β- arrestin, and the β1-AR receptor [196], it is less likely that Epac modulation of
CaMKII is via NOS1. These data indicate that the Ca2+ released from the SR (i.e.,
RyR) is mainly regulated by direct NOS1-mediated CaMKII activation in a highly organized and compartmentalized pathway.
86
3.4.2 Akt is the upstream activator of NOS1 during β-AR stimulation
The activation of NOS1 during β-AR stimulation is not well defined. It is known that increased cytosolic [Ca2+] can lead to increased binding of the
Ca2+/CaM complex to NOS1 and further activate NO production via utilization of
L-Arginine [57]. Thus, in the past elevated cytosolic [Ca2+] during β-AR stimulation was used to explain the activation of NOS1. Here I found a new potential activator of NOS1 during -AR stimulation is Akt. Akt, also known as protein kinase B (PKB), is a serine/threonine-specific protein kinase, which is activated during β-AR stimulation and involved in multiple cellular processes such as glucose metabolism, apoptosis, cell proliferation, and transcription [203].
In our study, Akt was able to phosphorylate NOS1 at Ser1416 to increase NO production and activate CaMKII to increase RyR activity (Figure 27). Further, I found that inhibition of Akt decreased the positive inotropic effect of -AR stimulation in WT myocytes but did not have any effect on the already blunted inotropic effect in NOS1-/- myocytes (Figure 28). Thus, these data indicate that
Akt is necessary for NOS1 and CaMKII activation to phosphorylate RyR, increase diastolic SR Ca2+ leak, and contribute to the positive inotropic effect of
-AR stimulation.
3.4.3 Relevance to cardiac diseases
A common finding in human and animal models of HF and hypertrophy is increased CaMKII activity [126, 204, 205]. Cellular [Ca2+] is lower in failing versus non-failing hearts. This seems paradoxical, as one may expect lower [Ca2+] to
87 lead to decreased CaMKII activity. However, Erickson and colleagues have proposed a plausible mechanism for the maintenance of CaMKII activity by ROS
[111]. Our studies were unable to demonstrate a role for ROS in activating
CaMKII in the NOS1 signaling cascade (Figures 23 & 24). I speculate that the
ROS-dependent activitation of CaMKII may only manifest itself under conditions of chronic β-AR stimulation, such as HF, where ROS production is increased and the uncoupling of NOS from NO to ROS production may exacerbate this condition [206]. Therefore, in the healthy myocardium, CaMKII activity may rely on RNS much more than on ROS in regulating Ca2+ handling and myocardial contraction.
The role of NOS-mediated CaMKII activitation in the pathological heart may also be of significance. Here we found that NOS inhibition via L-NAME attenuated the ISO induced SCaW generation in healthy myocytes, indicating a role of NOS1 in arrhythmogenesis. We also took a translational approach and investigated if this signalling cascade is present under disease conditions, since
HF patients are at high risk of arrhythmias and sudden death. Indeed we detected the protective effects of L-NAME by the disappearance of irregular Ca2+ aftertransients in human failing ventricular trabeculae. However, as I discussed in
Chapter 1, there is dysfunction of NOS isozymes in HF, with expression, location and function altered. Interestingly, a number of groups reported cardioprotective effects of NOS1 in HF. Therefore, the potential “risky” effect of NOS1 in heart failure is a novel aspect of NOS1 in disease. Thus, further study of this
NOS1/CaMKII/RyR signaling pathway is warranted in cardiac disease models.
88
Figure 22. NOS1-/- hearts show attenuated CaMKII-autophosphorylation.
Western blots showing total CaMKII normalized to GAPDH (bottom left, n=8) and
CaMKII phosphorylated at Thr286 normalized to total CaMKII (bottom right, ±ISO, n=5). *Statistically different from WT (p<0.05).
89
Figure 23. NO increases CaMKII activity via S-nitrosylation.
A) Purified CaMKII was preincubated with 200 μM Ca and CaM. H2O2 (Lane 2) or 500 μM SNAP (Lane 3) was added followed by EGTA. ATP32 was added along with purified β2a LTCC subunit on nickel beads. Incorporation of P32 was measured as an indicator of Ca2+-independent sustained kinase activity. Lane 1 is CaMKII without Ca, CaM, or ATP; Lane 4 is CaMKII without Ca, CaM, or ATP plus the addition of SNAP (500 μM) alone. Lane 5 is P32 incorporation in the continued presence of Ca and CaM. B) Cardiac myocytes were field stimulated at 0.5 Hz under the indicated conditions. CaMKII was then immunoprecipitated from cellular homogenates which were then blotted with an antibody to S-NO. * different from ISO, ** different from both ISO and control (p<0.05). (Data collected from Mohler & Shannon labs)
90
Figure 24. NADPH oxidase inhibitor is unable to decrease diastolic SR Ca2+ leak.
A) Leak/load relationship for all treatments. B) Data were matched such that
[Ca]SRT did not vary (left) between treatments, resultant leaks are shown (right, n=11-12). C) Data were matched such that leak did not vary between treatments
(left), resultant [Ca]SRT needed to induced that leak are shown (right, n=11-14). *
Statistically different from control (P<0.05). (Data collected from Shannon lab).
91
W T N O S 1 -/- IS O - + - + O x d iz e d C a M K II
G A P D H
T o ta l C a M K II
G A P D H
C a M K II O x id a tio n (n o rm a liz e d to to ta l C a M K II) 1 .2 C O N T IS O 1 .0
0 .8
. U
. 0 .6
A
0 .4
0 .2
0 .0 W T N O S 1 -/-
Figure 25. No difference in CaMKII oxidation between WT and NOS1-/- hearts.
Western blots showing CaMKII oxidization at Met281/282 normalized to total
CaMKII (±ISO, P=NS, n=4 mice).
92
M a tc h e d [C a ] 2 + A B S R T S R C a L e a k 0 .0 6 IS O s tim u la te d R y R A c tiv ity C O N T # 6 8 -C P T *
0 .0 4 *
W T 0
0 .1 4 0
F F 4 /
W T + 8 -C P T /
F F
- / - N O S 1 0 .1 2 0 .0 2 - / - N O S 1 + 8 -C P T 2 k *
a 0 .1 0 e
L 0 .0 0
0
0 .0 8 -/-
a W T N O S 1 C
0 .0 6
C M a tc h e d L e a k [C a ] S R T R
8 S 0 .0 4 C O N T 0 .0 4 8 -C P T * 0 .0 2 6
0 .0 3 0
0 * # F
0 .0 0 F /
/ 4 F F *
0 .0 2 .5 5 .0 7 .5 1 0 .0 0 .0 2
S R C a L o a d 2 0 .0 1
0 .0 0 0 W T N O S 1 -/- Figure 26. Epac activator 8-CPT has the same effects on diastolic SR Ca2+ leak in WT and NOS1-/- myocytes.
A) Leak/load relationship with 8-CPT in the presence of ISO. B) Data were matched such that [Ca]SRT did not vary (left) between treatments, resultant leaks are shown (right, n=10-11). C) Data were matched such that leak did not vary between treatments(left), resultant [Ca]SRT needed to induced that leak are shown (right, n=10-12). *Statistically different from WT control, # statistically different from NOS1-/- control. (P<0.05)
93
Figure 27. Akt activates NOS1.
A) ISO-dependent increase in p-Akt is blunted by Akt-Inhibitor X (top, right, * different from control, ** different from ISO, P<0.05). Cells were treated with ISO or ISO + Akt Inhibitor X. Akt Inhibitor X decreased the SR Ca leak and the activation of Akt (B & C). C) NOS1 phosphorylation at Akt phosphorylation site
S1416 (* different from control, ** different from ISO, P<0.05). (Data collected from Shannon lab).
94
C e ll S h o rte n in g A m p litu d e C a 2 + T ra n s ie n t A m p litu d e 1 8 6 IS O 1 6 IS O + A K T i-V 1 4 * *
1 2 4
0
L
F C
1 0 /
R
F
% 8 6 2 4 2
0 -/- 0 W T N O S 1 W T N O S 1 -/-
Figure 28. Effects of Akt inhibition on cell contraction in WT and NOS1-/- myocytes.
Pooled data (mean±SEM) of cell shortening and Ca2+transient amplitudes. Open bar: ISO, Dark bar: ISO+Akt inhibitor V (*P<0.05 vs. others, n=11-15 myocytes/3-
5 hearts).
95
Figure 29. Inhibition of NOS attenuates SCaW formation in ISO treated myocytes.
A) Average [Ca]SRT over all treatments in isolated rabbit myocytes (n=34-40) for each treatment (raw data at the top). B) Percentage of all myocytes which showed at least one SCaW. C) Average of the percent activity, data in B, normalized to each myocyte’s [Ca]SRT. D) Matched data such that the average
[Ca]SRT was the same for treatments (right) and the resultant average number of
SCaWs exhibited (left) are shown (n=13-15). *Statistically different (p<0.05).
(Data collected from Shannon lab).
96
Figure 30. Inhibition of NOS attenuates Ca2+ aftertransients in ISO treated failing human ventricular trabeculae.
(n=2 hearts) (Data collected from Janssen lab).
97
Figure 31: Summary of NOS1 signaling pathway during β-AR stimulation
98
Chapter 4: Discussion
4.1 Principle findings
This section summarizes the most novel findings of chapters 2 and 3.
1. NO signaling via NOS1 (but not NOS3) is essential for the enhanced
diastolic SR Ca2+ leak in rabbit and mouse myocytes during β-AR
stimulation.
2. NO–mediated increase in diastolic SR Ca2+ leak during β-AR stimulation is
via CaMKII phosphorylation of RyR at Ser2814.
3. NOS1/CaMKII enhancement of RyR activity contributes to the positive
inotropic effect of β-AR stimulation.
4. CaMKII mediated SR Ca2+ leak is independent of Ca2+, Epac, NADPH
oxidase or PKG.
5. S-nitrosylation of CaMKII is increased during β-AR stimulation and CaMKII
can be directly activated by NO in vitro, suggesting a novel activation
mechanism of CaMKII by NOS1 (S-nitrosylation).
6. Akt is the upstream activator of NOS1 signaling during β-AR stimulation.
7. NOS inhibition blunts SCaW generation in healthy myocytes and
arrhythmic Ca2+ aftertransients in human failing trabeculae, indicating a
novel role of NO in myocardial disease.
99
4.2 Future directions
4.2.1 Physiological implication of NOS1 signaling cascade during β-AR stimulation
In chapter 2, we illustrated that NOS1 is necessary for the increased diastolic SR Ca2+ leak during β-AR stimulation, which is mediated by CaMKII phosphorylation of RyR at Ser2814. I also ruled out other possible modifications on RyR including direct S-nitrosylation or PKA phosphorylation at Ser2808. The lab previously showed that RyR S-nitrosylation contributes to the positive inotropy of NOS1 under basal conditions. I believe the major effect of S- nitrosylation is to protect against oxidation on the same thiol group and protect
RyR from oxidative damage. When it comes to β-adrenergic stimulation, the importance of S-nitrosylation might be preceded by the increased kinase phosphorylation (i.e. CaMKII) to dominate RyR activation. However, it would still be interesting to investigate the S-nitrosylation levels of RyR during -AR stimulation. Since both NO bioavailability and ROS production are elevated, the levels of S-nitrosylation versus oxidation of RyR could reveal the status of the nitroso-redox balance at the SR microdomain during the β-AR response.
I also showed that the activation of CaMKII by Epac is still intact in NOS1-/- myocytes, and not involved in the β-AR stimulated RyR Ca2+ release. These data suggest that the activation of CaMKII is compartmentalized in the myocyte.
Currently, CaMKII has been shown to be localized and activated at three
100 microdomains: the CaMKII/NADPH oxidase complex on the cell membrane [111], the CaMKII/ EPAC/ β-arrestin complex coupled with β1-AR receptors [194, 196], and the CaMKII/NOS1/RyR complex at the SR (from this dissertation). Further studies are required to investigate the diverse function of CaMKII in these compartments.
The effects of S-nitrosylation on CaMKII are not fully understood. Here our data suggest that S-nitrosylation helps sustain the autophosphorylation of
CaMKII. As I mentioned above, S-nitrosylation usually works as a competitor of oxidation [89, 190]. There are no data showing if oxidation on cysteine residues of CaMKII affects its function. Despite the stimulatory oxidation on Met281/282 by NADPH oxidase, it is likely that oxidatized cysteine residues also exist. But the S-nitrosylation/oxidation interaction on CaMKII is not known. Furthermore,
ROS is produced from various sources in the heart besides NADPH oxidase, such as XOR and mitochondria. XOR is a prime candidate for CaMKII at the SR microdomain since it colocalizes with NOS1 and RyR. Thus, RyR and CaMKII activity can be measured in the presence of XOR inhibition (i.e. allopurinol) and
ISO.
Furthermore, the feedback system to regulate CaMKII activity such as phosphatases needs to also be considered. Currently, there are no specific phosphatases associated with CaMKII dephosphorylation. Previous studies have shown increased PP activity with oxidative stress [207]. Our group has also shown elevated PP activity in NOS1-/- vs. WT myocytes due to the lack of NO
- buffering of O2∙ [86]. Therefore, the increased PP activity in NOS1 deficiency
101 models may further decrease CaMKII activity and decrease contraction during β-
AR stimulation.
Another question is whether RyR is the only end target of NOS1/CaMKII signaling. Previous studies reported that neither PLB phosphorylation at Ser16 nor Thr17 is altered between WT and NOS1-/- myocytes during β-AR stimulation[77, 183, 208]. These results indicate that there are other targets than
PLB. Our results here showed that, in the specific RyR knockin mice (S2814A/D),
RyR activity was resistant to NOS1 inhibition. Further, Ca2+ transient amplitudes of these mice showed no response to NOS1 inhibition either (data not shown).
These results suggest that RyR and not PLB is the end target of NOS1/CaMKII signaling. However, limited research has studied the phosphorylation of myofilament proteins by CaMKII. Interestingly, a recent study has shown that
CaMKII can phosphorylate titin [125, 209]. Given that the classic β-AR signaling molecule, PKA, has been shown to phosphorylate a variety of myofilament proteins[34],[210, 211], a future direction of study is to investigate the effects of
NOS1/CaMKII on the myofilaments.
Another effect of β-AR signaling other than increasing contractility is to increase heart rate. As we mentioned above, FDAR is CaMKII dependent but
PLB independent[28]. Thus, I hypothesize that the NOS1 dependent CaMKII activation may also affect the FDAR. Further, NOS1/CaMKII is involved in the myocyte contraction during the frequency changes of β-AR stimulation. To test this hypothesis, I could perform FFR with CaMKII inhibition in NOS1-/- and WT myocytes. It is expected that NOS1-/- myocytes will have blunted Ca2+ transient
102 and shortening amplitudes during frequency changes compared to WT myocytes due to the lack of RyR activity, and CaMKII inhibition will only have effects on myocyte contraction and FDAR in WT myocytes, but not in NOS1-/- myocytes.
4.2.2 Pathological implications of the NOS1 signaling cascade
As I discussed in chapter 1, there is increased diastolic SR Ca2+ leak, decreased SR Ca2+ load, and a hyperadrenergic response in the early stages of
HF. A common finding in human and animal models of HF and hypertrophy is the increased activity of CaMKII [126, 204, 205]. Increased diastolic SR Ca2+ leak via hyperphosphorylation of RyR by CaMKII is thought to be highly arrhythmogenic and is a fatal factor in heart failure. In chapter 3, I have shown that ISO dependent NO production increases the SCaW in healthy myocytes and NOS inhibition attenuates the aftertransients in human failing trabeculae. Therefore it seems that the NOS1/CaMKII signaling increases the risk of arrhythmias in HF.
However, previous publications show a paradoxical role of NOS1 in heart failure.
That is, NOS1 signaling is cardioprotective in heart failure.
The expression and activity of NOS1 were found to be increased in HF.
However, NOS1 translocates from the SR to caveolae in the sarcolemmal membrane by binding to caveolin-3[150-152]. In particular, studies have shown that myocardial infarction in NOS1-/- mice display worse ventricular remodeling, contractile dysfunction, and mortality compared to WT mice [139, 140]. Whereas, mice with cardiac myocyte-specific NOS1 overexpression developed less dilation, maintained contractile function, and prevented the development of HF in a TAC model. It is believed that NOS1 is required to maintain the nitroso-redox balance 103 especially in the highly oxidative stressed heart (i.e. HF). In addition, NOS1 signaling is protective against arrhythmias via reducing ICa by PKG phosphorylation on LTCC [151, 153] acting similar as NOS3 at the caveolae. It is unclear whether NOS1 is able to regulate CaMKII in heart failure due to the down-regulated NOS1 levels at the SR. In this thesis, L-NAME is found to be protective in failing trabeculae, but it is not known if NOS1 is still the main source of NO to regulate CaMKII activity. Especially since NOS2 is induced in heart
- failure because it produces more NO as well as uncoupled O2∙ in the myocyte.
Since L-NAME is a non-specific NOS inhibitor, I cannot rule out NOS2 involvement in the Ca2+ aftertransients observed in failing trabeculae. In addition,
CaMKII has been reported to be oxidized by NADPH oxidase in HF. Since both
.- NOS3 and NOS2 can become uncoupled to produce O2 in HF, these isozymes may also contribute to the oxidation of CaMKII. Therefore, the oxidation of
CaMKII may succeed the effects of S-nitrosylation of CaMKII in disease conditions. Regardless, our finding of the potential effect of NO contributing to the CaMKII mediated arrhythmia in this dissertation is novel and provides a new perspective for future therapeutic approaches for heart failure.
It is also interesting to investigate the status of NOS1 activation via Akt in
HF. Recently, phosphorylation of Akt has been shown to be down regulated in skeletal muscle of HF mice, but the alteration of Akt in myocardium is still not known[212]. A study showed that phosphorylation of NOS1 at Ser1416 (Akt site) is antihypertrophic and decreases oxidative stess in rat myocytes[213]. Therefore, further investigation of the beneficial effects of Akt/NOS1 pathway is needed to
104 better understand the molecular mechanisms of NOS1 signaling in cardiac disease.
On the other hand, the translocation of NOS1 to the caveolae is likely to induce other CaMKII related events other than SR Ca2+ handling. NOS3 signaling is known to be protective for the hypertrophic remodeling via inhibition of calcineurin/NFAT pathway, whereas CaMKII is found to activate the calcineurin/NFAT pathway leading to hypertrophic gene expression. The downregulation and uncoupling of NOS3 and the compensatory up-regulation of
NOS1 at caveolae might shift the heart to severe damage by increasing adverse remodeling and apoptosis.
4.3 Therapeutic targets of NOS1 dysfunction in heart disease
The decreased NO bioavailability and oxidative stress in HF is directly the result of dysfunctional NOSs with the consequence of contractile dysfunction, hypertrophy, and adverse remodeling. Numerous therapeutic approaches have been tried to generally repair total NO signaling in human HF, but have had little success. The reason is that no current approaches are specific to target certain
NOS without influencing the other NOS isozymes. Given the complexity of NOS dysfunction in heart disease (i.e, isoforms, compartmentalized function, translocation, uncoupling, and ROS), the use of general NOS inhibitors, NO donors, and ROS scavengers/inhibitors may not represent the best option.
Further work is required to find “perfect” strategies to specifically target signaling pathway components and end targets to regain proper NO signaling. In the scale
105 of this dissertation, the β-AR-dependent increase in diastolic SR Ca2+ leak is predominantly CaMKII-dependent. This increased leak is also potentially arrhythmogenic [122, 123]. In order to prevent these effects, many HF patients are treated with β-AR blockers but it results in a decrease in the inotropic state of the tissue, preservation of which may be beneficial to the patient and NOS inhibition will limit the protective NO effects other than the CaMKII mediated arrhythmia. Therefore, to distinguish the exact NO effects on CaMKII dependent arrhythmogenesis (i.e. NO resources, compartment), and consequently develop specific therapeutics to target this process at certain intracellular microdomains or directly target the end point would be the future direction of research.
4.4 Final Remarks
Nitric oxide is a potent regulator of cardiac contractile function. The results presented in this dissertation provide evidence that NOS1-derived NO is a positive inotrope during β-AR stimulation in the heart. Furthermore, these results report the novel finding that this positive inotropy is activated by Akt, which mediates activation of NOS1 to regulate CaMKII dependent SR Ca2+ leak from
RyR. These findings suggest that NOS1 signaling is an important regulator of cardiac function in both health and disease. With further study, the results presented herein may help aid in the development of new therapeutic agents to treat heart failure.
106
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