CONTRACTILE DYSFUNCTION IN HEART FAILURE AND FAMILIAL HYPERTROPHIC CARDIOMYOPATHY
By
YI-HSIN CHENG
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy
Dissertation Adviser: Dr. Julian E. Stelzer
Department of Physiology and Biophysics CASE WESTERN RESERVE UNIVERSITY
January, 2014 CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES
We hereby approve the thesis/dissertation of
______Yi-Hsin Cheng ______candidate for the ______Doctoral______degree *.
(signed)______Corey Smith______
(chair of the committee)
______Julian Stelzer ______
______Thomas Nosek ______
______David Van Wagoner ______
______Brian Hoit ______
______Xin Yu ______
(date) ______September 10th, 2013______
*We also certify that written approval has been obtained for any proprietary material
contained therein.
ii DEDICATION
To my family overseas, who have supported me and allowed me to pursue anything I want.
To my boyfriend, who has helped me through the conflicts and struggles due to the nature of this work and led me on the right path.
To my vegan mentors and community, who continue to give me hope.
iii TABLE OF CONTENTS
List of Tables ix
List of Figures x
Acknowledgements xii
List of Abbreviations xv
Abstract xx
Chapter 1: Cardiac Health and Disease 22
1.1 Introduction 22
1.2 Cardiac Structure and Functions 23
1.2.1 Heart Structure and Functions 23
1.2.2 Cardiomyocyte and Myofilament Structure and Functions 26
1.2.3 The Electrical Properties of the Heart 37
1.2.4 Ca2+ Handling 39
1.2.5 Autonomic and -adrenergic Regulation 41
1.3 Cardiac Metabolism 44
1.4 Cardiac Dysfunction 47
1.4.1 LV Dysfunction 47
1.4.2 LV Remodeling 48
1.4.3 Heart Failure 51
1.4.4 Familial Hypertrophic Cardiomyopathy 59
1.5 Rationale and Hypothesis 66
iv Chapter 2: Changes in Myofilament Proteins, but not Calcium Regulation, are
Associated with a High Fat Diet-induced Improvement in Contractile Function in
Heart Failure 68
2.1 Introduction 69
2.2 Materials and Methods 71
2.2.1 Experimental Model 71
2.2.2 Plasma Metabolic Substrates 72
2.2.3 Echocardiography 72
2.2.4 Hemodynamic Measurements 73
2.2.5 Histological Assessment of Cardiac Morphology 73
2.2.6 Cardiomyocyte Isolation 74
2.2.7 In Vitro Cardiomyocyte Shortening and Ca2+ Transients 75
2.2.8 Protein Expression by Western Blot Analysis 76
2.2.9 L-type Ca2+ Current Measurements 77
2.2.10 Myosin Heavy Chain Protein Expression 77
2.2.11 Myofilament Protein Phosphorylation and Expression 78
2.2.12 Statistics 79
2.3 Results 79
2.3.1 Body Weight and Metabolic Substrates 79
2.3.2 Cardiac Morphology 80
2.3.3 Echocardiography and Hemodynamic Function 81
2.3.4 Cell Shortening 83
2.3.5 Ca2+ Transients 83
v 2.3.6 Ca2+ Regulatory Protein Expression 86
2.3.7 Correlations between in Vivo and in Vitro Function 86
2.3.8 ICa 88
2.3.9 Myofilament Protein Composition and Phosphorylation 89
2.4 Discussion 90
Chapter 3: Impaired Contractile Function Due to Decreased Cardiac Myosin
Binding Protein C Content in the Sarcomere 96
3.1 Introduction 97
3.2 Materials and Methods 100
3.2.1 Ethical Approval and Experimental Model 100
3.2.2 Echocardiography 100
3.2.3 Hemodynamic Measurements 101
3.2.4 Electrocardiogram 101
3.2.5 High Resolution Optical Mapping 102
3.2.6 Histological Assessment of Cardiac Morphology 103
3.2.7 Myocyte Isolation and in Vitro Shortening and Ca2+ Transients 103
3.2.8 Myofilament Contractile Function 104
3.2.9 Quantification of Protein Expression and Phosphorylation 109
3.2.10 Phosphatase Activity Assay 112
3.2.11 Statistical Analysis 112
3.3 Results 112
(Comparison between WT and MyBP-C−/− Models) 112
3.3.1 Myofilament Protein Content and Phosphorylation 112
vi 3.3.2 Isolated Myocyte Contractile Properties 114
3.3.3 Myocyte Ca2+ Handling Properties 116
3.3.4 Expression of Ca2+ Handling Proteins and PP1 Activity 116
3.3.5 Echocardiography 118
3.3.6 In Vivo Hemodynamic Function at BL and Following DOB Challenge 119
3.3.7 Optical Mapping and Cx43 Protein Expression and Phosphorylation 121
3.3.8 ECG 123
(Comparison between WT and MyBP-C+/− Models) 124
3.3.9 Myofilament Protein Content and Phosphorylation 124
3.3.10 Steady-state Force and Dynamic Cross-bridge Kinetics Prior to and
Following PKA Treatment 126
3.3.11 Isolated Myocyte Contractile Properties 131
3.3.12 Myocyte Ca2+ Handling Properties 132
3.3.13 Expression and Phosphorylation of Ca2+ Handling Proteins 133
3.3.14 In Vivo Hemodynamic Function 134
3.3.15 Echocardiography 135
3.3.16 Histological Assessment of Cardiac Morphology 136
3.3.17 ECG 137
3.4 Discussion 140
3.4.1 In Vitro Contractile Function and Ca2+ Handlingin MyBP-C−/− Myocytes
142
3.4.2 In Vivo Contractile and Hemodynamic Function in MyBP-C−/− Hearts 144
3.4.3 Electrical Conduction and ECG Recordings in MyBP-C−/− Hearts 145
vii 3.4.4 Myofilament Contractile Function in MyBP-C+/− Hearts 146
3.4.5 In Vitro Contractile Function and Ca2+ Handlingin MyBP-C+/− Myocytes
151
3.4.6 In Vivo Contractile and Hemodynamic Function in MyBP-C+/− Hearts 152
3.4.7 ECG Recordings in MyBP-C+/− Hearts 154
Chapter 4: Summary and Future Directions 156
4.1 Summary and Future Directions of “Changes in Myofilament Proteins, but not
Calcium Regulation, are Associated with a High Fat Diet-induced Improvement
in Contractile Function in Heart Failure” 156
4.1.1 Potential Factors Contributing to MHC Changes 156
4.1.2 Other Potential Mechanisms for Improved Contractility in HFSAT
Animals 160
4.1.3 Limitations 165
4.1.4 Fat vs. Carbohydrate 167
4.2 Summary and Future Directions of “Impaired Contractile Function Due to
Decreased Cardiac Myosin Binding Protein C Content in the Sarcomere” 170
4.2.1 Regional Differences in Expression of MyBP-C 171
4.2.2 Cardiac Energetic Alterations 174
4.2.3 Autonomic Regulations 175
4.2.4 Beyond Mutations? 178
Reference List 181
viii LIST OF TABLES
Table 1-1. Summary of Hypertrophic Cardiomyopathy Susceptibility Genes 61
Table 2-1. Plasma Substrates, Echocardiography and Hemodynamic Function 80
Table 3-1. Gravimetric Measurements and in Vivo LV Function by
Echocardiography between WT and MyBP-C−/− Models 119
Table 3-2. LV Hemodynamic Function at BL and Following -adrenergic
Stimulation between WT and MyBP-C−/− Models 120
Table 3-3. ECG at BL and Following -adrenergic Stimulation between WT and
MyBP-C−/− Models 123
Table 3-4. Steady-state Mechanical Properties of Skinned Fibers Isolated from WT and MyBP-C+/− Myocardium 127
Table 3-5. Cross-bridge Kinetics of Skinned Fibers Isolated from WT and
MyBP-C+/− Myocardium 129
Table 3-6. LV Hemodynamic Function at Baseline and Following -adrenergic
Stimulation between WT and MyBP-C+/− Models 135
Table 3-7. LV Morphology and in Vivo Function Measured by Echocardiography between WT and MyBP-C+/− Models 136
Table 3-8. Electrocardiographic Data Acquired by Radio Telemetry between WT and MyBP-C+/− Models 138
Table 3-9. Comparisons between MyBP-C−/− and MyBP-C+/− Models. 139
Table 4-1. A List of Pathophysiological Stimuli Controlling mRNA and/or Protein
Expression of Cardiac MHC Isoforms 158
ix LIST OF FIGURES
Figure 1-1. LV Pressure-volume Loop 24
Figure 1-2. Structure of the Sarcomere 27
Figure 1-3. Components of the Filaments 28
Figure 1-4. Cardiac Muscle Cross-bridge Cycling 29
Figure 1-5. Slack-restretch and stretch activation responses 33
Figure 1-6. General Domain Structure of Mouse MyBP-C 36
Figure 1-7. Cardiac Ionic Currents, Action Potentials, and ECGs in Humans versus
Mice 38
Figure 1-8. Ca2+ Transport in Ventricular Myocytesduring ECC 40
Figure 1-9. Determination of Resting Cardiac Parasympathetic and Sympathetic Tone
44
Figure 1-10. Cardiac Energy Metabolism 46
Figure 1-11. Patterns of LV Remodeling Based on EDV, Wall Mass, and RWT 50
Figure 2-1. Histological Assessment of Infarct Size and Myocyte Cross-sectional
Area 82
Figure 2-2. LV Cardiomyocyte Shortening 84
Figure 2-3. LV Cardiomyocyte Ca2+ Regulation 85
Figure 2-4. Correlations between in Vivo and in Vitro Functional Measurements 87
2+ Figure 2-5. L-type Ca Currents (ICa) 88
Figure 2-6. Myofilament Protein Composition and Phosphorylation 89
Figure 3-1. Stretch Activation Responses in Murine Myocardium 108
x Figure 3-2. Protein Expression and Phosphorylation of Myofibrillar Proteins between WT and MyBP-C−/− Models 113
Figure 3-3. Ventricular Cardiomyocyte Sarcomere Shortening between WT and
MyBP-C−/− Models 115
Figure 3-4. Ventricular Cardiomyocyte Ca2+ Transients between WT and
MyBP-C−/− Models 117
Figure 3-5. Protein Expression of Ca2+ Handling Proteins and PP1 Activity 118
Figure 3-6. Optical Mapping and Protein Expression of Cx43 122
Figure 3-7. Expression and Phosphorylation of Myofilament Proteins between WT and MyBP-C+/− Models 125
Figure 3-8. Rate of Force Development and Fiber Stiffness 130
Figure 3-9. Ventricular Cardiomyocyte Sarcomere Shortening between WT and
MyBP-C+/− Models 132
Figure 3-10. Ventricular Cardiomyocyte Ca2+ Transients between WT and
MyBP-C+/− Models 133
Figure 3-11. Morphology of the Hearts and Cardiomyocytes 137
Figure 3-12. ECG Waveforms 138
Figure 4-1. Regional Differences in Expression of MyBP-C 173
Figure 4-2. Sympathetic and Parasympathetic Tone 176
xi ACKNOWLEDGEMENTS
I have a special thank you for Cathy Carlin, who protected me through a conflict with a faculty member during my very first week in the program. I have followed her advice ever since and found it helpful, and I want to thank her for that.
I want to thank my first advisor, Margaret Chandler, for accepting me at the end of my fourth rotation and giving me flexibility to explore my project. I also want to thank my committee members (past and present), Brian Hoit, Corey Smith, David Van
Wagoner (Department of Molecular Cardiology, Cleveland Clinic), Edward Lesnefsky,
Julian Stelzer, Robert Harvey, Thomas Nosek, and Xin Yu (Department of Biomedical
Engineering, Case Western Reserve University) for the continuous guidance and support, both academically and personally. I especially want to thank Julian Stelzer for providing me a shelter when I needed it and serving as my advisor for the second half of my thesis.
I want to thank my labmates, (from Chandler lab) Julie Rennison, Cody Rutledge,
Xiaoqin Chen, Tracy McElfresh, Jessica Berthiaume, Sarah Stewart, and Bridgette
Christopher, and (from Stelzer lab) Brian Zeise, Arthur Coulton, Sergei Merkoulov, and
Kenneth Gresham for the scientific discussions and technical assistance. Additionally, I want to thank Julie Rennison for checking my reports and helping me through my initial cultural transition period.
There are several techniques used in this thesis that I had to learn and build from scratch through much collaboration with different labs, and I want to thank each and every person who helped me with these. I want to thank Ya Chen from Harvey lab, Wei
Li from Yu lab, Tony Prosdocimo from George Dubyak’s lab (Department of Physiology and Biophysics, Case Western Reserve University), Sarah Stewart (from Lesnefsky lab at
xii the time), and (remotely) Karen O’Shea from William Stanley’s lab (Department of
Medicine, University of Maryland) for providing their protocols for myocyte isolation and helping me with trouble shooting, and Cody Rutledge for establishing the technique with me. I want to thank Wei Li for teaching me the technique for cell shortening and
Ca2+ transient measurements and both Wei Li and David Van Wagoner for the subsequent trouble shooting. I want to thank Wen Li from Yu lab, Julie Wolfram and
Sandra Siedlak from Mark Smith’s lab (Department of Pathology, Case Western Reserve
University), and Hyoung-gon Lee (Department of Pathology, Case Western Reserve
University) for teaching me various histology techniques and tricks from tissue procurement, slicing, all the way to the many different staining methods. I want to thank
Scott Howell (The Visual Sciences Research Center, Case Western Reserve University) for his imaging expertise and coming up with the quantification methods for the histology data. I want to thank Chao Yuan (Proteomics Core, Case Western Reserve University) for the myofilament protein purification method, and both Chao Yuan and (remotely) Chad
Warren from John Solaro’s lab (Department of Physiology and Biophysics, University of
Illinois) for trouble shooting with myosin heavy chain separation. I want to thank Yee-
Hsee Hsieh from Ted Dick’s lab (Department of Medicine, Case Western Reserve
University) for assisting me with electrocardiogram recording and David Van Wagoner for lending me the Ponemah software for subsequent analysis. Last, I want to thank
Caroline El Sanadi and Andrea Boyd-Tressler from Dubyak lab for teaching and assisting me with the in vitro ATP assay.
I also wish to acknowledge everyone who collected data for me through collaborations. I want to thank Brain Hoit for allowing us to use the ultrasound machine,
xiii Laurie Castel from Van Wagoner lab and David Van Wagoner for collecting and analyzing the ICa data, Xiaoping Wan from David Rosenbaum’s lab (Heart and Vascular
Center, MetroHealth) for collecting the sarcomere shortening and Ca2+ transient data for the second project of this thesis, Maria Strom and Michelle Jennings from Rosenbaum lab for collecting and analyzing the optical mapping and action potential duration data, and the Tissue Resources Core of the Case Comprehensive Cancer Center for doing most of the histology for the second project of this thesis.
Last but not least, I want to thank Department of Physiology and Biophysics and the chairs (past and present), the staffs, and the many friends I have in it and around for their company and help along the way, however trivial they might seem, for that every kind gesture from them propelled me to complete this thesis.
xiv LIST OF ABBREVIATIONS
±dL/dt: peak velocity of cell relaxation and shortening
2D: two-dimensional
AR: -adrenergic receptors
L: stretch length
: the exponential or logistic decay constant
APD: action potential duration
BL: baseline
BMI: body mass index
BW: body weight
CaMKII: Ca2+/calmodulin kinase II
CETP: cholesteryl ester transfer protein
CICR: Ca2+-induced Ca2+ release
CL: cycle length
CO: cardiac output
CVD: cardiovascular disease
Cx43: connexin 43
DATD: N-N' diallyltartardiamide
DOB: dobutamine dP/dtmax/min: left ventricular maximal and minimal rate of pressure changes with time
ECC: excitation-contraction coupling
ECG: electrocardiogram
EDA: end-diastolic area
xv EDD: end-diastolic dimensions
EDP: end-diastolic pressure
EDV: end-diastolic volume
EF: ejection fraction
ESA: end-systolic area
ESD: end-systolic dimensions
ET: ejection time
ET-1: endothelin 1 f: the forward rate constant for the force-generating transition between the non-force generating and force-generating states
FFA: free fatty acids
FHC: familial hypertrophic cardiomyopathy
FS: fractional shortening g: the reverse rate constant for the force-generating transition between the non-force generating and force-generating states
GH: growth hormone
GLUT: glucose transporters
H&E: hematoxylin and eosin stain
HDL: high-density lipoproteins
HF: heart failure
HFpEF: heart failure with preserved ejection fraction
HPLC: high-performance liquid chromatography
HR: heart rate
xvi HRV: heart rate variability
HSC70: heat shock chaperone 70
2+ ICa: L-type Ca current
IGF-1: insulin-like growth factor-1
ISO: isoproterenol
IVRT: isovolumic relaxation time kdf: the rate constant of delayed force development after phase 2 after acute stretch krel: the rate constant of force decay after the peak of phase 1 after acute stretch ktr: the rate constant of force redevelopment after the slack-restretch maneuver
LDL: low-density lipoproteins
LTCC: L-type Ca2+ channel
LV: left ventricle
MHC: myosin heavy chains
MI: myocardial infarction
MRI: magnetic resonance imaging
MRLC: myosin regulatory light chains
MyBP-C: myosin binding protein C
MyBP-C+/−: heterozygous myosin binding protein C null mice
MyBP-C−/−: myosin binding protein C knockout mice
MyBP-CAllP−: transgenic mice that bear unphosphorylatable myosin binding protein C
MyBP-Ct/t: transgenic mice that bear a truncation mutation in myosin binding protein C
NC: normal chow
NCX: Na+-Ca2+ exchanger
xvii NFAT: nuclear factor of activated T cells nH: Hill coefficient
NIH: the National Institutes of Health
P: submaximal force
Po: maximal force pCa50: pCa required for 50% activation
PCr: phosphocreatine
PKA: protein kinase A
PLB: phospholamban pMyBP-C273: phosphorylated myosin binding protein C on Ser273 pMyBP-C282: phosphorylated myosin binding protein C on Ser282 pMyBP-C302: phosphorylated myosin binding protein C on Ser302
PP1: phosphatase 1 pPLB16: phosphorylated phospholamban on Ser16 pPLB17: phosphorylated phospholamban on Thr17 pTnI: phosphorylated TnI
PVDF: polyvinylidene difluoride
PWTd: posterior wall thickness at end diastole
QTc: the corrected QT interval
ROS: reactive oxygen species
RWT: relative wall thickness
RyR: ryanodine receptor
SAT: saturated fat diet
xviii SCD: sudden cardiac death
SERCA: sarcoplasmic reticulum Ca2+ ATPase
SH: sham surgery
SOCE: store-operated Ca2+ entry
SR: sarcoplasmic reticulum
STIM: stromal interaction molecule
SV: stroke volume
SVR: systemic vascular resistance
TG: triglyceride
TnC: troponin C
TnI: troponin I
TnT: troponin T
TRPC: canonical transient receptor potential proteins
T-tubule: transverse tubules
WT: wild-type
xix Contractile Dysfunction in Heart Failure and Familial Hypertrophic Cardiomyopathy
Abstract
by
YI-HSIN CHENG
Elevations in plasma and myocardial lipids can exacerbate the progression of heart failure (HF). However, our previous studies reported that high saturated fat (SAT) feeding improves in vivo myocardial contractile function after infarction. Since alterations in cardiomyocyte Ca2+ kinetics and myofilament proteins also contribute to contractile dysfunction in HF, in the first study we hypothesize that SAT improves contractile function by ameliorating these alterations in HF. Rats underwent coronary artery ligation or sham surgery (SH) and were fed normal chow (SHNC and HFNC) or
SAT (SHSAT and HFSAT) for 8 weeks. SAT reduced in vivo myocyte hypertrophy and improved in vivo (LV dP/dtmax/min) and in vitro contractility (–dL/dt) in HF. MHC isoform switched from fast MHC to slow MHC in HFNC but reversed in HFSAT. Alterations in Ca2+ transients, L-type Ca2+ currents and expression of Ca2+ handling proteins could not account for changes in in vivo contractile properties. Together, the cardioprotective effects associated with SAT in HF occur at the level of cardiomyocyte, specifically involving changes in myofilament function, but not Ca2+ handling properties.
xx Mutations in cardiac myosin binding protein C (MyBP-C) cause familial hypertrophic cardiomyopathy (FHC). Most MyBP-C mutations reduce MyBP-C expression, however, the consequences of MyBP-C deficiency are unclear. In the second study, we employed MyBP-C null (MyBP-C−/−) and heterozygous null (MyBP-C+/−) mice. Complete MyBP-C deficiency altered in vivo and in vitro contractile function, Ca2+ handling, electrical activity, and chamber remodeling of MyBP-C−/− hearts. Partial
MyBP-C deficiency and concomitant down-regulated MyBP-C phosphorylation in
MyBP-C+/− hearts altered cross-bridge function which contributed to in vitro and in vivo contractile dysfunction and ECG abnormalities in the absence of adaptations in Ca2+ handling or LV chamber remodeling. Contractile dysfunction in MyBP-C+/− myofilaments and intact hearts were normalized by β-agonists, suggesting that their basal contractile dysfunction is partly mediated by impaired MyBP-C phosphorylation.
Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere results in myofilament dysfunction, contributing to contractile dysfunction that precedes adaptations in Ca2+ handling and chamber remodeling. Perturbations in mechanical and electrical activity in these mice could also increase their susceptibility to arrhythmia.
xxi Chapter 1
Cardiac Health and Disease
1.1 INTRODUCTION
A human heart beats more than 2.5 billion times in an average life time, providing circulation throughout the body. To meet the demand and adjust to various situations that the body faces, the rate and the force of contraction and relaxation of the heart are tightly regulated to ensure optimal circulation. Failure to provide adequate circulation manifests as contractile dysfunction which can precipitate into severe heart diseases, some of which are genetically predisposed. Improving or preserving contractile function in the pathological state is often the main focus of disease management for cardiovascular disease (CVD).
CVD remains the number one cause of death and is responsible for one in every three deaths in America (275). On average every 39 seconds, someone dies from it (275).
CVD encompasses a variety of conditions, including heart failure (HF), myocardial infarction (MI), congenital heart disease, hypertension, atherosclerosis, and stroke, etc
(275). It is a large social and financial burden with the estimated total cost being ~$500 billion in 2010 (193). Currently there is no cure for most CVD. Reducing risk factors before the onset of CVD is recommended (13), however, once diagnosed most of the
22 available treatments can only slow down the progression of ventricular dysfunction with the exception of the rare heart transplant and drastic diet and lifestyle changes. Prognosis and the life quality of the patients are poor. Therefore, CVD is one of the main areas of research today. This dissertation focuses on two components— HF and familial hypertrophic cardiomyopathy (FHC).
1.2 CARDIAC STRUCTURE AND FUNCTIONS
1.2.1 Heart Structure and Functions
The mammalian heart consists of four chambers, including two atria and two ventricles. [For general review see (38).] The right atrium and ventricle receive relatively deoxygenated blood from the body and pump the blood through pulmonary circulation where oxygen exchange occurs. The left atrium and ventricle (LV) receive the oxygenated blood and provide systemic circulation throughout the body. The function of
LV has significant diagnostic value for CVD and can be measured through pressure- volume catheters among many other techniques. (The following discussion is centered on
LV unless otherwise specified.) The changes of LV pressure and volume during a complete cardiac cycle are described in Fig. 1-1, including four phases: diastolic filling
(segment a), isovolumic contraction (segment b), ejection (segment c), and isovolumic relaxation (segment d). Aspects of the LV function and parameters derived from the pressure-volume measurements that are of particular interest are listed below.
Preload: the stretching of cardiac muscle before contraction due to venous return
and filling of chamber, which is affected by venous tone and the volume of
circulating blood. Within the optimal range, increasing preload can increase
23 contraction, a principle of Frank-Starling mechanism. End-diastolic volume (EDV)
or pressure at the end of phase A can be used as indices of preload but are less
useful in disease state with increased LV chamber such as dilated cardiomyopathy.
Figure 1-1. LV Pressure-volume Loop. The standalone LV pressure and volume of a full cardiac cycle are shown on the left, and the pressure-volume relationship on the right. Diastolic filling starts at point 4 when mitral valve opens. Isovolumic contraction happens between point 1 when mitral valve closes and point 2 when aortic valve opens. Ejection follows and ends at point 3 when aortic valve closes. Isovolumic relaxation ensues until point 4. EDPVR: end-diastolic pressure-volume relationship; ESPVR: end- systolic pressure-volume relationship; LVP: left-ventricular pressure; LV Vol: left- ventricular volume; SV: stroke volume. (Reprinted from Cardiovascular Physiology Concepts at www.cvphysiology.com with permission from Dr. Richard E. Klabunde.)
Afterload: the tension or pressure that LV has to generate to eject blood into the
aorta and is affected by aortic pressure and LV volume. The greater the aortic
pressure (such as hypertension) or LV volume (such as dilated LV chamber), the
larger tension and pressure needed to eject blood. Therefore if the level of
inotropy (the properties of cardiac contractility) remains the same, higher
afterload would result in less amount of blood ejection.
24 Stroke work: the work done by the LV to eject the stroke volume (SV) (the
difference between EDV and end-systolic volume) into aorta. It is the area
enclosed by the pressure-volume loop, a product of the SV and the mean aortic
pressure (afterload).
Cardiac output (CO): the amount of blood pumped out by the ventricle in unit
time; a product of SV and heart rate (HR). CO increases when the demand for
oxygen increases, such as during exercise.
Ejection fraction (EF): the fraction of EDV that is ejected out of the ventricle
during each contraction. EF = SV/EDV. Increasing inotropy leads to an increase
in EF, and vice versa.
dP/dtmax/min: the maximum and minimum rate of pressure change in the ventricle,
indicators of contractility independent of afterload, wall motion abnormalities, or
ventricular anatomy and morphology. However, it can be influenced by preload
and HR. An increase in dP/dtmax indicates an increase in inotropy and/or the
ability to contract faster [chronotropy (the frequency properties of cardiac
contractions, heart rate) and klinotropy (the rate properties of cardiac contractions,
cross-bridge kinetics)] during phase B. Similarly, a decrease in dP/dtmin (which is
a negative value) indicates an increase in diastolic function or the ability to relax
faster (lusitropy, the properties of cardiac relaxation) during phase D.
Tau (): the exponential or logistic decay constant of the LV pressure during
phase D, an index of LV relaxation. is independent of preload, and an increase
in indicates negative lusitropy.
25 There are various causes of contractile dysfunction (see Chapter 1.4). Each hemodynamic parameter reflects a different aspect of contractile function, and utilizing cardiac hemodynamic measurements allows us to characterize and examine the pathophysiology of CVD. These measurements can be used in conjunction with stress such as sympathetic agonists or exercise, which are useful in revealing symptoms that are absent in the steady state.
1.2.2 Cardiomyocyte and Myofilament Structure and Functions
Along with other cell types, a heart consists of millions of cardiomyocytes, which are grouped into myofibers by perimysium and align in various angles depending on their location in the chamber wall. [For review see (295).] The specific pattern of cardiomyocyte orientation is supported by the sieve-like collagen bed and is designed for orchestrated contractions, in a wringing motion. The design possesses energetic and mechanical advantages (356), allowing the heart to be more efficient, and hence it has functional significance. Disruption of the alignment is termed myocardial disarray, and it can contribute to contractile dysfunction (see below).
Cardiomyocytes contain series of sarcomeres (Fig. 1-2), the basic unit of contraction, ranging from 1.8 to 2.3 m in length. [For review see (121, 233, 302, 358).]
Each sarcomere consists of repeated groups of thin and thick myofilaments that span from Z line to Z line, which gives the muscles a striated pattern. The thick filament is a polymer of several hundreds of myosin (Fig. 1-3), which exists as a dimer due to the alpha-helical structure of the tail region, resulting in one myosin rod, two myosin heavy
26 Figure 1-2. Structure of the Sarcomere. Schematic drawing of sarcomere structure. Tropomyosin forms an -helical coiled double strand lying along the grooves of actin double strands through head-to-tail polymerization. Troponin complex attaches to a specific region of each tropomyosin and is distributed at regular intervals of 38 nm in the thin filament. Myosin heavy chain (MyHC) has a globular head domain containing actin binding and ATP hydrolytic sites. Myosin-binding protein C (MyBP-C) is a thick filament associated protein, which forms 7–9 transverse stripes at regular intervals of 43 nm in the C-zone of the sarcomere A-band. Titin is a giant protein spanning entire half of the sarcomere from Z-disc to M-line. [Reprinted from (232) by permission of Oxford University Press.] chains (MHC) and four light chains [two essential and two regulatory light chains
(MRLC)] per myosin dimer. The thin filament is composed of actin and its regulatory proteins, such as tropomyosin and the troponin complex [troponin C (TnC), I (TnI), and
T (TnT)]. Globular actin molecules form long filamentous polymers which dimerize as alpha-helical chains that serve as a scaffold for the troponin-tropomyosin regulator
2+ complex. In the relaxed state with low [Ca ]i, the tropomyosin blocks the binding sites
2+ 2+ for myosin on actin. When [Ca ]i increases in the beginning of a contraction cycle, Ca
27 Figure 1-3. Components of the Filaments. Thin filament (on the top) consists of actin, tropomyosin, TnC, TnI, and TnT. Thick filament (on the bottom) consists of myosin, compartmentalized into myosin head which contains heavy chains and light chains, and myosin rod. Myosin binding protein C spans across thin and thick filaments. [The image is reproduced with minor modifications with permission from (317), Copyright Massachusetts Medical Society.] binds to TnC, and there is a conformation change in TnI that reduces the inhibition of tropomyosin, allowing the binding sites to be exposed and cross-bridges formed in a weakly-bound, “closed state”. The weakly-bound cross-bridges then transition into strongly-bound but non force-generating state, which furthers the transformation of the thin filament, allowing the strongly-bound cross-bridges to go into the force-generating,
“open state”. The myosin head has ATPase activity and moves along actin and slide towards Z lines when hydrolyzing ATP, generating forces (Fig. 1-4). When ATP binds to the myosin head, it causes the detachment of cross-bridges. When ATP is hydrolyzed into
28 Figure 1-4. Cardiac Muscle Cross-bridge Cycling. The cross-bridges go through several states as the myosin heads hydrolyze ATP. Descriptions on the ATP utilization and the associated conformational changes are outlined and numbered 1-5. The same cycle repeats several times in a contraction. [Reprinted from (37), Copyright © Elsevier (2009).]
ADP and inorganic phosphate, the myosin head position is restored and ready to bind to the next actin. The releasing of phosphate from myosin produces a change in conformation, the power stroke, which slides the two filaments in opposite direction. The
ADP is then released, and the actomyosin complex stays attached, awaiting the next ATP binding for separation. The same process repeats multiple times during a contraction.
29 Contraction shortens the sarcomere by increasing the overlap of the thin and thick filaments, therefore cardiac muscle contraction occurs in a parallel plane to sarcomere organization.
Within the optimal sarcomere length, the longer it is the more room for cross- bridge sliding and hence stronger the contraction, which is the molecular basis of Frank-
Starling relationship. The distance between the thin and thick filaments also needs to be optimal for proper cross-bridge attachments and the following dissociation, and the timing of these events is crucial for contractile function (see below). The length- dependent increase in contractility is also mediated by increased myofilament Ca2+ sensitivity associated with increased sarcomere length, which is regulated in part by cross-bridge formation. Both Ca2+ and strongly-bound cross-bridges can facilitate the activation of thin filament by changing the conformation in the adjacent previously inactive units and hence increase the probability of new cross-bridge formation, a
2+ mechanism of cooperative activation. As [Ca ]i decreases, dissociation of cross-bridges also impacts adjacent units in a similar fashion and hence cooperative inactivation of force. Cross-bridge binding has allosteric effects on tropomyosin and troponin, and the activation spreads within and between 7 actins (there are 7 actins in one regulatory unit) and the associated tropomyosin and troponin molecules. Strongly-bound cross-bridges also induce cooperative activation by increasing the affinity of TnC to Ca2+ through conformational changes in TnC, therefore strongly-bound cross-bridges and Ca2+ synergistically activate thin filaments. Additionally, the increased Ca2+ sensitivity associated with increased sarcomere length could be due to shorter distance between the thin and thick filament assuming the sarcomeres operate in a constant volume, although
30 this hypothesis has generated controversial results. Cooperativity also affects Frank-
Starling relationship as conditions that hinder cooperativity also abolishes the length dependence of the myofilaments. Cooperative activation can happen with almost no Ca2+ and a small number of cross-bridges in vitro, and this strong sensitivity to activation factors contributes to the fine tuning of force regulation. Together, the length dependence force generation of cardiac muscle gives the heart the ability to “pump what it receives”, and multiple activation mechanisms would allow for more precise regulations of force generation.
The force generated by myofilaments is influenced by many factors, such us
2+ [Ca ]i, cross-bridge kinetics, and the composition of myofibril proteins. The relationship
2+ 2+ between isometric force and [Ca ]i, if plotted between force and pCa (-log[Ca ]), is sigmoidal. The Hill equation is used to fit this relationship:
P [Ca ] H 0 = where P is force, P0 is maximumP force,pCa nH is +the[ HillCa coefficient,] and pCa50 is the pCa required for 50% activation. The nH is the slope of the force-pCa relationship, and it predicts the number of Ca2+ binding sites on TnC involved in force generation. The fact
2+ that in cardiac muscle nH is usually greater than 3 yet there is only one regulatory Ca binding site on TnC highlights the cooperativity in cardiac muscle activation. Because of high cooperativity in cardiac muscle activation, force generation is greater as if there is more than one Ca2+ binding site on TnC. However, the contribution of cooperativity at
2+ force generation becomes less significant at higher [Ca ]i as more thin filaments get switched on by Ca2+.
31 Cross-bridge kinetics can affect the rate of force generation and decay. To study cross-bridge kinetics, isolated permeabilized myofibrils are often used to bypass influences from the sarcoplasmic reticulum (SR). Protocols such as slack-restretch and stretch activation response are useful for discerning the individual components in a contraction cycle (e.g. the rate of cross-bridge attachment/detachment) and certain properties of myofilament that influence its contractile function (e.g. stiffness and Ca2+ sensitivity). Fig. 1-5 illustrates the typical responses of slack-restretch (on the left) and stretch activation (on the right) protocols (55, 323). For the slack-restretch maneuver, the skinned fibers are held at ~2.1 m and incubated in various Ca2+-activating solutions (e.g. pCa 4.5). Once isometric force reaches steady state, the preparation is rapidly slackened by 20% in length. The cross-bridges detach as a result, and the force drops to near zero.
After 15 ms of unloaded shortening, the preparation is then stretch back to its original length.∼ Force redevelops as the cross-bridges reform in a cooperative manner. The rate constant of force redevelopment is termed ktr and is a direct measurement of cross-bridge cycling rate, which is the sum of forward (f) and reverse (g) rate constants for the force- generating transition between the non-force generating and force-generating states.
For the stretch activation maneuver (Fig. 1-5), the skinned fibers are held and incubated in various Ca2+-activating solutions the same way as the slack-restretch protocol. Once the fiber reaches steady-state, a series of instantaneous (≤ 2 ms) stretches ranging from 0.5 to 2.5% of fiber length are imposed and each held for 5 s before returning the fiber to pCa 9. There is an initial peak of force during phase 1, representing the tension build up in the fibers in response to the acute stretch. The force decreases
(phase 2) at which point the cross-bridges can no longer hold and thus detach, and it
32 builds back up as the cross-bridges re-attach in phase 3. All amplitudes are normalized to pre-stretch isometric force to allow comparisons between different levels of activation.
Amplitudes measured are as follows: P1: measured from pre-stretch steady-state force to the peak of phase 1; P2: measured from pre-stretch steady-state force to the minimum force decay; P3: measured from pre-stretch steady-state force to the peak value of delayed force; and Pdf: difference between P3 and P2. Apparent rate constants are derived for
−1 phase 2 (s , krel) from the force decay after the peak of phase 1 as cross-bridges detach
−1 (the released state in the cross-bridge cycle), and for phase 3 (s , kdf) from the point of force reuptake after phase 2 to the completion of delayed force development as cross- bridges transition into the strongly-bound cross-bridge state. Also, the slope of the amplitude of P1 against the degree of stretch represents the tension/stiffness of the fibers.
The steeper the slope, the stiffer the fibers.
Figure 1-5. Slack-restretch and stretch activation responses. The slack-restretch response is shown on the left (55) and the stretch activation response on the right (323), depicting the changes in force recorded before, during, and after a step change in length of skinned fibers. See text for description.
Stretch activation not only allows us to distinguish the detachment and reattachment rate of a cross-bridge cycle, it also has physiological significance during
33 contractions. It has been shown that the epicardium produces more force compared to endocardium due to molecular differences in these regions, and as a result the endocardium is stretched during a contraction (78). [The greater force in epicardium also comes from its greater helical radius due to the alignment of cardiomyocytes (301).] This stretch and the resulting activation of force take place during the ventricular torsion twist in systole and could contribute to the speed and force generation and hence sustaining the power output, and it involves thin filament activation and cooperative recruitment of weakly bound non-force generating cross-bridges into strongly bound force-generating states (323).
2+ Both strong-binding cross-bridges and Ca accelerate ktr (233). However, cooperative activation by strong-binding cross-bridges slows ktr. Reducing cooperativity through experimental approaches such as directly supplying a strong-binding derivative of myosin subfragment-1 or altering the composition or structure of troponins and
2+ tropomyosin accelerates ktr. Therefore, ktr at diastolic or sub-maximal [Ca ]i (where
2+ cooperative activation is dominant) is slower compared to systolic or saturating [Ca ]i
2+ (where Ca influence is dominant). Other factors affecting ktr include the rate of delivery of Ca2+ to the myofilaments, distance between the thin and thick filaments, the expression and phosphorylation of several myofibril proteins.
Several myofilament proteins have different isoforms, each with distinct properties. For example, MHC has - and -isoforms. The relative abundance of MHC
- and -isoforms correlates with in vivo contractile function (328). An increase in
MHC contributes to decreased contractile function as a result of its lower intrinsic
ATPase activity and hence reduced ktr and myofilament shortening velocity (351). On the
34 other hand, phosphorylation of the myofilament proteins has a major impact on regulating myofilament function. TnI can be phosphorylated on Ser 23 and/or 24 by protein kinase A (PKA) and several other kinases (312). (TnI also has several other residues that can be phosphorylated by several kinases, and each of the phosphorylations has different functional consequences.) When phosphorylated on Ser 23 and/or 24, Ca2+ dissociates from TnC faster and hence myofilament Ca2+ sensitivity is decreased, leading to faster myofilament relaxation and increased cross-bridge kinetics. This gives the heart positive chronotropy and lusitropy reserve and the ability to maintain power during ejection, in addition to proper length dependence of activation.
Myosin binding protein C (MyBP-C) spans across thin and thick filaments at the
C-zone of the A-band (Fig. 1-3). [For review see (17, 18, 149, 179, 257).] It binds to titin and the light meromyosin in the thick filament backbone and helps the formation and stabilization of the sarcomeres. It serves as an internal load to optimize the distance and hence interaction between the thin and thick filaments and the formation of cross-bridges.
It has multiple phosphorylation sites including Ser 273 (in mice, or 276 in human), 282
(in mice, or 285 in human), and 302 (in mice, or 304 in human), which are substrates of
PKA, Ca2+/calmodulin kinase II (CaMKII), protein kinase C, and/or protein kinase D
(Fig. 1-6). There might be unique properties to each individual phosphorylation site, most of which are still under investigation. Generally, MyBP-C binds to myosin when unphosphorylated and releases this constraint and binds to actin instead when phosphorylated. Phosphorylation of MyBP-C increases the proximity of myosin and actin and hence promotes cross-bridge formation and increases cross-bridge cycling and the velocity of force development without alterations in lattice spacing (66). This allows the
35 contractile machinery to increase power output and adapt under stress. However, phosphorylation of MyBP-C decreases Ca2+ sensitivity of force, perhaps due to increased krel more so than increased kdf, therefore there is less force-generating cross-bridges and
2+ less force production at submaximal [Ca ]i. Phosphorylation of MyBP-C also maintains the structural integrity of sarcomere and stabilizes MyBP-C from proteolysis.
Phosphorylation of Ser 282 (by CaMKII) is pre-requisite for the rest of the phosphorylation (215), and proper expression and phosphorylation of MyBP-C contribute to normal contractile function as mutations in MyBP-C can cause FHC. (See 1.4.4 for more discussion.)
PKD
Figure 1-6. General Domain Structure of Mouse MyBP-C. Domains, phosphorylation sites, site-specific kinases, and regions that interact with other myofibrillar proteins are shown. The amino acid sequences in the M domain are conserved between species. [Reprinted with a minor modification from (18). Copyright © 2010, with permission from Elsevier.]
36 1.2.3 The Electrical Properties of the Heart
The cardiac cells are electrically active and perform contractions upon receiving action potentials. [For general review see (36).] In order to contract effectively, the heart is wired with a special conduction system, and the myocytes are linked by gap junctions which allow electrical signals to pass through. The action potential originates in sinoatrial node in the right atrium, a group of cells that depolarize simultaneously. This triggers the atria to contract, corresponded to the P wave on the electrocardiogram (ECG) (Fig. 1-7).
The action potential then reaches the atrioventricular node, where it subsequently travels down the His-Purkinje fiber to the apex of the heart and initiates ventricular contraction, shown as the QRS complex on ECG. The repolarization and relaxation of the ventricles reflect as the T wave on ECG. The pattern and duration of the individual segments (e.g.
QRS and QT intervals) signify the conduction and contractile properties of the stages of contraction they represent. However, due to the intrinsic differences in the makeup of ion channels that are responsible for the ECG waveforms between humans and mice, the interpretations of QRS complex and QT interval are different, as detailed in Fig. 1-7.
There are several factors that affect the conduction efficiency, one of which is gap junctions. Gap junctions are located at the intercalated disc along with desmosomes and fasciae adherentes junctions. [For review see (296).] Each gap junction is composed of 2 connexons, each consisting of 6 connexins from each cell. Among the many connexin isoforms, connexin 43 (Cx43) is the predominant one in the ventricles. Under basal condition, Cx43 is highly phosphorylated. During acute stress or in diseased myocardium, Cx43 is dephosphorylated and either get internalized or moved from the intercalated disc to the lateral side of the myocytes, a process called lateralization. A
37 decrease in Cx43 expression and/or its phosphorylation are correlated with decreased conduction velocity of the electrical signals and impaired ventricular contraction. Also, heterogeneous expression of Cx43 is associated with higher risk of arrhythmia.
P P
Figure 1-7. Cardiac Ionic Currents, Action Potentials, and ECGs in Humans versus Mice. Major depolarizing and repolarizing currents are shown for the human and mouse hearts. The size of the arrow for each current is roughly proportional to its magnitude, and arrows for outward currents point upward. For each heartbeat, action potentials of the first cells to depolarize are depicted as continuous lines and action potentials of the last cells to depolarize are depicted as dotted lines. APD90 is the time until 90% repolarization of the action potential. The ECG of the mouse is the signal average of five consecutive beats, and the ECG of the human is a simulation. Note that the apparent QRS duration (‘QRS’) in the mouse corresponds to both depolarization and early repolarization. [The figure is adapted from (284) with minor modifications. Copyright © 2006, John Wiley and Sons.]
38 1.2.4 Ca2+ Handling
Ca2+ acts as an inotropic agent that directly impacts contractility, independent of changes in preload or afterload. It plays a key role in excitation-contraction coupling
(ECC) in muscle cells and is regulated by several Ca2+ handling proteins. [For reviews see (29, 52, 168)]. The pathway of Ca2+ during a cycle of contraction is depicted in Fig.
2+ 1-8. During the relaxation state of the cycle, [Ca ]i is kept extremely low (~100 nM) compared to ~1-2 mM in the extracellular space. Upon receiving an action potential down the transverse tubules (T-tubule) — the invaginations of the muscle cell external membrane enriched in ion channels involved in ECC — the membrane depolarizes and
Ca2+ enters the cytosol through the voltage gated L-type Ca2+ channel (LTCC). This triggers a larger-scale of Ca2+ release from the SR, a process known as Ca2+-induced Ca2+ release (CICR). The SR functions as the Ca2+ store of the myocytes, containing
2+ 2+ millimolar level of Ca that are bound to calsequestrin. The initial elevation of [Ca ]i activates ryanodine receptor (RyR) on SR and releases Ca2+ through it, further increasing
2+ 2+ [Ca ]i up to ~1 M. The cytosolic Ca then binds to TnC on the myofilament and initiates actin-myosin cross-bridge interactions and muscle contractions.
For relaxation, Ca2+ needs to dissociate from TnC. The majority of Ca2+ is either actively pumped back into the SR by SR Ca2+ ATPase (SERCA) or extruded by sarcolemmal Na+-Ca2+ exchanger (NCX). A small portion of Ca2+ is pumped out of the cells by sarcolemmal Ca2+ ATPase or taken up into the mitochondria by the mitochondrial Ca2+ uniporter. SERCA is the main Ca2+ clearing mechanism, accounting for 70-90% of the removal. Phospholamban (PLB) binds to SERCA as an inhibitor but releases its inhibition when phosphorylated. PLB can be phosphorylated by PKA on Ser
39 16 and by CaMKII on Thr 17. The ability of SERCA to restore Ca2+ into the SR in a beat- to-beat fashion is crucial for proper relaxation and the subsequent contractions.
Figure 1-8. Ca2+ Transport in Ventricular Myocytes during ECC. Ca2+ entry pathway during contraction is delineated by red arrows, and Ca2+ clearing pathway during relaxation by green arrows. The inset shows the time course of an action potential (AP), 2+ o Ca transient ([Ca]i), and contraction measured in a rabbit ventricular myocyte at 37 C. 2+ + 2+ ATP: ATPase; ICa: L-type Ca currents; NCX: Na -Ca exchanger; PLB: phospholamban; RyR: ryanodine receptor; SR: sarcoplasmic reticulum. [Reprinted by permission from Macmillan Publishers Ltd: Nature (30), copyright © 2002.]
The specialized T-tubule structure plays an important role for ECC. The invaginations in the center of myocytes by T-tubules provide easier and faster propagation of electrical signal to every sarcomere, and hence faster excitation and more effective ECC (143). The close proximity of LTCC and RyR brought together by T- tubule ensures CICR, and simultaneous CICR in multiple sarcomeres produces a larger
40 force compared to unsynchronized CICR as in area with lower T-tubule density. T-tubule is not only rich in Ca2+ entry ion channels such as LTCC but also Ca2+ extrusion proteins such as NCX and sarcolemmal Ca2+ ATPase. Therefore, T-tubule is important for both speedy excitation and relaxation of the cycle. T-tubule also contains several key signaling proteins that regulate contractile function such as the ones involved in-adrenergic signaling.
Apart from ECC, Ca2+ can also enter the cell through store-operated Ca2+ entry
(SOCE), which involves stromal interaction molecule (STIM), Orai, and canonical transient receptor potential (TRPC) proteins. STIM functions as a Ca2+ sensor on SR membrane, and upon SR Ca2+ store depletion, it oligomerizes and translocates to activate
Ca2+ channels Orai and non-selective cation channels TRPC on the plasma membrane
2+ with the help of several other proteins (28, 234). SOCE help substantiates [Ca ]i
2+ especially when [Ca ]i increase from ECC is not sufficient, and hence SOCE becomes essential to SR Ca2+ store refilling and the activation of certain cellular processes such as gene transcription.
1.2.5Autonomic and -adrenergic Regulation
The autonomic system consists of sympathetic and parasympathetic divisions and is responsible for the involuntary control of heart and vasculature. [For review see (339).]
In the heart, both sympathetic and parasympathetic innervations control chronotropy and conduction velocity (dromotropy), whereas inotropy and lusitropy are mainly controlled by sympathetic nerves. Sympathetic nerves release norepinephrine and have positive effects on all four properties via -adrenergic receptors (AR), whereas parasympathetic
41 nerves release acetylcholine and have negative chronotropic and dromotropic effects via muscarinic receptors. In the blood vessels, sympathetic nerves exert vasoconstriction via
-adrenergic receptors, whereas parasympathetic nerves cause vasodilation although to a lesser extent compared to simply withdrawing sympathetic tone.
Under stress condition such as during exercise, the heart increases its performance
(e.g. increased CO) in order to meet the demand. It does so primarily through the neurohormonal activation of adrenergic receptors in a compartmentalized, location- specific manner. [For reviews see (158, 376).] Adrenergic receptors belong to the family of G-protein coupled receptor and play the major roles in the classical fight-or-flight response in the heart. Activation of ARs by sympathetic input initiates a cascade of signaling mediated by cyclic AMP and PKA. PKA phosphorylates the downstream targets including LTCC, RyR, PLB, MyBP-C, TnI, and titin among others, facilitating the various strategies for improving cardiac performance. Phosphorylation of LTCC, RyR, and PLB result in larger Ca2+ transients during systole, together with MyBP-C phosphorylation which releases its inhibition on myosin, result in positive inotropy.
Phosphorylation of TnI accelerates Ca2+ dissociation and cross-bridge detachments and hence positive lusitropy. Activation of AR at the sinoatrial node increases the slope of depolarization and hence increased firing rate, while activation of AR at the atrioventricular node increases conduction velocity, both resulting in positive chronotropy and dromotropy. Desensitizing ARs by its internalization terminates the surface signaling and can initiate a second wave of signaling at the nucleus level which is less well understood (353).
42 The activity of sympathetic and parasympathetic (vagal) systems is in dynamic balance and can be adjusted quickly in response to environmental factors.
Parasympathetic modulations are faster than sympathetic ones, allowing fluctuations
[measured as HR variability (HRV)] to occur on a beat-to-beat basis. However, if one dominates the other, the system losses its flexibility and can lead to various pathological conditions (335). This autonomic imbalance is common in CVD patients, typically hyperactive sympathetic/ hypoactive parasympathetic activity (see below).
Parasympathetic activity also has well-appreciated anti-inflammatory and anti-arrhythmic effects (277, 335), and poor vagal tone is positively correlated with mortality. There are several methods to measure autonomic control, and pharmacological blockage is one of the most common ways, as illustrated in Fig. 1-9 (53). Notice the intrinsic HR is higher than resting HR, reflecting the stronger parasympathetic influence in basal state, which favors energy preservation. Autonomic imbalance in the sympathetic direction not only increases the energy demand on the system, but is also associated with a range of metabolic and hemodynamic abnormalities in CVD. The common mechanism seems to be that sympathetic hyperactivity is proinflammatory while parasympathetic activity counteracts it (335).
43 Figure 1-9. Determination of Resting Cardiac Parasympathetic and Sympathetic Tone. The increase in mean HR after administering a muscarinic cholinergic receptor (M-ChR) blocker (e.g., atropine, tachycardia) reflects the cardiovagal tone present under baseline resting conditions. Conversely, the decrease in mean HR after -adrenergic receptor (-AdR) blockade (e.g., propranolol, bradycardia) reflects cardiac sympathetic tone. Subsequent administration of the second autonomic blocking agent during the peak HR response to the first agent identifies the intrinsic HR (double blockade) and enables calculation of residual sympathetic tone in absence of vagal tone (HR after M-ChR blockade – intrinsic HR) and residual vagal tone in absence of sympathetic tone (intrinsic HR – HR after -AdR blockade). [Reprinted with kind permission from Springer Science and Business Media. Springer and (53), copyright © 2011.]
1.3 CARDIAC METABOLISM
The heart requires a constant supply of energy both at rest and during stress for steady contractions, however, the heart has limited capacity for storing substrates, and therefore it relies on constant uptake of them. [For reviews see (194-196, 237, 319).]
Energy metabolism in the heart has three components, depicted in Fig. 1-10. The first is substrate utilization. Glucose is taken up by the glucose transporters (GLUT) that are constantly present on the plasma membrane such as GLUT1 and/or translocation
44 inducible such as GLUT4. It is then broken down through glycolysis, without the use of oxygen. Fatty acids, on the other hand, can be taken up by several transporters such as fatty acid translocase CD36 or simple diffusion/flip-flopping through the plasma membrane. They can then be esterified for storage or broken down through -oxidation which consumes oxygen. Both -oxidation and glycolysis produce acetyl coenzyme A, which goes into Krebs cycle, producing NADH and CO2. The second component is oxidative phosphorylation which produces energy while consuming oxygen. Respiratory- chain complexes I through IV in mitochondria transfer electrons from NADH to oxygen and create NAD and water, as well as a proton electrochemical gradient across the inner membrane of mitochondria. The proton gradient is then used by the ATP synthase to phosphorylate ADP, synthesizing ATP. (Activation of uncoupling proteins causes mitochondria to produce heat instead of ATP.) One molecule of glucose produces two
ATP, where as one cycle of -oxidation produces 14 ATP. Therefore, fatty acids that have longer carbon chains (which require more cycles of -oxidation to break down) generate more ATP. The heart demands aerobic production of ATP through -oxidation because it has limited anaerobic capacity such as glycolysis (195). In fact, the heart has the highest oxygen consumption per weight at rest among all major organs and tissues
(276), and it can increase up to twenty folds during intense exercise (42). The heart is designed with certain metabolic flexibility, which is facilitated by the molecular regulations of the different metabolic pathways. The fetal heart relies on carbohydrate as the primary energy source. In the adult heart, however, about 70% of its energy comes from fatty acids and the remaining ~30% from carbohydrates such as glucose and lactate
(319). These changes are facilitated by upregulation of proteins responsible for fatty acid
45 oxidation such as peroxisome proliferator-activated receptor and its coactivator and downregulation of proteins involved in carbohydrate metabolic pathway such as glucose transporters and pyruvate dehydrogenase. The heart can adjust the utilization of the substrates depending on the availability, and the oxidation of one substrate can inhibit the utilization of the others (43). The metabolism of the heart is tightly regulated so that its overall ATP level normally remains constant in any physiological situation (319).
Figure 1-10. Cardiac Energy Metabolism. Energy metabolism in the heart has three components, including substrate utilization (outlined in red), oxidative phosphorylation (outlined in blue), and energy transfer and utilization (outlined in green). See text for details. Δμ H+: proton electrochemical gradient, ANT: adenine nucleotide translocase, CKmito: mitochondrial creatine kinase isoenzyme, CKMM: myofibrillar creatine kinase isoenzyme, Cr: free creatine, GLUT: glucose transporter, PCr: phosphocreatine, Pi: inorganic phosphate, and UCP: uncoupling proteins. [Reproduced with permission from (237), Copyright Massachusetts Medical Society.]
46 Apart from substrate utilization and oxidative phosphorylation which is essential for producing ATP, the third component, which is the system involved in ATP transfer and utilization is also very important (237). ATP transfer is achieved by the creatine kinase energy shuttle, which transports the energy to myofibrillar ATPase and other ATP consumers, such as sarcolemmal and SR ion pumps. Creatine is produced by the liver and kidney but not the heart, and hence it has to be taken up from the blood stream by the creatine transporter. Mitochondrial creatine kinase transfers the high-energy phosphate bond in ATP to creatine, producing phosphocreatine (PCr) and ADP. Myofibrillar creatine kinase, on the other hand, catalyzes PCr back to ATP and creatine and thus releases ATP for utilization. Creatine and PCr diffuse faster than ATP due to their smaller size and function as the “energy shuttle” between mitochondria and myofilaments. They also function as an energy buffer, keeping ATP concentration normal while decreasing PCr level when energy demand is greater than energy supply.
However, due to its essential function in transporting ATP, dysfunction can occur while
PCr is decreased while ATP level remains relatively unchanged.
1.4 CARDIAC DYSFUNCTION
1.4.1 LV Dysfunction
Given the heart’s most important role being providing adequate perfusion throughout the body, LV contractile dysfunction is the main problem for the majority of
CVD. There are many causes that can affect the ability of LV to function as an effective pump, and they can generally be classified as reversible or irreversible. Myocardial ischemia for example, a result of coronary blockage, can be transient and reversible if it
47 lasts for a short period of time and is followed by reperfusion. The resulting LV dysfunction is transient and the time to recovery is positively correlated with the duration of ischemia (35, 263). A complete coronary block, however, results in total ischemia and subsequent MI. Irreversible contractile dysfunction like this can progress further into HF, and the LV dysfunction is often considered as the preclinical stage of HF.
LV contractile dysfunction can also be classified as systolic or diastolic dysfunction, namely the inability to contract or relax properly. It goes without saying that one affects the other, however, distinguishing between the two allows us to diagnose different subtypes of certain cardiac diseases and to target treatments better. (The differences between the two will be discussed further in chapter 1.4.3.)
1.4.2 LV Remodeling
The heart undergoes chamber remodeling in response to the chronic hemodynamic changes. [For review see (101).] There is physiological hypertrophy such as in athletes and women during pregnancy, whose LV chamber enlarges and myocardial fibers thicken and elongate. Their LV mass-to-volume ratio remains within a physiological range, and the hypertrophy facilitates their LV to accommodate the increased demand. On the other hand, pathological hypertrophy develops under triggers such as pressure (e.g. hypertension) and/or volume overload (e.g. mitral regurgitation). At the cellular level, hypertrophy of myocytes and nonmuscular cells as well as hyperplasia of fibroblasts and endothelial cells all contribute to LV hypertrophy. The pathological LV hypertrophy is classified into concentric and eccentric hypertrophy. There have been several attempts to classify LV remodeling to accommodate as many different
48 physiological and pathological conditions as possible based on indices such as EDV, LV mass, and relative wall thickness (RWT). The latest classification is shown in Fig. 1-11.
The term “concentric” refers to no chamber dilation and “eccentric” with dilation, and the degree/pattern of hypertrophy (normal, remodeling, and hypertrophy) is classified based on the extent of increases in LV mass and RWT. The initial thickening of the wall and enlargement of the chamber are an attempt to compensate and balance the increased LV systolic wall stress according to the law of Laplace (circumferential wall stress = transmural distending pressure × radius of chamber ÷ wall thickness). However, the corrected LV function is only temporary and the imbalance of LV mass-to-volume ratio or abnormal RWT becomes a substrate for LV dysfunction in the long run. Based on the high incidence of subsequent HF, adverse LV remodeling (most commonly concentric hypertrophy) is often considered as an intermediate phenotype of HF (57). Classifying
LV geometry has its prognostic value in addition to predicting the chamber functions.
Among all categories, the concentric hypertrophy has the worst mortality outcome, followed by eccentric hypertrophy and concentric remodeling.
Another common pathological LV remodeling is fibrosis, an alteration in the interstitial or extracellular matrix (79). Collagen is the main content of the matrix secreted by fibroblasts. It provides structural support and is the main determinant of diastolic stiffness. Excessive buildup of extracellular collagen type I due to increased collagen synthesis and cross-linking as well as decreased turnover and degradation all lead to the formation of fibrosis. There are two types of fibrosis: the reactive or interstitial fibrosis which occurs between viable myocytes with thickened existing collagen fibers, and the reparative or replacement fibrosis which replace damaged myocytes after
49 necrosis or apoptosis. Fibrosis increases the stiffness of LV wall, impeding its motion and causing diastolic dysfunction. Replacement fibrosis and scarring such as post MI causes decreased contractile mass and systolic dysfunction as well. Fibrosis also causes altered geometry, reduced coordination of myocyte contraction, resulting in unsynchronized electrical activation and contraction. The risk of developing cardiac arrhythmia and contractile dysfunction is positively correlated with the degree of fibrosis.
Figure 1-11. Patterns of LV Remodeling Based on EDV, Wall Mass, and RWT. A normal LV chamber size/EDV indicates a concentric or normal geometry; differences in relative wall thickness (RWT) distinguish concentric from normal remodeling. A dilated chamber dictates an eccentric geometry; those with left ventricular hypertrophy (LVH) are distinguished by differences in RWT. [Reprinted from (101). Copyright © 2011, with permission from Elsevier.]
50 1.4.3 Heart Failure
HF is the fastest growing subclass of CVD over the past decade and currently affects more than six million Americans (275). Survival after diagnosis has improved but remains poor; the mortality rate in five years is ~50% (275). HF patients have poor quality of life and huge financial burdens. In 2010, the estimated total cost for HF is ~$40 billion (193). There is currently no cure other than heart transplants, although a variety of drugs and treatments are available to manage the symptoms and progression of HF.
HF is “a chronic, progressive condition in which the heart muscle is unable to pump enough blood through to meet the body's needs for blood and oxygen” according to
American Heart Association. It has a variety of causes and risk factors, such as coronary artery disease, hypertension, and diabetes (275). It is a progressive disorder traditionally characterized by deteriorating LV function (e.g. decreased EF and CO). Also, as a result of insufficient pumping and circulation, there is edema in the lung and limbs. The patients experience tiredness and shortness of breath, which worsen during exercise.
Based on the LV function and tolerance of daily activity and exercise, the New York
Heart Association classifies HF patients into four groups (265). The four classes range from no limitations in ordinary physical activity (class I) to symptoms at rest and severe limitations in any physical activity (class IV). Class I patients have an 81% survival rate four years after diagnosis, while class IV patients have only a 36% survival rate one year after diagnosis (336, 337). HF can also be classified based on objective assessment (such as angiography) into stage A to D, with stage A being no objective evidence to stage D severe evidence. The two classifications do not necessarily parallel each other, reflecting
51 the complexity of the disease. Other than contractile dysfunction, HF patients usually exhibit adverse LV remodeling including hypertrophy and fibrosis.
Contrary to the traditional view on HF, it is now well established that about 50% of the HF patients have preserved systolic function, known as HF with preserved EF
(HFpEF) (345). HFpEF patients have different predictors including hypertension, atrial fibrillation, and female gender, compared to HF patients with reduced EF having previous MI and QRS abnormality (185). Some drugs used for HF do not work well on
HFpEF, such as angiotensin receptor blockers (345) and -blockers (348). It is therefore very important that the patients undergo LV functional assessment when planning treatment strategies. Compared to HF patients who have systolic dysfunction (and frequently also diastolic dysfunction), HFpEF patients have mainly diastolic abnormalities and chronotropic incompetence during exercise stress, yet with a higher mortality and morbidity. The symptoms are usually accompanied by ventricular inflammation, fibrosis, and stiffness. While the molecular mechanisms of diastolic dysfunction have been intensely studied (see below), much is unknown in regards to what sets HFpEF apart from HF at this point.
There are many alterations at the cellular level that contribute to LV dysfunction, including decreased contractility, prolonged relaxation time, and blunted responsiveness to -adrenergic stimulation (141). Abnormalities in both Ca2+ handling and myofilaments can account for the cellular contractile dysfunction. Defects in Ca2+ handling include structural alterations in the T-tubule, Ca2+ overload in the cytosol, and SR Ca2+ pool depletion among others. Energy deficiency can also play a role since some of the Ca2+ handling proteins require ATP to function.
52 In HF no matter the etiology, the detubulation, decreased T-tubule density, and T- tubule disarray all cause uncoupling of LTCC and SR, leading to reduced Ca2+ release synchronicity, a longer time to peak Ca2+ release, and a lower peak concentration of Ca2+
(143). The uncoupled Ca2+ release machinery also contributes to Ca2+ spark/leak independent of contractions (see below). Several proteins are involved in the composition and/or formation of T-tubule such as amphiphysin 2, junctophilin 2, Tcap, and caveolin
(143), and the disrupted signaling (e.g. -adrenergic pathway) micro domain maintained by T-tubule proteins also plays a role in contractile dysfunction.
Ca2+ overload in the cytosol is largely due to upregulation of the Na+/H+ exchanger and sarcolemmal NCX (29, 52). Cellular acidosis resulting from insufficient blood perfusion in ischemia and/or MI strongly activates Na+/H+ exchanger and causes
Na+ overload in the cytosol. Because the driving force of NCX depends on Na+ gradient,
Ca2+ gradient, and the membrane potential, elevated Na+ concentration in the cytosol and prolonged action potential duration (APD) in HF trigger NCX to work in the reverse mode, which extrudes Na+ but brings Ca2+ into the cytosol. Together with reduced Na+ extrusion by Na+/K+ ATPase due to down-regulated protein expression and activity in
HF, up-regulated NCX (both their mRNA and protein level), and decreased sarcolemmal
Ca2+ ATPase activity due to insufficient ATP supply (146, 153, 154), a significant amount of Ca2+ can builds up in the cytosol and results in decreased ECC fidelity and cellular injury such as mitochondrial dysfunction and apoptosis (29, 52). Several long- chain fatty acids and intracellular long-chain acyl coenzyme A esters are also reported to directly activate voltage dependent Ca2+ channels and reverse-mode NCX activity, respectively, accounting partly for Ca2+ overload during ischemia, a condition with
53 2+ elevated free fatty acid in the serum (138). Elevation of [Ca ]i also can saturate calmodulin and activate calcineurin and the subsequent dephosphorylation of nuclear factor of activated T cells (NFAT), which induces signaling involved in hypertrophic growth (368).
SR Ca2+ pool depletion is a result of increased Ca2+ leak through RyR and a
2+ 2+ decrease in Ca reuptake through SERCA (29, 52). The L-type Ca current (ICa) on the other hand, is relatively unchanged under basal condition. PKA and/or CaMKII mediated hyperphosphorylation of RyR itself and/or its auxiliary inhibitor protein FKBP12.6 increase the open probability of RyR, uncoupled to ECC (8, 363). The resulting Ca2+ leak leads to decreased ECC fidelity and SR Ca2+ pool depletion. SERCA works in a futile cycle and has a high energy waste in this situation. On the other hand, the reuptake of
Ca2+ by SERCA is downregulated in HF. In fact, the mRNA, protein expression, and activity of SERCA are all down-regulated, the SERCA/PLB ratio is decreased, and the
PLB phosphorylation (either by PKA and/or by CaMKII) is also decreased. All of these factors together with decreased ATP supply, impede Ca2+ reuptake following muscle contraction, resulting in prolonged relaxation and SR Ca2+ pool depletion and hence a progressive decrease in contractility. These factors also exacerbate Ca2+ overload in the cytosol (29, 52). Also, NCX can enhance Ca2+ extrusion through the forward mode
+ during relaxation in situations which [Na ]i is not overloaded (e.g. nonischemic cardiomyopathy), indirectly leading to SR Ca2+ pool depletion overtime (259).
Alterations in SOCE also contribute to abnormalities in Ca2+ handling. Increased
2+ 2+ STIM is associated with increased [Ca ]i in hypertrophy, reducing STIM can cause Ca oscillation and arrhythmia, the lack of Orai decreased SOCE and SR Ca2+ store, and
54 STIM, Orai, and TRPC channels all mediate the development of hypertrophy from various stimuli (63). SOCE is also involved in ischemia reperfusion injury induced Ca2+ overload, which contributes to contractile dysfunction as discussed above (63).
Abnormal intracellular Ca2+ homeostasis can reversely affect excitation, a mechanism termed reversed ECC (40, 333). Through both simple diffusion and gap junctions, Ca2+ ion build-up can propagate in forms of Ca2+ wave through the myocardium, unrelated to the electrical input and sinus rhythm. Ca2+ wave of significant scale can initiate CICR and hence “triggered propagated contractions”. The Ca2+ transients produced in this manner can also depolarize the cell membrane through the electrogenic extrusion of Ca2+ by means of NCX and cause delayed after polarization, which can be responsible for action potential if reaching the threshold. The Ca2+ build-up come from leaky SR and/or sarcolemma of diseased myocardium and myofilaments that contract weaker (such as ischemic myocardium) during the rapid decline of stretch- induced contraction by stronger neighboring myofilaments (333). (Rapid release of activated sarcomeres causes Ca2+ dissociation from myofilaments.) Reversed ECC therefore serves as a mechanism linking damaged myocardium with arrhythmia and decreased contractile function.
HF has blunted responsiveness to -adrenergic stimulation (decreased contractile reserve) both under baseline (BL) and more so during stress, therefore HF patients have low exercise tolerability (95). Chronic elevation of sympathetic tone in HF (intended to compensate for the reduced function) leads to downregulation of -adrenergic signaling due to AR desensitization and a decrease in the functional coupling of AR to the stimulatory Gs and its downstream targets, a result of disrupted caveolin structure at the
55 T-tubule (320). G protein–coupled receptor kinase 2 which phosphorylates and desensitizes the ARs, is increased in HF. The Gs to Gi (the inhibitory G protein) ratio is decreased in HF, presetting the 1/2 signaling pathway to be less effective at increasing contractile function. The decreased phosphorylation of the downstream effector proteins (e.g. PLB) causes a decrease in contractile function. On the other hand, the reduced phosphorylation on TnI in end-stage HF causes increased Ca2+ sensitivity, which impedes relaxation and causes diastolic dysfunction (212) and arrhythmia (139).
There is also a decrease in frequency dependent Ca2+ desensitization due to decreased frequency dependent TnI phosphorylation (348). The alteration of Ca2+ sensitivity appears to depend on the degree and etiology of the disease and might be species specific
(212). The long-term activation of -adrenergic signaling also has detrimental effects including the induction of hypertrophy, apoptosis, and fetal gene program (158). The plasma level of -adrenergic agonists correlates positively with the prognosis of HF (95,
334).
Other than the decreased phosphorylation associated with increased demand, decreased phosphorylation on certain myofilament proteins at BL also contributes to contractile dysfunction in HF, including smaller force production and decreased cross- bridge kinetics due to reduced myosin light chain 2 phosphorylation, and high passive force (stiffness) resulted from decreased titin phosphorylation (348). Additionally, increased TnT and TnI phosphorylation on protein kinase C sites (due to upregulation of multiple isoforms of protein kinase C in HF) might result in decreased maximum force production (120).
56 The composition of myofilament protein isoforms is also different in the disease state. MHC isoform switch back to the fetal profile, from the fast to slow isoform, and there are other isoform switches including TnT and TnI (213, 352). Moreover, there is disarray of several cytoskeletal and extracellular proteins which is worse in fibrotic area (300). Cytoarchitecture disorganization which involves decreased mitochondria and creatine kinase content and their contact to myofibrils also exacerbate the contractile dysfunction from the energy supply/transfer standpoint (153).
On the metabolism side of the story, there are also many alterations in regards to substrate utilization (264). The injured heart switches to fetal heart metabolism profile, utilizing carbohydrates as the primary energy source. This is achieved by changes in the expression of the enzymes involved in the respective metabolic pathways. The metabolic flexibility that the heart once had is also lost in the disease state (327). The switch to fetal heart metabolism is thought to be cardiac protective, at least at first, due to higher oxygen utilization efficiency with carbohydrates. However, the adaptation transits into cardiac dysfunction at some point during the disease progression yet the mechanism remains unknown. Current dietary guidelines recommend a low-fat/high-carbohydrate diet
[except -3 (329)] for patients at risk of HF (175) because high-fat (especially saturated) has long been indicated to increase peripheral resistance in blood vessels which leads to hypertension, LV remodeling (e.g. hypertrophy), obesity, insulin resistance, and diabetes, all of which are risk factors for HF (103, 287, 299). Although hearts prefer fatty-acids to carbohydrates as a source of energy, cardiac myocytes have a limited capacity for lipid storage (192, 287). Moreover, -oxidation is reported to be down regulated in HF, which can lead to an imbalance between fatty-acid uptake/storage and utilization (319). This
57 results in a condition known as lipotoxicity, which is characterized by ceramide accumulation, mitochondrial dysfunction, reactive oxygen species (ROS) generation, and apoptosis (287). ROS increases oxidative stress in cells, causing a loss in membrane integrity and damage to Ca2+ handling proteins including L-type Ca2+ channel, RyR,
SERCA, and NCX, which together with other lipotoxic effects of high-fat, may contribute to LV dysfunction, cardiomyopathy, and subsequent HF (52, 382).
Despite the known lipotoxic effects by high-fat and obesity being a risk factor for
HF, some reports challenged the idea of increased dietary fat on LV function deterioration during the pathological state. The Candesartan in Heart Failure: Assessment of Reduction in Mortality and Morbidity study showed that in patients with symptomatic
HF, higher body mass index (BMI) was actually associated with decreased all-cause mortality in a step-wise fashion (BMI 30-34.9 < 25-29.9 < 22.5-24.9 < 22.5) (162). The mechanism was unclear, although increased metabolic reserve associated with higher
BMI was one of the hypotheses. In animal models of various cardiac dysfunctions, high dietary fat has also been reported to have beneficial effects. In a Dahl Salt-Sensitive rat model of hypertension, high-fat/low-carbohydrate feeding prevented the hypertension induced LV hypertrophy and improved contractile function despite elevated plasma fatty acids and myocardial triglycerides (244). The authors suggested that the decreased insulin stimulation caused by high-fat diet could be responsible for the improved contractile function and anti-hypertrophy response considering that elevated insulin signaling is a known trigger for hypertrophic growth in the heart. Howarth et al (2005) has also reported that in a diabetic-prone mouse model, isolated LV cardiac myocytes had improved contractility and reduced relaxation time when animals were fed a high-fat
58 compared to normal chow (134). The amplitude and kinetics of the Ca2+ transient were not different, but the authors pointed out that increased myofilament Ca2+ sensitivity might account for these improvements. Fauconnier et al (2007), using isolated LV cardiac myocytes, has shown that the saturated fatty acid palmitate accelerated the Ca2+ transient and increased cell shortening in obese, insulin-resistant ob/ob mice (93). Palmitate impaired Ca2+ handling in wild-type (WT) mice in a ROS dependent manner. In contrast, palmitate did not increase ROS production in ob/ob mice and its beneficial effects on contractility were not altered by antioxidants. The authors speculated that the increased fatty acid oxidation typically associated with obesity and type-2 diabetes may result in increased ATP production thereby promoting SERCA function, but it remains to be determined whether this situation might apply to conditions with decreased fatty acid oxidation such as HF (319). Whether cardiac myocytes in HF with high-fat feeding can adapt to a persistent increase in fatty acid levels as seen in obesity and type-2 diabetes and return to the adult phenotype in which fatty acids are the preferred energy source has yet to be determined (142).
1.4.4 Familial Hypertrophic Cardiomyopathy
FHC is the genetic form of hypertrophic cardiomyopathy and is the most common inherited heart disease, currently affecting ~0.5 million Americans and more than 60 million people worldwide (275). It is a relatively benign disease in terms of progression, and it is estimated that most of FHC carriers can live a full life unaware of their condition. However, FHC has some of the most devastating symptoms including arrhythmia and sudden cardiac death (SCD). In fact, FHC is the leading cause of SCD in
59 young people and competitive athletes without precedent symptoms which usually occurs during intense exercise, thus participation in competitive sports is often prohibited once diagnosed (207). FHC is inherited as an autosomal dominant trait with incomplete penetrance with identified mutations in more than 19 genes currently (Table 1-1), most of which encodes sarcomeric proteins (39). Thus, FHC is described as a disease of the sarcomere. Given the genetic heterogeneity of the disease, it is not surprising that the pathology of FHC is diverse. However, the extent of symptoms and overall prognosis of
FHC vary widely even within family members carrying the same mutations (248), presumably a summation of other modifier genes and environmental factors.
Symptomatic patients exhibit various degrees of chest pain, dyspnea, palpitations, arrhythmias, diastolic dysfunction, and LV remodeling including LV outflow tract obstruction, asymmetric hypertrophy (primarily interventricular septum), interstitial fibrosis, and myocardial/myofilament disarray. A small portion of the patients possess systolic dysfunction and are more likely to progress into HF. Patients with more mutations (e.g. homozygous) in more genes (e.g. compound heterozygous) exhibit earlier onset with more severe phenotypes (9). There is currently no cure for FHC, and treatments for relieving symptoms are limited. Understanding the disease mechanism is important because treating symptoms alone might not affect the pathophysiology of FHC and can have adverse outcome (14).
60 Table 1-1. Summary of Hypertrophic Cardiomyopathy Susceptibility Genes.
Gene Protein Frequency (%) Myofilament proteins MYH7 -myosin heavy chain 15–25 MYBPC3 Cardiac myosin-binding protein C 15–25 TNNT2 Cardiac troponin T <5 TNNI3 Cardiac troponin I <5 TPM1 -tropomyosin <5 MYL2 Ventricular regulatory myosin light chain <2 MYH6 -myosin heavy chain <1 MYL3 Ventricular essential myosin light chain <1 TTN Titin <1 ACTC -cardiac actin <1 TNNC1 Cardiac troponin C <1 Z-disc proteins LBD3 LIM binding domain 3 (alias: ZASP) 1–5 CSRP3 Muscle LIM protein <1 TCAP Telethonin <1 VCL Vinculin/metavinculin <1 ACTN2 -actinin 2 <1 MYOZ2 Myozenin 2 <1 Calcium-handling proteins JPH2 Junctophilin-2 <1 PLN Phospholamban <1
[Reprinted from (39) with minor modifications. Copyright © 2009, with permission from Elsevier.]
Patients carrying mutations in MyBP-C gene usually are heterozygous (with one functional allele), however, the amount of functional protein is reduced as a result of haploinsufficiency and no mutant protein is detected in the sarcomere (288). Unlike
61 mutations in other FHC causing genes that result in production and incorporation of dysfunctional proteins (so-called poison peptides), it is predicted that ~70% of MyBP-C mutations are frameshift ones and generate mRNA with a premature termination codon.
Therefore, the transcription of MyBP-C is decreased via nonsense-mediated mRNA decay (288), and the translated proteins have C-terminal truncation which are also premature and get degraded primarily in the ubiquitin-proteasome system (285). The one functional allele, however, fails to produce enough MyBP-C, resulting in a 24-33% loss of full length MyBP-C (148, 210, 349). This leads to the hypothesis that haploinsufficiency is the culprit of MyBP-C related FHC, and the greater loss of MyBP-C observed with homozygous (236) and compound heterozygous (188) mutations is indeed associated with severer phenotypes.
It has been proposed that haploinsufficiency in MyBP-C causes FHC by
2+ increasing Ca sensitivity (at pCa50) like many other FHC mutations (139, 166, 211).
Increased Ca2+ sensitivity causes faster cross-bridge attachment and incomplete relaxation, which render the heart in a hypercontractile state with diastolic dysfunction. If the dysfunction is not uniform, local stretch induced mechanical stress can cause arrhythmia through mechano-electric feedback (266). Increased Ca2+ sensitivity can also decrease peak Ca2+ transients and slow Ca2+ transient decay due to increased Ca2+- binding affinity and hence Ca2+ buffering effect of TnC (167, 273). Excess diastolic
2+ [Ca ]i can activate hypertrophic signaling through calcineurin-NFAT signaling (368), induces reversed ECC (as discussed in 1.4.3), and contributes to arrhythmia through activation of CaMKII (374). Reduced peak Ca2+ transients and prolonged Ca2+ transient decay also promote arrhythmia through changes in action potential, such as prolong APD,
62 triangulation, early afterdepolarizations, and alternans (139). Additionally, increased Ca2+ sensitivity induces arrhythmia due to increased spatial dispersion of ventricular activation time during high HR, which is related to shorter effective refractory periods in action potential and greater beat-to-beat variability of its durations, all of which can happen without histological abnormalities (22). Furthermore, the occurrence of ventricular tachycardia in the myofilament Ca2+ sensitized animals is directly proportional to the degree of increase in Ca2+ sensitivity, and the pacing frequency required to induce tachycardia is lower with increased Ca2+ sensitivity (139). Since diastolic dysfunction, hypertrophy, and arrhythmia are all classic traits of FHC, these findings have prompted the research in understanding the role of decreased MyBP-C in sarcomeric function.
Many genetically altered mice models have been created to understand the role of
MyBP-C in contractile performance, including MyBP-C knockout (MyBP-C−/−) which lacks MyBP-C completely, transgenic mice that bear a truncation mutation in MyBP-C
(MyBP-Ct/t) which express very little if any full-length MyBP-C, and ones with fully expressed but unphosphorylatable MyBP-C (MyBP-CAllP−), among others. Based on information gathered from these animals, it is generally agreed that MyBP-C serves as an internal load that optimize the distance between the thin and thick myofilaments and inhibit cross-bridge formation. The lack of MyBP-C in the sarcomere brings the filaments closer and increases cooperative activation, leading to accelerated cross-bridge kinetics
(ktr, krel, and kdf) (172, 288, 321). Also, the rigidity and stiffness of the sarcomere once ensured by MyBP-C is lost in MyBP-Ct/t animals (242, 251), resulting in shortened cross- bridge lifetime in the myofilament (252). Reduced myofilament stiffness contributes to systolic dysfunction in vivo, such as reduced LV systolic stiffness (216), shortened
63 ejection time (ET) (250), and decreased fractional shortening (FS) (48, 124). Also,
2+ increased ktr and kdf at sub-maximal Ca concentration promotes diastolic dysfunction in vivo, including elevated Pmin, blunted dP/dtmin, prolonged and isovolumic relaxation time (IVRT) (41, 48, 124, 250). Structurally, MyBP-C deficiency also leads to myocyte disarray, fibrosis, and profound LV hypertrophy, especially in the septum (41, 48, 124,
250, 260).
Apart from the expression of MyBP-C, the phosphorylation of MyBP-C is also important for cardiac function. MyBP-C is one of the downstream targets of -adrenergic stimulation, a substrate of PKA as well as several other kinases such as CaMKII and PKC
(17, 18). Phosphorylation of MyBP-C is decreased in FHC patients (68, 133, 148). Also,
MyBP-CAllP− mice have poor contractile function (281), whereas the ones with mimetic constitutively phosphorylated MyBP-C were rescued from the null mutant phenotype
(282). MyBP-CAllP− mice have reduced contractile reserve when challenged with
Dobutamine (DOB), and their cardiomyocytes respond less to PKA (342). At the myofilament level, phosphorylation of MyBP-C reduces myofilament lattice spacing/ increases the proximity of myosin head to actin, thereby promoting activation of the sarcomere (18, 65, 66, 252, 324, 364). Furthermore, MyBP-CAllP− mice showed similar decrease in lattice rigidity as well as cross-bridge lifetime as MyBP-Ct/t mice, indicating that proper phosphorylation of MyBP-C is crucial for maintaining sarcomere order and function (252). Last but not least, phosphorylation of MyBP-C is protective against
MyBP-C degradation during ischemic insult (81, 282) as well as ischemic injury to the cardiac tissue (279, 282).
64 Besides direct myofilament dysfunction caused by decreased MyBP-C expression and phosphorylation, it has been shown that disruption in Ca2+ homeostasis also occurs in
FHC and contributes further to the pathology. This has been shown in MHC, TnT, and
MyBP-C mutant mice models of FHC. The MHC mutant mice exhibited decreased SR
Ca2+ content and decreased expression of Ca2+ handling proteins including RyR and calsequestrin before the onset of hypertrophy (294). Their hypertrophy might be due to
Ca2+ overload as Ca2+ shift from SR to cytosol and the subsequent activation of calcineurin-NFAT signaling. Correcting Ca2+ overload in this model partially prevented the hypertrophy. The TnT mutant models of FHC revealed mutation specific slower rates of Ca2+ rise and decline, prolonged relaxation, reductions in SR Ca2+ load and uptake, and decreased SERCA2/PLB ratio (110, 118). The MyBP-C mutant mice with a truncated form of MyBP-C had decreased SERCA2 content and prolonged Ca2+ transient duration
(313). Alterations in these Ca2+ handling properties can contribute to contractile dysfunction as previously described.
Decreased contractile function can also be a result of alterations in ventricular conduction properties such as in HF (296). Alterations in the conduction system can manifest as abnormal waveforms on ECG. In FHC patients carrying MyBP-C mutations, the QT interval was longer and it correlated with the degree of LV hypertrophy (155).
However, in patients carrying MHCmutations, the prolonged QT interval was present in both symptomatic and asymptomatic (non-penetrant) groups (155). This suggests that there are mutation related alterations in dromotropy not secondary to the LV remodeling
(at least in the case of MHC mutations), however, the primary defect(s) that cause these
65 alterations in dromotropy in these populations are unknown (e.g. ion channel dysfunction, gap junction abnormalities, etc).
1.5 RATIONALE AND HYPOTHESIS
CVD continues to pose a threat to public health. It is important to understand the pathogenesis of the individual types and causes of CVD as it helps with forming new therapies and adjusting the current treatment strategies and options. Here we focus on the fastest growing subclass of CVD — HF, and the most common inherited CVD — FHC.
Taking into consideration the well documented effects of high-fat diets, we originally hypothesized that a high-fat diet would exacerbate the progression of HF and inhibit mitochondrial respiration by increasing myocardial ceramide (271). We tested this hypothesis in a rat model of coronary artery ligation induced HF. Immediately following sham (SH) or ligation surgery, animals were fed saturated fat diet (SAT) or normal chow
(NC) for eight weeks. Surprisingly, no further progression of HF was evident in HFSAT compared to HFNC, and mitochondrial respiration was improved in HFSAT compared to
HFNC, despite elevated tissue ceramide content. Furthermore, HFSAT exhibited an improved LV contractility compared to HFNC, measured by LV dP/dtmax (270) and LV maximal power (31) under steady state conditions. These results demonstrating improved
LV contractility in HF animals fed SAT have been a consistent finding in our laboratory.
The mechanism by which high-fat diet improves LV contractility in HF was unknown.
Since there are defects in Ca2+ handling in HF which contributes to its contractile dysfunction, we hypothesized that high-fat diet induced improvements in LV contractile function in HF result from improvements in myocardial Ca2+ handling properties.
66 Truncation mutations in MyBP-C are some of the most common causes of FHC, however, the link between MyBP-C deficiency and the manifestation of FHC symptoms and disease progression is not clear. In our laboratory we have MyBP-C−/− mice that lack
MyBP-C completely, and MyBP-C+/− mice that express a similar decrease in MyBP-C content and phosphorylation as heterozygous patients (48, 82). Although MyBP-C−/− mice have been created for more than a decade and are known to have severe in vivo and myofilament dysfunction, there are currently limited studies on their cellular functions
(i.e. cell shortening and Ca2+ transients) (41, 260) and none on their dromotropy properties. The MyBP-C+/− model on the other hand, has much milder phenotypes (48,
124). However, through sensitive technique such as magnetic resonance imaging (MRI), we have confirmed that they present early-onset mechanical dysfunctions as well (82).
Because of their unique representation of MyBP-C expression and phosphorylation, we believe that they are more clinically relevant and hence provide more accurate information on the pathophysiology of MyBP-C mediated FHC. We investigated the in vivo and in vitro contractile functions as well as myofilament function of both MyBP-C−/−
(pure MyBP-C deficiency) and MyBP-C+/− (partial MyBP-C deficiency) mice and ex vivo electrophysiological properties of MyBP-C−/− mice. We hypothesized that there are abnormalities in myofilaments of MyBP-C+/− mice and there are defects in electrical properties and/or Ca2+ handling in MyBP-C−/− and/or MyBP-C+/− mice that could contribute to the pathogenesis of FHC in addition to their myofilament dysfunction.
Together, these two studies will further our understanding of specific sub-groups of HF and FHC, their disease mechanisms, and how different causes of LV dysfunction contribute to their disease progression.
67 Chapter 2
Changes in Myofilament Proteins, but not
Calcium Regulation, are Associated with a
High Fat Diet-induced Improvement in
Contractile Function in Heart Failure
Y. Cheng1, W. Li2, T. A. McElfresh1, X. Chen1, J. M. Berthiaume1, L. Castel3, X. Yu2,
D. R. Van Wagoner3 and M. P. Chandler1
From: 1 Department of Physiology and Biophysics, Case Western Reserve University,
Cleveland, OH 44106;USA; 2 Department of Biomedical Engineering, Case Western
Reserve University, Cleveland, OH 44106, USA; 3 Department of Molecular Cardiology,
Cleveland Clinic, Cleveland, OH 44106, USA.
(Expanded from: Am J Physiol Heart Circ Physiol. 301: H1438-H1446, 2011.)
68 2.1 INTRODUCTION
HF is a progressive disorder often associated with hypertension, obesity, insulin resistance, and diabetes (140). At the whole heart level, HF is characterized by deteriorating LV function and at the cellular level, by decreased cell shortening, a prolonged relaxation time, and blunted responsiveness to -adrenergic stimuli. In consideration of the comorbidities associated with HF (e.g. obesity, hypertension, diabetes), dietary guidelines have traditionally recommended a low-fat/high-carbohydrate diet for coronary artery disease patients (175), although these recommendations are currently under revision (135, 329). Nonetheless, high dietary fat, particularly saturated fat, has long been implicated with myocardial lipid accumulation, contractile and mitochondrial dysfunction, apoptotic cell death, enhanced ventricular remodeling, and cardiac hypertrophy (287, 299).
Given the heart’s limited capacity to store lipids, coupled with a decrease in the - oxidation of fatty acids in HF, it has been suggested that excessive fat intake may increase the propensity for lipotoxicity (192, 196, 287), ultimately leading to contractile dysfunction. In contrast to the proposed lipotoxic effects on contractile function, we have previously reported no further progression of HF/LV dysfunction in rats fed SAT diet post-MI compared with rats fed NC. Rather, HF animals fed a SAT diet showed improvements in LV contractility (assessed by LV dP/dtmax) (270), LV maximal power
(31), and mitochondrial function despite elevations in myocardial triglyceride (TG) and ceramide content (270). These results demonstrate that the administration of a high-fat diet immediately after coronary ligation surgery may ameliorate the deterioration in myocardial contractility typically associated with the progression of HF/LV dysfunction.
69 However, the mechanism by which a high fat diet exerts this cardio-protective effect and improves LV contractility in HF has yet to be determined.
At the level of cardiomyocytes, Ca2+ plays a critical role in myocyte contractility.
Ca2+-handling properties are regulated by several proteins, including LTCC, SERCA, and
PLB, all of which participate in coordinating Ca2+ movement between cellular compartments (29). Likewise, changes in Ca2+ regulation contribute to the contractile dysfunction associated with HF. In HF, the SR Ca2+ pool is often depleted due to downregulated SERCA activity, a decreased SERCA/PLB ratio, decreased PLB phosphorylation, and increased SR Ca2+ leak, resulting in decreased Ca2+ transients and impaired cardiomyocyte contractility (29). Similarly, at the myofilament level, the composition and phosphorylation of contractile proteins also impact contractility. The relative abundance of MHC- and -isoforms correlates with in vivo contractile function
(328). Specifically, an increase in MHC contributes to decreased in vivo contractile function as a result of lower intrinsic ATPase activity and reduced myofilament shortening velocity (351). Likewise, phosphorylation of other myofilament proteins, e.g.,
MyBP-C, TnI, and MRLC, have also been shown to alter Ca2+ sensitivity and/or cross- bridge kinetics, impacting both in vitro and in vivo contractility (249, 311). Alterations in the ratio of MHC isoforms and the phosphorylation status of other myofilament proteins have been implicated in the contractile dysfunction characteristic of HF in a variety of animal models (189, 223, 249, 311, 326). However, the mechanism(s) by which dietary lipids impact the Ca2+ regulation of contractile function and myofilament protein composition in HF has not been investigated.
70 Prior studies from our laboratory have established that SAT feeding is associated with improvements in global contractile function in coronary ligation-induced HF/LV dysfunction; however, a clear mechanism of action has yet to be identified. Given that cardiomyocyte contractility is largely dependent on Ca2+-handling properties and myofilament protein composition, this study examined the impact of high-fat diet feeding on Ca2+ regulation of contractile properties and myofilament protein expression in isolated cardiomyocytes from rats with ligation-induced HF/LV dysfunction. We hypothesized that in infarcted rat hearts, improvements in intrinsic contractile performance associated with high dietary fat are the result of changes in Ca2+ regulatory properties and/or myofilament protein expression. Taken together, these results will contribute to our understanding of the cardio-protective effects of high-fat diet feeding in post-MI hearts.
2.2 MATERIALS AND METHODS
2.2.1 Experimental Model
This study was conducted in accordance with the National Institutes of Health
(NIH) Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 85-23, Revised
1996) and was approved by the Institutional Animal Care and Use Committee of Case
Western Reserve University. The model used in this study has been previously described
(270). Animals were maintained on a reverse 12h:12h light:dark cycle, and all procedures and tissue harvests were performed in the fasted state between 3 and 6 hr into the dark phase of the cycle. Male Wistar rats (300-350g) were anesthetized with isoflurane (1.5-
2.0%) and ventilated during infarction surgery. HF/LV dysfunction was induced by
71 ligation of the left main coronary artery as previously described (230). The lungs were inflated, the ribs were then approximated, and the chest was closed. SH animals were subjected to the same surgical procedure without coronary artery ligation. After ligation or SH surgery, rats were randomly assigned a dietary group and fed ad libitum either a
NC diet (SHNC and HFNC groups: 14% fat, 26% protein, and 60% carbohydrate) or a
SAT diet [SHSAT and HFSAT groups: 60% fat (25% palmitic, 33% stearic, 33% oleic acid), 20% protein, and 20% complex carbohydrates] (Research Diets, New Brunswick,
NJ) for 8 wk.
2.2.2 Plasma Metabolic Substrates
Glucose, free fatty acids (FFA) and TG concentrations in plasma (n = 8-10) were measured using enzymatic spectrophotometric kits (Wako Chemicals, Richmond, VA)
(253).
2.2.3 Echocardiography
Myocardial function was evaluated by echocardiography 7 wk post-ligation. A
Sequoia C256 System (Siemens Medical, Malvern, PA) with a 15-MHz linear array transducer was used as previously described (230) with anesthetized rats (1.5-2.0% isoflurane). The animal was situated in the supine position on a warming pad with shaved chest, and ECG limb electrodes were placed. Two-dimensional (2D), 2D-guided M- mode, and Doppler echocardiographic analyses of aortic and transmitral flows were performed via parasternal and foreshortened apical windows. End-diastolic (EDA) and end-systolic area (ESA) were measured using software resident on the ultrasonograph,
72 and the EF was calculated based on volume and presented as a percentage of EDV (n =
10-11). All data were analyzed in an investigator-blinded fashion. The criteria for a successful coronary artery ligation were an EF below 70% (based on the average EF of
~90% in SH animals) and clear evidence of a scar (necrotic and thinned LV wall below the ligation suture).
2.2.4 Hemodynamic Measurements
In vivo LV contractile properties were assessed 8 wk after coronary artery ligation surgery (n = 11-14). Rats were anesthetized (1.5-2.0% isoflurane), intubated, and ventilated. A Millar 3.5-Fr microtip pressure transducer catheter was introduced via the right carotid artery as previously described (270). Endpoints related to heart rate, LV maximum pressure, and dP/dtmax/min were determined with the aid of PVAN/Chart 5 software (Millar Instruments, Houston, TX). Systemic vascular resistance (SVR) was calculated as maximal pressure/cardiac output.
2.2.5 Histological Assessment of Cardiac Morphology
A separate set of hearts was harvested (n = 5-6) for the histological evaluation of myocyte and infarct size. All hearts were perfused with cardioplegic buffer [containing
(in mM): 110 NaCl, 16 MgCl2, 16 KCl, 10 NaHCO3, 5 dextrose, and 1.2 CaCl2] to ensure a relaxation state and then formalin fixed and paraffin embedded. The base, mid-LV, and apex of the hearts were sliced into 6-m-thick horizontal sections (~1/4 of the distance from the apex to the base of the heart) using a slicer matrix. For infarct size measurements, samples from the apex were stained with Masson’s trichrome (Sigma,
73 Saint Louis, MO). Infarct size was determined by quantifying the area of the entire LV
(including the septum) and the area defined by the scar (evidenced by the thinned area in blue) using MetaMorph software (Molecular devices, Sunnyvale, CA), and the scar size was expressed as a percentage of the total LV area. Due to the extreme thinning of the wall in the area of the infarction, the calculation of area of infarction underestimates the extent of the infarction in terms of actual surface area. For cross-sectional area measurements, all three locations of samples were stained with fluorescein conjugated wheat germ agglutinin (Invitrogen, Carlsbad, CA), which recognizes cell membranes, and
4’,6-diamidino-2-phenylindole (Invitrogen), which recognizes nuclei. The cross-sectional area of the myocytes was determined by measuring the area of the cells with the aid of
MetaMorph software (Molecular Devices).
2.2.6 Cardiomyocyte Isolation
LV cardiomyocytes were isolated as previously described with modifications
(24). Rats were anesthetized (1.5% isoflurane), heparin (200 units) was injected intravenously, and hearts were quickly excised and chilled in ice-cold Ca2+-free Tyrode buffer containing (in mM): 136 NaCl, 5.4 KCl, 1 MgCl2, 10 HEPES, 1.2 NaH2PO4(H2O),
5.6 D-glucose, 2 L-glutamine, and 5 taurine (pH 7.4). The coronary arteries were perfused
o 2+ at 37 C via the aorta with Tyrode buffer containing 2 mM CaCl2, followed by Ca -free
Tyrode buffer and then Ca2+-free Tyrode buffer containing collagenase type II
(Worthington, Lakewood, NJ). The entire LV from SH rats and the nonscar LV tissue from HF rats were removed and minced in modified Kraft-Brühe solution containing (in mM) 110 potassium glutamate, 10 KH2PO4, 25 KCl, 2 MgSO4, 20 taurine, 5 creatine, 0.5
74 EGTA, 20 glucose, and 5 HEPES (pH 7.4). Cells were filtered and resuspended in Ca2+- free Tyrode buffer containing 0.5 mg/ml BSA. Ca2+ was reintroduced in a graded fashion to a final concentration of 1 mM. Cells were used within 4 h after isolation.
Cardiomyocyte isolation procedures typically yielded 2 million cells with 50% viability.
2.2.7 In Vitro Cardiomyocyte Shortening and Ca2+ Transients
Cardiomyocyte shortening and Ca2+ transients were measured as previously described with modifications (269). Cardiomyocytes were field stimulated at 1 Hz using a Grass stimulator (Astro-Med, West Warwick, RI). A video edge detector (Crescent
Electronics, Windsor, Ontario, Canada) with 60-Hz temporal resolution was used for cell shortening measurements. Cell shortening (absolute shortening/maximum relaxation cell length, presented as percent cell shortening) and peak velocity of cell shortening and relaxation (±dL/dt) were calculated (n = 117-137). For Ca2+ transient measurements, cells were incubated with 1 µM fura-2 AM (Invitrogen) at room temperature for 15 min; this concentration of fura-2 had no effect on cell shortening. The remaining dye was washed out twice after 10 min of gravity cell settling. Fura-2 was excited at 340 and 380 nm through a computer-controlled high-speed random access monochromator, and the fluorescent signals were detected at 510 nm by an analog/photon counting photomultiplier detector with background fluorescence measured with an unloaded myocyte from the same treatment group beforehand and automatically subtracted by
FeliX32 software (Photon Technology, Birmingham, NJ). Ca2+ transients were recorded with a sampling frequency of 120 Hz and expressed as the ratio of fluorescence at 340- to
75 380-nm wavelength (n = 130-168). Only myocytes with clear edges that remained viable throughout the entire recording were included. All experiments were performed at 37ºC under continuous buffer flow conditions. All data was analyzed using Matlab.
2.2.8 Protein Expression by Western Blot Analysis
Non-scar LV tissue (n = 6-8) was homogenized in Mammalian Protein Extraction
Reagent buffer (Thermo Scientific, Lake Barrington, IL) containing protease and phosphotase inhibitor cocktails (Sigma). Protein concentration was quantified by BCA protein assay (Thermo Scientific). Fifteen micrograms of protein were loaded onto 7.5%
Tris-HCl gels (Bio-Rad, Hercules, CA) for SERCA2. Forty-five micrograms of protein were loaded onto 18% Tris-HCl gels for pPLB16 and pPLB17 as well as PLB. Heat shock chaperone 70 (HSC70) was chosen to serve as a loading control. Gels were run at
200 V for 1 hr and then transferred to polyvinylidene difluoride (PVDF) membranes at
100 V for 45 min. Membranes were incubated overnight with 1:2000 anti-SERCA2
(MA3-919, Thermo Scientific), 1:1000 anti-PLB (05-205, Millipore, Billerica, MA),
1:1000 anti-pPLB16 (07-052, Millipore), 1:5000 anti-pPLB17 (A010-13, Badrilla, Leeds,
UK), and 1:10000 HSC70 (sc-7298, Santa Cruz Biotechnology, Santa Cruz, CA), followed by incubations with the appropriate secondary antibodies. Membranes were incubated with chemiluminescence reagents (Thermo Scientific) and exposed to films.
Densitometry of bands was determined using Image J (NIH). A standardizing sample was run on each gel to allow for normalizing densitometry across individual gels.
76 2.2.9 L-type Ca2+ Current Measurements
ICa were acquired using whole cell patch-clamp techniques as previously described (47) with modifications. The pipette solution contained (in mM) 125 CsCl, 20 tetraethylammonium chloride, 5 MgATP, 3.6 creatine phosphate, 10 EGTA, and 10
HEPES (pH 7.2). The bath solution contained (in mM) 157 tetraethylammonium chloride, 1 CaCl2, 0.5 MgCl2, and 10 HEPES (pH 7.4). ICa recordings began 3 min after patch rupture. After BL ICa was acquired, 1 μM isoproterenol (ISO) was applied, and the
β-adrenergic response was recorded at steady state. The maximal β-adrenergic response at 4 min after exposure was analyzed (n = 6-9). All experiments were performed under continuous flow conditions at room temperature, and the ICa amplitude was normalized to myocyte capacitance (in pA/pF). Clampfit and Origin software (OriginLab, Northampton,
MA) were used for electrophysiologic data analysis.
2.2.10 Myosin Heavy Chain Protein Expression
Protein contents of cardiac MHC and were determined via gel electrophoresis as previously described with modifications (361). Nonscar LV tissue (n = 5) was homogenized in RIPA buffer. The protein concentration was quantified by BCA protein assay (Thermo Scientific). Ten micrograms of protein were loaded onto a 6% big format
(16 x 18 cm) acrylamide gel cross-linked with N-N' diallyltartardiamide (DATD). The resolving gel (17 ml) consisted of 6.56 ml ddH2O, 3.4 ml 50% glycerol (v/v), 4.25 ml 1.5
M Tris (pH 8.8), 2.55 ml 40% acrylamide (37.5:1 cross-linked with DATD), 170 μl 10% sodium dodecyl sulfate (w/v), 50 μl 10% ammonium persulfate (w/v), and 20 μl TEMED.
The stacking gel (5.085 ml) consisted of 1.5 ml 10% acrylamide (5.6:1 cross-linked with
77 DATD), 1.3 ml 0.5 M Tris (pH 6.8), 1.15 ml ddH2O, 1 ml 50% glycerol (v/v), 50 μl 10% sodium dodecyl sulfate (w/v), 30 μl 10% ammonium persulfate (w/v), and 25 μl TEMED.
The upper buffer reservoir had 600 ml of 0.05 M Tris-base, 0.384 M glycine, 0.1% (w/v)
SDS, and 10 mM 2-mercaptoethanol (no pH adjustment). The lower buffer chamber had
4 liters of the same buffer except without 2-mercaptoethanol. Gels were run at 10-mA constant current for 19 h at 4oC. Bands were detected by silver staining (Bio-Rad) per the manufacturer’s protocol. The densitometry was analyzed using the GelBandFitter analysis program as previously described (227).
2.2.11 Myofilament Protein Phosphorylation and Expression
LV tissue (n = 6) was harvested, and myofilament proteins were purified using a previously described protocol (184) with modifications. The tissue was homogenized in standard PBS buffer with phosphotase inhibitor (Roche), protease inhibitor (Thermo
Scientific), and 0.02% Triton X-100. The samples were centrifuged at 12500 g at 4oC for
1 min, and the pellets were homogenized again in the same buffer. The samples were then centrifuged at maximal speed at 4oC for 1 min, and the pellets were resuspended in the same buffer without Triton X-100. The samples were centrifuged again at maximal speed at 4oC for 1 min, and the pellets were solubilized in buffer containing 8 M urea, 2
M thiourea, and 4% Chaps (w/v) on a 1:20 (mg/l w/v) ratio. The samples were then sonicated twice with a 30 min incubation at room temperature in between, and then centrifuged at maximal speed at room temperature for 30 min. The protein concentration of the supernatant was quantified by RC-DC protein assay (Bio-Rad). Ten micrograms of protein were loaded onto a 4-12% XT Bis-Tris gel (Bio-Rad), followed by Pro-Q
78 Diamond Phosphoprotein Gel staining (Invitrogen) per the manufacturer’s protocol. The same gel was then stained with Coomassie Blue for total protein expression.
Densitometry of bands was determined using Image J (NIH). The degree of protein phosphorylation was quantified as Pro-Q density/Coomassie Blue density.
2.2.12 Statistics
All statistical analyses were performed using SigmaStat software. Differences were determined using two-way ANOVA followed by Bonferroni post hoc analysis for multiple pairwise comparisons unless otherwise noted. Significance was established at P
< 0.05. Data are expressed as means ± SE.
2.3 RESULTS
2.3.1 Body Weight and Metabolic Substrates
High-fat diet feeding was associated with increased body weight (BW) in the
SHSAT group but not in the HFSAT group, despite there being no significant difference in total caloric intake between groups (SHNC group: 574 ± 28 kcal/wk, SHSAT group:
645 ± 19 kcal/wk, HFNC group: 576 ± 25 kcal/wk, and HFSAT group: 617 ± 29 kcal/wk,
P = 0.069). Although BW in the HFSAT group was significantly lower than in the
SHSAT group, coronary artery ligation surgery had no impact on BW between the two
HF groups (SHNC group: 509 ± 16 g, SHSAT group: 561 ± 11 g, HFNC group: 489 ± 12 g, and HFSAT group: 506 ± 15 g).
79 Plasma FFA and TG were increased with SAT feeding in both SHSAT and
HFSAT groups. Plasma glucose was increased in both surgical (HFNC and HFSAT)
groups compared with their SH controls (Table 2-1).
Table 2-1. Plasma Substrates, Echocardiography and Hemodynamic Function.
SHNC SHSAT HFNC HFSAT
Plasma Substrates
Glucose (mg/dl) 243±16 261±10 321±19 * 279±9 *
Free Fatty Acids (mol/ml) 0.14±0.02 0.25±0.01 † 0.18±0.03 0.26±0.03 †
Triglycerides (mg/ml) 0.53±0.05 0.87±0.12 † 0.48±0.09 0.68±0.08 †
Echocardiography
2 End-diastolic area (cm ) 1.03±0.04 1.02±0.03 1.18±0.03 ‡ 1.25±0.05 ‡ 2 End-systolic area (cm ) 0.43±0.02 0.43±0.02 0.73±0.03 ‡ 0.82±0.06 ‡
Ejection fraction (%) 90.1±0.5 90.8±1.2 58.2±2.4 ‡ 56.3±3.1 ‡
Hemodynamic
Max LV pressure (mmHg) 135±3 129±3 123±4 132±3
LV dP/dtmax (mmHg/sec) 8900±477 8751±315 6168±121 ‡ 7633±379 ‡¥
LV dP/dtmin (mmHg/sec) -9045±570 -8615±369 -5686±172 ‡ -6866±400 ‡¥
Values are means ± SE for plasma substrates (n = 8-10), echocardiography (n = 10-11), and hemodynamic results (n = 11-14). * P < 0.05, main effect for surgery; † P < 0.05, main effect for diet; ‡ P < 0.05, HF vs. SH within diet; ¥ P < 0.05, diet effect within HF.
2.3.2 Cardiac Morphology
Representative color images of infarct size at the apex of the LV are shown in Fig.
2-1A. Infarct size was not different between the HFNC and HFSAT groups (Fig. 2-1B).
Representative fluorescent images of LV horizontal sections are shown in Fig. 2-1C. The
80 cross-sectional area of the myocytes indicated hypertrophy in both HF groups compared with SH groups. Although high-fat diet feeding was associated with mild hypertrophy in the SHSAT group, the degree of hypertrophy was significantly decreased in the HFSAT group compared with the HFNC group (Fig. 2-1D).
2.3.3 Echocardiography and Hemodynamic Function
The progression of contractile dysfunction and LV remodeling was assessed by echocardiography at 7 wk and by direct LV cannulation at 8 wk after ligation. ESA and
EDA were increased in both HF groups compared with SH groups, indicative of LV remodeling subsequent to ligation surgery (Table 2-1). In addition, EF, an indirect measure of LV contractile function, were decreased in both HF groups compared with SH groups (Table 2-1). There was no evidence of LV dysfunction or remodeling due to high- fat diet feeding in the SH groups.
Hemodynamic assessment via direct LV cannulation confirmed that myocardial contractility was depressed in the HFNC group compared with the SHNC group, as measured by LV dP/dtmax and dP/dtmin (Table 2-1). However, LV dP/dtmax and dP/dtmin were significantly improved with high-fat diet feeding in the HFSAT group compared with the HFNC group. High-fat diet feeding in the SH groups did not alter LV dP/dtmax or dP/dtmin (Table 2-1). HR (SHNC group: 324 ± 9 beats/min, SHSAT group: 332 ± 6 beats/min, HFNC group: 318 ± 6 beats/min, and HFSAT group: 329 ± 6 beats/min), maximum LV pressure (Table 2-1), and SVR (SHNC group: 1.28 ± 0.13 mmHg·ml−1·min−1, SHSAT group: 1.19 ± 0.09 mmHg·ml−1·min−1, HFNC group: 1.41 ±
81 0.18 mmHg·ml−1·min−1, and HFSAT group: 1.47 ± 0.15 mmHg·ml−1·min−1) were not different between groups.
Figure 2-1. Histological Assessment of Infarct Size and Myocyte Cross-sectional Area. A: representative color images of stained apical cardiac slices with fibrotic tissue in blue and the remaining (viable) myocardium in red. B: infarct size expressed as a percentage of the total LV area (n = 5–6). Due to the extreme thinning of the wall in the area of the infarction, the calculation of area of infarction underestimates the extent of the infarction in terms of actual surface area. C: representative fluorescent images at the apex of the LV with membranes shown in green and nuclei shown in blue. D: average of the cross-sectional area of the LV cardiomyocytes (n = 70/section/animal) from all sections (base, mid-ventricular, and apex) of the heart (n = 5–6). * P < 0.05, HF vs. SH groups within diet; † P < 0.05, diet effect within SH or HF groups.
82 2.3.4 Cell Shortening
To assess whether improvements in contractile function in the HFSAT group were related to individual cardiomyocyte contractility, unloaded cell shortening was assessed in freshly isolated LV cardiomyocytes that had been field stimulated at 1 Hz.
Representative recordings of cell shortening are shown in Fig. 2-2A. LV cardiomyocytes from HF hearts were hypertrophied ( 17% longer) compared with SH hearts (Fig. 2-2B).
Cell shortening (expressed as a percentage∼ after being normalized to cell length; Fig. 2-
2C) was decreased in both HF groups compared with SH groups. Interestingly, −dL/dt was decreased in the HFNC group compared with the SHNC group but was not different in the HFSAT group (Fig. 2-2D). In contrast, +dL/dt was not different between groups
(Fig. 2-2E).
2.3.5 Ca2+ Transients
Representative Ca2+ transients are shown in Fig. 2-3A. Ca2+ transient measurements with fura-2-loaded LV cardiomyocytes revealed an unexpected trend relative to our measurements of in vivo contractile function. The mean amplitude of Ca2+ transients was increased in the HFNC group compared with the SHNC group and was decreased in both SH and HF groups fed the SAT diet (Fig. 2-3B). Although the time to peak Ca2+ was prolonged in the HFSAT group compared with the HFNC group (Fig. 2-
2+ 3C), the time to half Ca decay (τ50%) was prolonged in both HF groups compared with
SH groups (Fig. 2-3D).
83 Figure 2-2. LV Cardiomyocyte Shortening. A: representative tracings of cell shortening at 1 Hz averaged from 8 peaks from the same cell from each group. B–E: cell length (B), percent cell shortening (C), velocity of cell shortening (−dL/dt; D), and velocity of relaxation (+dL/dt; E) were measured on fresh isolated LV cardiomyocytes at 1 Hz. n = 117–137. * P < 0.05 HF vs. SH groups within diet.
84 Figure 2-3. LV Cardiomyocyte Ca2+ Regulation. A: representative raw tracings of Ca2+ transients at 1 Hz (left) and tracings averaged from 10 peaks from the same cell from each group (right). B–D: Ca2+ transients (B), time to peak Ca2+ (C), and time to half Ca2+ decay (τ50%; D) were measured in fresh isolated LV cardiomyocytes loaded with fura-2 at 1 Hz. n = 130–168. E: representative bands of SERCA2, PLB, pPLB16, pPLB17, and HSC70. Protein expression was quantified using Western blot analysis. F: ratio of SERCA2 to PLB (both proteins normalized to HSC70). G: ratio of pPLB16 to PLB. H: ratio of pPLB17 to PLB. n = 6–8. * P < 0.05 HF vs. SH groups within diet; † P < 0.05 diet effect within SH or HF groups.
85 2.3.6 Ca2+ Regulatory Protein Expression
Representative Western blots for proteins involved in Ca2+ regulation are shown in Fig. 2-3E. SERCA2 normalized to our loading control HSC70 was significantly increased in the HFSAT group compared with the HFNC group (SHNC group: 0.14 ±
0.05, SHSAT group: 0.46 ± 0.13, HFNC group: 0.24 ± 0.07, and HFSAT group: 0.61 ±
0.21), but the PLB-to-HSC70 ratio was not different (SHNC group: 2.25 ± 0.69, SHSAT group: 3.59 ± 0.98, HFNC group: 2.26 ± 0.88, and HFSAT group: 2.52 ± 0.57). The ratio of SERCA2 to total PLB (both proteins normalized to HSC70; Fig. 2-3F) and pPLB16 phosphorylation of PLB (Fig. 2-3G) were not different between groups. However, the pPLB17-to-PLB ratio was downregulated in the SHSAT group compared with the SHNC group (Fig. 2-3H).
2.3.7 Correlations between in Vivo and in Vitro Function
A significant correlation was reported between in vivo and in vitro measures of systolic (cardiomyocyte −dL/dt and LV dP/dtmax, r = 0.37, P < 0.05) and diastolic
(cardiomyocyte +dL/dt and LV dP/dtmin, r = 0.34, P < 0.05) contractility (Fig. 2-4, A and
B). The changes reported in LV dP/dtmin also correlated with cardiomyocyte τ50% (r =
0.46, P < 0.05; Fig. 2-4C). However, no relationship was found between functional changes in unloaded isolated cardiomyocytes (e.g., percent cell shortening and −dL/dt) and Ca2+ measurements (e.g., Ca2+ transients and time to peak Ca2+).
86 Figure 2-4. Correlations between in Vivo and in Vitro Functional Measurements. A– C: scatterplots showing significant correlations between LV cardiomyocyte −dL/dt and LV dP/dtmax (A), LV cardiomyocyte +dL/dt and LV dP/dtmin (B), and LV cardiomyocyte τ50% and LV dP/dtmin (C). This analysis was performed using 1 data point/animal comparing in vivo and in vitro responses from the same animal.
87 2.3.8 ICa
ICa was measured using whole cell voltage-clamp techniques and normalized to cell size. Peak ICa at −10 V was analyzed. There was no effect of ligation surgery or diet at BL (Fig. 2-5A) or with ISO stimulation (Fig. 2-5B). ICa was larger with ISO stimulation compared with BL in the HFNC group by Student's t-test (P < 0.05).
2+ Figure 2-5. L-type Ca Currents (ICa). ICa was measured on fresh isolated LV cardiomyocytes using whole cell voltage-clamp techniques in the absence (A) or presence (B) of 1 μM ISO. n = 6–9.
88 2.3.9 Myofilament Protein Composition and Phosphorylation
A representative gel silver stained for MHC is shown in Fig. 2-6A. (Bands for each protein were from the same gel, just reorganized according to group order.) The
MHC isoform switch from α to β was evident only in the HFNC group. The slow isoform
MHCβ content was elevated (both as a ratio of β to α and as a percentage; Fig. 2-6B) in the HFNC group but was unchanged in the HFSAT group. A representative Pro-Q- stained gel for myofilament protein phosphorylation and a Coomassie blue-stained gel for myofilament protein expression are shown in Fig. 2-6C. The degree of phosphorylation of MyBP-C, desmin, TnT, TnI, and MRLC were not different between groups.
Figure 2-6. Myofilament Protein Composition and Phosphorylation. A: representative bands of MHC- and -isoforms from the same gel. Protein expression was quantified using electrophoresis followed by silver staining. B: MHC expressed as a percentage of total MHC. n = 5. C: representative Pro-Q- and Coomassie blue-stained gels. * P < 0.05, HF vs. SH groups within diet; † P < 0.05, diet effect within HF groups.
89 2.4 DISCUSSION
The results of this study extend our previous findings and demonstrate that the initiation of SAT diet feeding after the induction of mild-moderate HF improves cardiomyocyte contractility, specifically, –dL/dt, and is associated with decreased MHC content. Changes in isolated ventricular myocyte systolic (–dL/dt) and diastolic (+dL/dt and τ50%) function and reductions in in vivo myocyte hypertrophy correspond to improvements in in vivo myocardial contractile function (dP/dtmax/min) but not changes in
Ca2+ regulatory properties. Ca2+ transients revealed an opposite trend to in vivo myocardial contractile function but were not due to changes in protein expression of
SERCA and PLB or the phosphorylation of PLB. In summary, these data suggest that the improvement in myocardial contractile function associated with SAT diet feeding in HF occurs primarily at the level of myofilament composition and function.
An important goal of this study was to relate in vivo contractile functional and cellular/molecular changes associated with high-fat diet feeding. Consistent with our previously published studies, in vivo contractile function (specifically, LV dP/dtmax and dP/dtmin) was significantly improved with high fat diet feeding in HF. Ventricular cardiomyocytes from both HF groups had decreased % cell shortening compared with SH groups. However, –dL/dt was depressed in HFNC group but not in the HFSAT group, a result that correlated with in vivo LV contractility as assessed by LV dP/dtmax.
Furthermore, in vivo diastolic function (LV dP/dtmin) correlated with cardiomyocyte diastolic properties (+dL/dt and τ50%). These results suggest that in vitro myocyte function reflects the improvements reported in in vivo contractile function.
90 Results similar to those presented here have been reported by Howarth et al. in a diabetic-prone mouse model. In their study, ventricular myocytes isolated from high-fat diet-fed animals had improved contractility and shortened relaxation times (134).
Likewise, high palmitate levels resulted in accelerated Ca2+ transients and increased cell shortening in obese, insulin-resistant ob/ob mice (93). These authors speculated that increased fatty acid oxidation typically associated with obesity and type-2 diabetes may result in increased ATP production, thereby promoting better SR Ca2+ handling and improved contractile function. Whether myocytes from failing hearts can restore their metabolic flexibility and adapt to an increase in fatty acids, thereby reverting to an adult phenotype where fatty acids remain the primary energy source, is a current subject of debate (142, 330). In terms of clinical relevance, these results appear to be contradictory to the traditional diet-heart hypothesis that relates dietary fat to coronary heart disease.
However, this diet-heart hypothesis has been under increasing scrutiny by the medical community, and dietary recommendations for patients with heart disease are undergoing considerable revisions (135, 329).
A marked switch from MHC to MHC was observed in the HFNC group, but this switch was prevented in HF animals fed the SAT diet. A MHC isoform switch is a key determinant of shortening velocity (which is primarily due to the intrinsic ATPase activity of fast -MHC compared with slower -MHC); this isoform shift translates to a decrease in contractility (332) and myocyte power output (127) and has been well documented in HF (189, 223, 326, 347). The increased expression of MHC in the
HFNC group could account for the decreased cell shortening velocity observed in isolated cardiomyocytes. In contrast, the SAT diet prevented the increased expression of
91 MHC and maintained normal cell shortening velocity. Okere et al. also reported that a high-fat diet prevented a Mhc- to Mhc- isoform switch that was accompanied by reductions in LV remodeling and contractile dysfunction in hypertension-induced hypertrophy (244). In addition, increased glucose availability has been reported to promote the Mhc- to Mhc- isoform switch, whereas high fatty acid levels blocked the changes in myosin isoforms (328, 379), considered a hallmark for a progressive decline in cardiac function. Thus, there may be a metabolic link that functions to protect the heart under pathological conditions through alterations in gene and protein expression involved in regulating contractile function; however, a potential cardio-protective effect may be unique to our model of mild-moderate HF/LV dysfunction. Evaluation of this hypothesis in this and other HF models of varying severity requires further study.
Other studies have reported that a reduction in cardiac growth and LV hypertrophy is associated with a high fat diet-induced attenuation in contractile dysfunction and inductions of molecular markers of hypertrophy, e.g., MHC (243, 244).
Our results support this concept given that in vivo myocyte hypertrophy (cross-sectional area) was reduced in our HFSAT group compared with our HFNC group. A low- carbohydrate/high-fat diet has been associated with a reduced stimulation of the insulin signaling pathway, resulting in decreased cardiac growth, contractile dysfunction, and gene expression in response to hypertension (299). Interestingly, we recently reported that HF animals fed a SAT diet exhibited preserved myocardial contractile function under conditions of myocardial insulin resistance and alterations in insulin signaling, specifically, diminished activation of Akt and increased total glycogen synthase kinase
3 (60). Similarly, a high-fat diet has also been reported to downregulate insulin-like
92 growth factor-1 (IGF-1) (50), a factor previously implicated in upregulating MHC (126) and increasing cardiac hypertrophy (220). Although plasma IGF-1 levels were not different (data not shown), it remains to be determined whether tissue IGF-1 is decreased in a manner similar to our reports of insulin resistance accompanied by decreased insulin signaling. A concomitant downregulation of the insulin/IGF-1 signaling pathway could account for changes in physiological and molecular markers of hypertrophy that, in turn, contribute to improvements in myocardial contractile function.
At the level of the SR, the Ca2+ transient amplitude was increased in the HFNC group compared with the SHNC group and was decreased in both SAT groups.
Differences in Ca2+-handling proteins, specifically, the expression of SERCA2, PLB, and phosphorylation of PLB, cannot account for the reported differences in Ca2+ transients, suggesting that other Ca2+-handling proteins could be involved. These are intriguing findings in that they contradict our in vivo LV contractility and in vitro cell shortening velocity results. However, conflicting reports of Ca2+ transients and total SERCA and total/phosphorylated PLB have been reported in the same model of ligation-induced HF
(11, 96, 112, 130, 199, 380). While the increased Ca2+ transients in the HFNC group might represent a compensatory response for the loss of viable myocardial tissue after infarction, it is contradictory to a decrease in myocyte shortening velocity. Furthermore, no correlations were reported between functional changes in isolated cardiomyocytes and
Ca2+ measures. These results demonstrate that Ca2+ regulation of isolated cardiomyocyte contractile function may be dissociated from global in vivo function. In our model, factors such as alterations at the level of the myofilaments and/or an increased exposure to cellular lipids may affect the contractile function of isolated cardiomyocytes.
93 ICa was not different between HF and SH groups, a result confirming previous reports in the literature (130, 308, 381). Given that ICa is a function of many factors (e.g., number of functional channels, open probability, and availability), one explanation for this result is a combined effect of decreased channel protein expression and increased open probability, as reported in human HF (290) and canine tachycardia-induced HF
(125). However, ICa was also not different with high-fat diet feeding. Long-chain fatty acids have been reported to directly activate voltage-dependent Ca2+ channels, resulting in Ca2+ overload during ischemia, possibly through modifications of the lipid-protein membrane interface (138). However, BL Ca2+ concentrations were not different with HF or high-fat diet feeding (data not shown). Our results do suggest, however, that defects in
ICa do not account for the observed differences in cardiomyocyte contractile function.
In this study, we focused on correlating in vivo cardiac function with in vitro cellular and molecular changes. We reported some discrepancies between in vivo hemodynamic and in vitro cardiomyocyte functional measurements that might be explained by structural differences (e.g., cardiomyocyte from the remote vs. border zone of the viable myocardium and alterations at the tissue level) and circulating factors (e.g., circulating adipokines and hormones) that were not examined in this study. Future studies should be directed at investigating additional mechanisms that could play a role in regulating in vivo functional differences in our model of HF/LV dysfunction, e.g., alterations in the responsiveness and regulation of -adrenergic-mediated changes in contractile function.
In conclusion, we report that SAT diet feeding after the induction of mild- moderate HF/LV dysfunction can improve cellular contractility (cell shortening velocity);
94 this change was associated with decreased MHC content. Furthermore, changes in isolated ventricular myocyte systolic and diastolic function correspond to improvements in in vivo myocardial contractile function and reductions in myocyte hypertrophy. These results suggest that the “cardio-protective” effect attributed to SAT feeding in HF may result from metabolic changes in the cardiac myocyte that lead to changes in myofilament function without affecting SR Ca2+ regulatory properties.
95 Chapter 3
Impaired Contractile Function Due to
Decreased Cardiac Myosin Binding Protein C
Content in the Sarcomere
Y. Cheng1, X. Wan2, T.A. McElfresh1, X. Chen1, K.S. Gresham1, D.S. Rosenbaum2*,
M.P. Chandler1, and J.E. Stelzer1
From: 1 Department of Physiology and Biophysics, Case Western Reserve University,
Cleveland, OH 44106, USA; 2 The Heart and Vascular Research Center, MetroHealth
Campus, Case Western Reserve University, Cleveland, OH 44109, USA.
*deceased
(Expanded from: Am J Physiol Heart Circ Physiol. 305(1): H52-H65, 2013. All figures
and tables reprinted with permission )
96 3.1 INTRODUCTION
Mutations in MyBP-C are among the most common causes of FHC, an autosomal dominant disease with variable disease onset, penetrance, and phenotypic presentation
(248). Millions of people worldwide carrying MyBP-C mutations are at a significantly higher risk for developing cardiac disease and SCD (293), and the majority of MyBP-C mutations are expected to produce C-terminal truncated proteins. However, to date these truncated proteins have not been found in myocardial samples isolated from patients with
MyBP-C mutations (148, 210, 349), but rather a decrease in the total amount of MyBP-C has been reported, likely due to degradation of unstable mutant MyBP-C by cell surveillance mechanisms (49). Despite an intense research effort in the last few years, the mechanisms by which mutations in MyBP-C cause disease, and specifically mutations which cause a decrease in MyBP-C expression in the heart, are still not understood.
MyBP-C is a thick filament protein localized in regular intervals in the C-zone of the A-band of the myofilament, and regulates myofilament contractile mechanics through its phosphorylation via interactions with actin and myosin [reviewed by (17, 18, 149,
257)]. The absence of MyBP-C leads to accelerated cross-bridge kinetics at the myofilament level (172, 321), and myocyte disarray and fibrosis at the tissue level (48,
124). At the organ level, MyBP-C−/− mice exhibit profound LV hypertrophy (41, 48, 124,
250, 260) and early-onset contractile dysfunction (41, 48, 124). In contrast, heterozygous
MyBP-C (MyBP-C+/−) null mice which express ~25% less MyBP-C than WT mice present with very mild or no hypertrophy (48, 124), yet still display early-onset mechanical dysfunction (82). Thus, the mechanisms by which altered MyBP-C
97 expression in the sarcomere contributes to altered whole organ contractile dysfunction remain elusive.
The primary effect of MyBP-C mutations is expected to manifest as a defect in the regulation of myofilament function in response to Ca2+ activation, and indeed, mechanical experiments on myocardial preparations isolated from hearts of patients carrying MyBP-C mutations have shown decreased force generating capacities at maximal Ca2+-activations and increased force generating capacities at low levels of Ca2+- activation (210, 349). Furthermore, there is also evidence that MyBP-C is dephosphorylated in hearts expressing MyBP-C mutations (68, 349). However, it is not clear how this dephosphorylation, along with decreased MyBP-C expression, contributes to cardiac dysfunction. Also, it is not known if altered myofilament behavior in MyBP-C deficient hearts causes downstream compensatory changes in signaling cascades that modulate the phosphorylation state of other key regulatory myofilament and Ca2+- handling proteins in the myocyte that could contribute to functional deficits observed at the organ level. Ca2+ kinetics and handling are essential regulatory components of LV contractile function and are responsible for modulating the availability of Ca2+ to the myofilaments. Thus it is possible that alterations in expression and/or phosphorylation of key Ca2+ handling proteins, such as NCX, SERCA, and PLB, which participate in coordinating Ca2+ movement between cellular compartments and the kinetics of the Ca2+ transients (10, 30), can contribute to contractile dysfunction in MyBP-C related FHC.
Also, because MyBP-C phosphorylation is crucial for modulating cardiac contractility
(17, 18, 149, 257) it is important to investigate whether MyBP-C deficiency impairs the contractile response to β-adrenergic stimulation.
98 Furthermore, abnormal Ca2+ homeostasis along with defective electrical coupling and propagation in myocardium are associated with increased risk of cardiac arrhythmia and SCD in FHC patients irrespective of the presence of cardiac hypertrophy (139, 177,
218, 341). The propagation of electrical signals from the pacemaker cells throughout the rest of the myocardium is coordinated through cardiac gap junctions which consist of connexin protein complexes (241, 296). The primary connexin protein responsible for electrical coupling in the LV is Cx43, and under pathological conditions the overall expression and phosphorylation levels of Cx43 are decreased, thereby contributing to abnormal electrical conduction and increased susceptibility to cardiac arrhythmia (241,
296). However, it is unknown whether electrophysiological defects and altered Cx43 expression and/or phosphorylation contribute to the pathogenesis of MyBP-C related
FHC.
The goal of the present study is to define the functional link between MyBP-C deficiency at the sarcomere and changes in the molecular mechanisms that modulate the function of the myofilament, Ca2+-handling machinery, and electrical conduction in the heart, which altogether, regulate in vivo cardiac contractile function. We employed adult
MyBP-C−/− and MyBP-C+/− mice, which served as models of total and partial MyBP-C insufficiency, respectively, and measured in vitro contractile function in skinned myocardium and intact cardiomyocyte and in vivo cardiac hemodynamic function and electrical activity. We also performed studies to characterize ventricular conduction properties in ex vivo intact hearts, and in vivo hemodynamic properties and electrical activity in response to -adrenergic stress to assess whole organ function under increased
99 workload. The utilization of a comprehensive integrative approach in this study allowed us to gain a deeper insight into the pathogenesis of MyBP-C related FHC.
3.2 MATERIALS AND METHODS
3.2.1 Ethical Approval and Experimental Model
This study was conducted in accordance with the Guide for the Care and Use of
Laboratory Animals (NIH Publication No. 85-23, Revised 1996), and the procedures of anesthesia, surgery, and general care of the animals were approved by the Institutional
Animal Care and Use Committee at Case Western Reserve University. Adult male WT,
MyBP-C−/−, and MyBP-C+/− mice of the SV/129 strain at ~6 month of age were used
(124).
3.2.2 Echocardiography
Myocardial function was evaluated by echocardiography at ~6 month of age (n =
7-11 per group). A Sequoia C256 System (Siemens Medical) with a 15 MHz linear array transducer was used as previously described (230) with anesthetized mice (1.5-2.0% isoflurane). 2D, 2D-guided M-mode, and Doppler echocardiographic studies of aortic and transmitral flows were performed via parasternal and foreshortened apical windows. Wall thickness, end-systolic (ESD) and end-diastolic dimensions (EDD) were measured, and
EF was calculated based on volume and presented as a percentage of EDV.
100 3.2.3 Hemodynamic Measurements
In vivo LV contractile properties were assessed at ~6 month of age (n = 7-9 per group). Mice were anesthetized (1.5-2.0% isoflurane), intubated, and ventilated. A Millar
1.4 F microtip pressure-volume transducer catheter (Millar Instruments) was introduced via the right carotid artery as previously described (270). After BL recording, 10 g/g
BW of DOB was administered through the jugular vein and the peak -adrenergic response was analyzed at 5min post-injection. Endpoints related to HR, LV pressure, EF, dP/dtmax, and , were determined with the aid of PVAN/Chart5 software (Millar
Instruments).
3.2.4 Electrocardiogram
The telemetric ambulatory ECG recordings were obtained at ~6 months of age (n
= 4-11 per group) with implantable transmitters (ETA-F10, DataSciences International,
St. Paul, MN). Mice were anesthetized with isoflurane and a midline incision was made along the spine. The implantable wireless radiofrequency transmitter was aseptically inserted into a subcutaneous tissue pocket in the back, and the leads were placed at the right shoulder and lower left chest and sutured to the muscle. Following a recovery period from the transmitter implant surgery the mice were placed in cages overlying a receiver for transmission of ECG signals to a computer for display and analysis.
Subsequent to a 1-2 hr acclimatization period when mice rested quietly in their cages, telemetered ECG tracings were obtained in conscious mice during quiet awake time at rest and after DOB injection (10 g/g BW) for a total period of 1-3 hrs. Measurements of durations and intervals of waveforms taken from ~8 min of undisturbed tracings
101 (monitored by activity channel recorded simultaneously) were performed with the aid of
Chart5 software (Millar Instruments) (for MyBP-C−/− data) or Ponemah software (Data
Sciences International) (for MyBP-C+/− data). Determination of QRS and QT segments was done according to previous studies (61, 284). The corrected QT interval (QTc) was calculated using the Bazett formula: QTc = QT/(RR/100)1/2, which is specifically modified for mouse ECG as previously described (226).
3.2.5 High Resolution Optical Mapping
High-resolution optical mapping was performed as previously described (88).
Briefly, mice (n = 5-6 per group) were anesthetized (0.1 ml Nembutal and 0.1 ml
Heparin) and hearts were rapidly excised. Hearts were then perfused as Langendorf
o preparations with warmed (37 C) oxygenated Tyrode’s solution (1.25 mM CaCl2, 140 mM NaCl, 4.5 mM KCl, 5.5 mM dextrose, 0.7 mM MgCl2, 10 mM HEPES, pH 7.4).
Optical action potentials were recorded simultaneously from 256 sites on the anterior surface of the ventricle using a 16×16 photodiode array (C4675-102, Hammamatsu
Corporation, Bridgewater, NJ). To measure transmembrane potentials, hearts were perfused with Di-4-ANEPPS at a final concentration of 10 M for 30min, and 7 M of cytochalasin D (Sigma) was used to ensure that motion artifact did not influence measurements. Constant cycle length (CL) stimulation was performed at CLs of 170 to
100 msec, by 10 msec decrements, with a stimulus current 2X diastolic threshold. Data analysis was performed using custom software designed specifically for the analysis of action potentials recorded optically from the mouse heart. Conduction velocity was quantified at CL of 170 msec, both parallel and perpendicular to fiber axis using a vector
102 summation technique described previously (88). APD was quantified at 85% repolarization.
3.2.6 Histological Assessment of Cardiac Morphology
Hearts were harvested (n = 7) for histological evaluation of chamber modifications, potential fibrosis, and myocyte hypertrophy. All hearts were washed in cardioplegic buffer (containing in mM: 110 NaCl, 16 MgCl2, 16 KCl, 10 NaHCO3, 5 dextrose, and 1.2 CaCl2), formalin fixed, and paraffin embedded. The hearts were sliced at mid-LV level using a slicer matrix, and the sections were cut at 5 m thickness using a microtome. The cross sections were stained with either Masson’s trichrome or hematoxylin and eosin stain (H&E) for gross morphology and fibrosis or cellular hypertrophy, respectively.
3.2.7 Myocyte Isolation and in Vitro Shortening and Ca2+ Transients
Ventricular cardiomyocytes were isolated enzymatically from mouse hearts (n =
4-5 per group) using enzymatic dispersion technique described previously (340).
Sarcomere shortening was assessed using a video-based sarcomere length detection system (IonOptix, Milton, MA) (86). To measure intracellular Ca2+ transients, myocytes were loaded with 2 M of indo-1AM (Invitrogen) and 0.025% (w/w) Pluronic F-127
(Invitrogen) for 20 min at room temperature. The intracellular indo-1 was excited at 355 nm, and the fluorescence emitted at 405 and 485 nm was collected by two matched photomultiplier tubes. Data were filtered at 200 Hz and sampled at 1 kHz. The ratio of the intensity of fluorescence emitted at 405 nm over that at 485 nm was calculated after
103 subtraction of background fluorescence and used to compare the time course and amplitude of intracellular Ca2+ transients between groups of myocytes. The emission field was restricted to a single cell with the aid of an adjustable window (359). Sarcomere shortening and Ca2+ transients were initiated by field stimulation at 2 Hz at room temperature with 1.8 mM Ca2+ and were measured simultaneously. All analysis of sarcomere shortening (n = 47-69 per group) and Ca2+ transients (n = 42-68 per group) was done using IonOptix software (IonOptix).
3.2.8 Myofilament Contractile Function
Solutions for skinned myocardium experiments
Solution compositions were calculated using the computer program of Fabiato
(92) and stability constants listed by Godt & Lindley (105) corrected to pH 7.0 and 22°C.
All solutions contained (in mM) 100 N,N-bis (2 hydroxy-ethyl)-2-aminoethanesulfonic acid, 15 creatine phosphate, 5 dithiothreitol, 1 free Mg2+, and 4 MgATP. pCa 9.0 solution contained (in mM) 7 EGTA and 0.02 CaCl2; pCa 4.5 contained 7 EGTA and 7.01 CaCl2; and pre-activating solution contained 0.07 EGTA. Ionic strength of all solutions was adjusted to 180 mM with potassium propionate. Solutions containing different amounts
2+ of Ca free were prepared by mixing appropriate volumes of solutions of pCa 9.0 and pCa
4.5.
Apparatus and experimental protocols
Skinned multicellular ventricular myocardium for mechanical experiments was prepared and attached to the arms of a position motor and force transducer as previously
104 described (56). Motor position and force signals were sampled using SLControl software
(45) and saved to computer files for later analysis. All mechanical measurements were performed at 22°C and sarcomere length was set to 2.1 µm. Data presented for all of the
BL mechanical experiments were the average of 20 fibers from 5 mice per group, and 12 fibers from 4 mice per group for PKA experiments.
Force-pCa relationships
Methods for obtaining and analysis of force-pCa relationships are described in detail elsewhere (56). Briefly, each myocardial preparation was allowed to develop steady force in solutions of varying free [Ca2+]. The difference between steady-state force and the force BL obtained after the 20% slack step was measured as the total force at that free [Ca2+]. Active force was then calculated by subtracting Ca2+-independent force in solution of pCa 9.0 from the total force and was normalized to the cross-sectional area of the preparation, which was calculated from the width of the preparations assuming a cylindrical cross-section. Force-pCa relationships were constructed by expressing submaximal force (P) at each pCa as a fraction of maximal force (Po) determined at pCa
4.5, i.e., P/Po. The apparent cooperativity in the activation of force development was inferred from the steepness of the force-pCa relationship and was quantified using a Hill plot transformation of the force-pCa data. The force-pCa data were fit using the Hill equation described in Chapter 1.
105 Rate of force development
The ktr was measured in skinned myocardium as a measure of the rate of transitions of cross-bridges between weak-binding, non-force-generating states and strong-binding, force-generating states, using a release-restretch protocol (322). Each skinned preparation was transferred from relaxing to an activating solution containing
Ca2+ (sufficient for activation of ~10-25% of maximal force) and allowed to generate steady-state force. The myocardial preparation was rapidly (< 2 msec) slackened by 20% of its original length, resulting in a rapid reduction of force which was followed by a brief period of unloaded shortening (10 msec) after which the preparation was rapidly restretched to its original length. ktr was estimated by linear transformation of the half- time of force redevelopment, i.e., ktr = 0.693/t1/2, as described previously (322).
Stretch activation experiments
For stretch activation experiments, fiber length in relaxing solution was adjusted to achieve a sarcomere length of ~2.1 m for measurement of initial isometric force and for subsequent imposition of stretch. To evoke stretch activation a rapid stretch (~10 muscle lengths sec-1) of 1% of muscle length was imposed on fibers that were activated to develop submaximal forces equivalent to ~10-25% maximal force (Po). Submaximal
2+ Ca -activated force (P) was expressed as a fraction of Po generated at pCa 4.5, i.e., P/Po.
The method used for measuring the stretch activation variables have been described in detail (323). The amplitudes of the phases of the stretch activation responses were measured as follows: P1, measured from pre-stretch steady-state force to the peak of phase 1, P2, measured from pre-stretch steady-state force to the minimum force value at
106 the end of phase 2, P3, measured from pre-stretch steady-state force to the peak value of delayed force, and Pdf, difference between P3 and P2. All amplitudes were normalized to the pre-stretch Ca2+ activated force to allow comparisons between preparations. Apparent
-1 rate constants were calculated for phase 2 (krel, s ) between the peak of phase 1 and the
-1 minimum of phase 2 and for phase 3 (kdf, s ) from the beginning of force re-uptake following phase 2 to the completion of delayed force development. krel was obtained by fitting the time course trace with a single exponential and kdf was estimated by linear transformation of the half-time of force redevelopment as previously described (323).
A representative stretch activation response in WT skinned myocardium is illustrated in Fig. 3-1, in which a stretch of 1% of initial length was imposed on a muscle generating Ca2+-activated force that was ~25% maximal. The characteristic features of the stretch activation response have been elucidated in detail elsewhere (44, 106, 323).
Briefly, following achievement of steady-state force generation, acute stretch elicits an immediate increase in force (P1), due to distortion of attached cross-bridges. The force transient then begins to rapidly decay due to the detachment of strained cross-bridges and reaches a minimum amplitude (P2) which can be positive or negative (i.e., below initial pre-stretch force). Next, force begins to increase due to a recruitment of new cross- bridges into strongly-bound states mediated by an increase in muscle length and eventually reaches a new steady-state level (P3) which is sustained over a period of several seconds before decaying back to the original isometric force.
107 In experiments assessing the effects of PKA (catalytic subunit of bovine PKA;
Sigma) on mechanical properties, skinned preparations were first incubated for 1 h
(22°C) in pCa 9.0 solution containing PKA (0.25 U/l). Mechanical properties were then measured as described above. Because PKA treatment decreased the Ca2+ sensitivity of force in both WT and MyBP-C+/− myocardium, it was necessary to use a pCa solution with a slightly higher [Ca2+] to match the PKA untreated BL isometric force.
P1 0.3
0.2
0.1 krel
kdf Pdf P3
Force (Normalized to pre-stretch force) topre-stretch (Normalized Force 0.0 P2 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Time (s)
Figure 3-1. Stretch Activation Responses in Murine Myocardium. A representative force transient following a stretch of 1% of muscle length recorded from WT skinned myocardium activated with Ca2+ to a pre-stretch isometric force of ~25% maximal. The transient is normalized to pre-stretch force, and the recorded variables are labeled on the trace and described in the text.
Muscle fiber stiffness
Skinned myocardium was incubated in a pCa solution that yielded forces of approximately half of maximal, and when the muscle fiber reached a steady-state isometric force, the muscle length was increased in a step-like fashion by 0.25, 0.5, 1.0,
108 1.5, 2.0, and 2.5% of initial muscle length (∆L). Cross-bridge stiffness associated with the number of strongly bound cross-bridges prior to stretch was estimated from the relationship between length change in ∆L and the peak force response following stretch
(P1). Stiffness is represented as the slope of the linear regression of the relationship between P1 and ∆L.
3.2.9 Quantification of Protein Expression and Phosphorylation
Ventricular tissue (n = 5-10 per group) was homogenized in buffer containing 20 mM Tris-base, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2-6H2O, 1 mM (K2)EDTA, 10% glycerol, 1% Triton X-100, pH 7.8, and with protease and phosphatase inhibitor cocktails
(Sigma). Protein concentration was quantified by BCA protein assay (Thermo Scientific).
Laemmli buffer (62.5 mM Tris-HCl, 10% glycerol, 2% SDS, 0.01% bromophenol blue,
5% -mercaptoethanol, pH 6.8) was added to the samples. Ten micrograms of non-boiled protein were loaded onto 7.5% Tris-HCl gels (Bio-Rad) for analysis of SERCA2 and
NCX1 levels, and 14 g of non-boiled protein were loaded onto 3-8% Tris-Acetate gels
(Bio-Rad) for RyR2. Samples were boiled for 10 min and 10 g of protein were loaded onto 10% Tris-HCl gels (Bio-Rad) for phosphorylated (p-)Ser273/Ser282/Ser302 MyBP-C
(pMyBP-C273, pMyBP-C282, and pMyBP-C302) as well as MyBP-C (69). Twenty micrograms of boiled samples were loaded onto 12.5% Tris-HCl gels (Bio-Rad) for
Cx43, and 10 g of boiled samples were loaded onto 15% Tris-HCl gels (Bio-Rad) for phosphorylated (p-)Ser23/24 TnI (pTnI) as well as TnI, and phosphorylated (p-)Ser16/Thr17
PLB (pPLB16 and pPLB17) as well as PLB. HSC70 was chosen to serve as a loading control. For RyR2, gels were run at 150V for 1 hr 45 min then transferred to PVDF
109 membranes at 30 V at 4°C for 30 hr and 70 V for 1hr. For Cx43, gels were run at 150V for 2 hr 10 min then transferred to PVDF membranes at 100 V for 1hr 10 min. For the rest of the target proteins, gels were run at 200 V for 1 hr then transferred to PVDF membranes at 100 V for 45 min. Membranes were incubated at 4oC overnight with
1:1000 anti-MyBP-C (sc-67353, Santa Cruz Biotechnology), 1:1000 anti-pMyBP-C273,
1:1000 anti-pMyBP-C282, 1:1000 anti-pMyBP-C302 (custom-made from 21st Century
Biochemicals, Marlborough, MA), 1:1000 anti-TnI (4002S, Cell Signaling Technology,
Danvers, MA), 1:1000 anti-pTnI (4004S, Cell Signaling Technology), 1:1000 anti-
SERCA2 (MA3-919, Thermo Scientific), 1:1500 anti-NCX1 (MA1-4672, Thermo
Scientific), 1:1000 anti-PLB (05-205, Millipore), 1:1000 anti-pPLB16 (07-052,
Millipore), 1:5000 anti-pPLB17 (A010-13, Badrilla), 1:1000 anti-Cx43 (71-0700,
Invitrogen), at room temperature for 30 min with 1:2000 anti-HSC70 (sc-7298, Santa
Cruz Biotechnology), and at room temperature overnight with 1:300 anti-RyR2 (MA3-
916, Thermo Scientific), followed by incubations with the appropriate secondary antibodies. Membranes were incubated with chemiluminescence reagents (Thermo
Scientific) and exposed to films. Densitometry of bands was determined using Image J
(NIH).
To assess regional MyBP-C expression in the hearts of MyBP-C+/− mice, LV tissues were isolated from the apex, mid-LV, and base (5-6 hearts per region). Tissues were then prepared and loaded onto gels for quantification of MyBP-C expression by
Western blot as described above.
The protein content of cardiac MHC and were determined via gel electrophoresis as previously described (361). Ventricular tissue (n = 5) was
110 homogenized in RIPA buffer. The protein concentration was quantified by BCA protein assay (Thermo Scientific). Ten micrograms of protein were loaded onto a 6% acrylamide gel cross-linked with DATD as previously described (361). The gel was run at 10 mA constant current for 19 hrs at 4 oC. Bands were detected by Silver Stain Plus (Bio-Rad) per manufacturer’s protocol. The densitometry was analyzed using GelBandFitter analysis program as previously described (227).
For the rest of the myofibrillar proteins, the ventricular tissue (n = 4) was harvested and purified for myofibrillar proteins using a protocol previously described
(184). Ten micrograms of protein were loaded onto a 10% Tris-glycine mini gel, and the gel was run at 150V for 1.5 hr, followed by Pro-Q Diamond Phosphoprotein Gel Staining
(Invitrogen) per manufacturer’s protocol. The same gel was then stained with Silver Stain
Plus (Bio-Rad) per manufacturer’s protocol for total protein expression. Densitometry of bands was determined using Image J (NIH). The degree of phosphorylation of a given protein was determined by the slope of a first order linear regression generated based on the Pro-Q optical density and protein content loaded as previously described (324).
To determine the effect of PKA treatment on the level of phosphorylation of
MyBP-C and other myofilament proteins, skinned myocardium was isolated from WT and MyBP-C+/− hearts and incubated with PKA (1 U/l) for 1 hr at 30°C as previously described (342). The samples were then denatured with Laemmli buffer and the protein content was quantified by BCA protein assay. Ten micrograms of boiled samples were loaded onto 4-20% Tris-glycine mini gels, and the gels were run at 150V for 1.5 hr, followed by Pro-Q Diamond Phosphoprotein Gel Staining per manufacturer’s protocol.
The same gel was then stained with Coomassie Blue for total protein expression.
111 3.2.10 Phosphatase Activity Assay
Phosphatase 1 (PP1) activity was measured using a fluorescent-based enzymatic assay with RediPlate 96 EnzChek Serine/Threonine Phosphatase Assay Kit (R-33700,
Molecular Probes, Grand Island, NY). Reaction buffers specifically for measuring PP1 activity were added per manufacturer’s protocol.
3.2.11 Statistical Analysis
All statistical analyses were performed using SigmaStat software. Regional differences in MyBP-C expression between the apex, mid-LV, and base of MyBP-C+/− myocardium were determined using one-way ANOVA. Differences between the BL measurements and DOB treatment of WT vs. MyBP-C−/− data were determined via two- way ANOVA, followed by Fisher LSD post-hoc analysis for multiple pairwise comparisons. All the other analyses were done via Student’s t-test. Differences between the BL measurements and DOB treatment of WT vs. MyBP-C+/− data were not tested.
Significance was established at P < 0.05. Data are expressed as the means ± SE.
3.3 RESULTS
Comparison between WT and MyBP-C−/− Models
3.3.1 Myofilament Protein Content and Phosphorylation
To evaluate the molecular changes in myofilament composition, the expression and phosphorylation levels of MyBP-C and other regulatory myofibrillar proteins were quantified in adult animals at age ~6 month. As expected MyBP-C was not detected in
MyBP-C−/− hearts (Fig. 3-2A). TnI expression (data not shown) and phosphorylation
112 Figure 3-2. Protein Expression and Phosphorylation of Myofibrillar Proteins between WT and MyBP-C−/− Models. A: representative blots of MyBP-C, TnI, pTnI, and the loading control HSC70 by Western blot. The expression of pTnI (normalized to TnI) (A) were quantified via densitometry. n = 6-7. B: representative silver stained gel of MHC and is shown on the top and the quantified relative MHC expression via densitometry on the bottom. n = 5. C: representative Pro-Q stained gel (phosphorylation) is shown on the left and the silver stained gel (total protein content) on the right. Slopes of phosphorylation signals of TnT (D) and MRLC (E) were determined from regression analysis of Pro-Q optical density versus protein content loaded. n = 4. * P < 0.05 compared to WT.
113 (normalized to TnI expression) were not different between groups (Fig. 3-2A). Consistent with previous studies (82), we observed an increase in the expression of MHC in
MyBP-C−/− myocardium compared to WT myocardium (Fig. 3-2B). No differences between groups were detected in the expression and phosphorylation of TnT (Fig. 3-2D) or MRLC (Fig. 3-2E) which were quantified by silver staining and Pro-Q phospho- staining of SDS-PAGE gels.
3.3.2 Isolated Myocyte Contractile Properties
To evaluate the in vitro contractile properties, we assessed sarcomere shortening in ventricular cardiomyocytes isolated from WT and MyBP-C−/−. Representative tracings of sarcomere shortening are shown in Fig. 3-3A. Diastolic sarcomere length in myocytes isolated from MyBP-C−/− hearts was shorter than WT myocytes (Fig. 3-3B). The % of sarcomere shortening (normalized to diastolic sarcomere length) was depressed in MyBP-
C−/− myocytes compared to WT myocytes (Fig. 3-3C), and the rate of sarcomere shortening, −dL/dt, was also depressed in MyBP-C−/− myocytes compared to WT myocytes (Fig. 3-3D). The rate of sarcomere relengthening, +dL/dt, was not different between groups (Fig. 3-3E), however, the time to peak shortening was reduced in MyBP-
C−/− myocytes compared to WT myocytes (Fig. 3-3F). Time to 50% relaxation was shorter in MyBP-C−/− myocytes compared to WT myocytes (Fig. 3-3G), however, time to
90% relaxation (data not shown) and the relaxation time constant, , were not different between groups (Fig. 3-3H).
114 Figure 3-3. Ventricular Cardiomyocyte Sarcomere Shortening between WT and MyBP-C−/− Models. A: representative tracings of sarcomere shortening at 2 Hz with 1.8 mM Ca2+ averaged from 8 peaks from the same cell from each group. Diastolic sarcomere length (B), % of sarcomere shortening (C), the rate of sarcomere shortening (−dL/dt) (D), the rate of sarcomere relengthening (+dL/dt) (E), time to peak (F), time to 50% relaxation (G), and the relaxation time constant, (H) were measured on fresh isolated intact ventricular cardiomyocytes at 2 Hz with 1.8 mM Ca2+. n = 47-67. * P < 0.05 compared to WT.
115 3.3.3 Myocyte Ca2+ Handling Properties
Since myofilament function and SR Ca2+ handling can influence and regulate each other, in vitro Ca2+ handling properties were evaluated by measuring the amplitude and kinetics of Ca2+ transients recorded from freshly isolated ventricular cardiomyocyte loaded with indo-1. The representative superimposed Ca2+ transient tracings are shown in
Fig. 3-4A. There were no differences between groups in BL Ca2+ levels (F405/485 ratio:
WT: 1.07 ± 0.06; MyBP-C−/−: 1.10 ± 0.07). Ca2+ transient amplitude (as % BL) was decreased in MyBP-C−/− myocytes compared to WT myocytes (Fig. 3-4B). The time to peak Ca2+ was not different between groups (Fig. 3-4C), however, the time to 50% Ca2+ decay (Fig. 3-4D) and (Fig. 3-4E) were both reduced in MyBP-C−/− myocytes by 13% and 31%, respectively, compared to WT myocytes. Time to 90% Ca2+ decay was not different between groups (data not shown).
3.3.4 Expression of Ca2+ Handling Proteins and PP1 Activity
Differences in Ca2+ handling properties were related to expression and phosphorylation of several key Ca2+ handling proteins by Western blot analysis. The representative blots of Ca2+ handling proteins are shown in Fig. 3-5A. The expression of
RyR2, the Ca2+ releasing channel on SR, was not different between groups (Fig. 3-5B).
The expression of NCX1, which contributes to extrusion of Ca2+ into the extracellular space, was not different between groups (Fig. 3-5C). The expression of SERCA2
(normalized to HSC70), the primary protein responsible for sequestering Ca2+ from the cytosol back to the SR, was increased in MyBP-C−/− hearts compared to WT hearts (WT:
1.16±0.60, MyBP-C−/−: 3.19±0.20). Expression of PLB (normalized to HSC70), the
116 inhibitor of SERCA2, was not different between groups (WT: 1.86±0.11, MyBP-C−/−:
1.82±0.09), therefore the SERCA2 to PLB ratio (both normalized to HSC70) was increased in MyBP-C−/− hearts compared to WT hearts (Fig. 3-5D). Both pPLB16 (Fig. 3-
5E) and pPLB17 (Fig. 3-5F) were decreased in MyBP-C−/− hearts compared to WT hearts. The expression (Fig. 3-5G) and activity (Fig. 3-5H) of PP1, the main phosphotase in the heart, were both increased in MyBP-C−/− hearts compared to WT hearts.
Figure 3-4. Ventricular Cardiomyocyte Ca2+ Transients between WT and MyBP- C−/− Models. A: representative superimposed Ca2+ transients tracings at 2 Hz with 1.8 mM Ca2+ averaged from 8 peaks from the same cell from each group. Ca2+ transient amplitude (% BL) (B), time to peak (C), time to 50% Ca2+ decay (D), and the relaxation time constant, (E) were measured on fresh isolated intact ventricular cardiomyocytes loaded with indo-1 at 2 Hz with 1.8 mM Ca2+. n = 42-68. * P < 0.05 compared to WT.
117 Figure 3-5. Protein Expression of Ca2+ Handling Proteins and PP1 Activity. A: representative bands of RyR2, NCX1, SERCA2, PLB, pPLB16, pPLB17, PP1, and HSC70 by Western blot. Ratio of RyR2 to HSC70 (B), NCX1 to HSC70 (C), SERCA2 to PLB (both normalized to HSC70) (D), pPLB16 to PLB (E), pPLB17 to PLB (F), and PP1 to HSC70 (G) were quantified via densitometry. n = 6-9. PP1 activity (H) was assessed by a fluorescent ELISA plate. n = 8. * P < 0.05 compared to WT.
3.3.5 Echocardiography
We investigated in vivo LV contractile function and morphology of WT and
MyBP-C−/− hearts by echocardiography. MyBP-C−/− hearts were hypertrophied as evident by increased posterior wall thickness at end diastole (PWTd), resulting in increased LV mass/BW compared to WT, and had increased LV chamber size as evidenced by larger
118 ESD and EDD (Table 3-1). Consistent with previous studies (41, 48, 124, 250), contractile function was compromised in MyBP-C−/− hearts compared to WT hearts, as indicated by decreased EF and FS (Table 3-1). Moreover, the MyBP-C−/− hearts had shortened ET and IVRT (the time between aortic valve closing and mitral valve opening) compared to WT (Table 3-1).
Table 3-1. Gravimetric Measurements and in vivo LV Function by Echocardiography between WT and MyBP-C−/− Models.
WT MyBP-C−/− BW (g) 31.1 ± 1.2 30.0 ± 1.5 LV Mass/BW 3.95 ± 0.21 6.03 ± 0.58 * HR (bpm) 427 ± 17 400 ± 9 PWTd (mm) 0.86 ± 0.03 1.10 ± 0.03 * ESD (mm) 2.13 ± 0.20 3.25 ± 0.07 * EDD (mm) 3.78 ± 0.07 4.28 ± 0.12 * EF (%) 80.7 ± 4.4 55.7 ± 2.5 * FS 0.44 ± 0.04 0.24 ± 0.01 * ET (msec) 60.9 ± 2.6 42.1 ± 1.2 * IVRT (msec) 22.9 ± 2.2 44.4 ± 3.6 * Values are means ± SE. BW: body weight; HR: heart rate; PWTd: posterior wall thickness in diastole; ESD: end systolic dimension; EDD: end diastolic dimension; EF: ejection fraction; FS: fractional shortening; ET: ejection time; IVRT: isovolumic relaxation time. n = 7-10. * P < 0.05 compared to WT.
3.3.6 In Vivo Hemodynamic Function at BL and Following DOB Challenge
In vivo LV hemodynamic function at BL and following DOB stimulation was assessed in WT and MyBP-C−/− hearts by pressure-volume catheterization. The BL HR
119 was not different between groups and increased similarly in both groups following DOB treatment (Table 3-2). The LV maximal pressure (Pmax) was not different between groups
−/− at BL, however, following DOB treatment Pmax was decreased in MyBP-C hearts
(Table 3-2). Consistent with previous studies (41, 48, 250), BL dP/dtmax was not different in MyBP-C−/− hearts compared to WT hearts, however, following DOB treatment MyBP-
−/− C hearts displayed a decrease in dP/dtmax in contrast to the increased dP/dtmax displayed by WT hearts (Table 3-2). The EF % was depressed in MyBP-C−/− hearts compared to
WT hearts at BL, and the increase in EF % following DOB treatment was blunted in
MyBP-C−/− hearts compared to WT hearts (Table 3-2). Diastolic function in MyBP-C−/− hearts was also impaired as evidenced by prolonged LV relaxation time constant, , at BL and following DOB compared to WT hearts, and an elevation in minimal LV pressure
(Pmin) at the end of diastole at BL (Table 3-2).
Table 3-2. LV Hemodynamic Function at BL and Following -adrenergic Stimulation between WT and MyBP-C−/− Models.
WT MyBP-C−/− BL DOB BL DOB HR (bpm) 410 ± 16 441 ± 16 408 ± 14 448 ± 21
Pmax (mmHg) 100 ± 3 102 ± 8 91.8 ± 3.9 69.1 ± 5.7 ¥*
Pmin (mmHg) 2.5 ± 0.6 2.6 ± 1.1 8.7 ± 2.8 * 6.2 ± 2.3
dP/dtmax (mmHg/sec) 7964 ± 273 11072 ± 961 ¥ 7819 ± 633 5623 ± 744 ¥* EF (%) 75.0 ± 4.9 90.2 ± 3.2 ¥ 51.8 ± 5.0 * 58.8 ± 8.5 * (msec) 9.5 ± 0.9 8.2 ± 0.6 19.0 ± 1.3 * 20.2 ± 1.8 *
Values are means ± SE. BL: baseline; DOB: dobutamine response at 5 min post injection; HR: heart rate; Pmax: maximal pressure; Pmin: minimal pressure; dP/dtmax: peak rate of pressure rise; EF: ejection fraction; : relaxation time constant. n = 7-9. ¥ P < 0.05 compared to the individual BL; * P < 0.05 compared to WT BL or DOB.
120 3.3.7 Optical Mapping and Cx43 Protein Expression and Phosphorylation
To investigate the potential arrhythmogenic phenotype associated with FHC, the electrical conduction velocity was examined in ex vivo hearts isolated from WT and
MyBP-C−/− mice by high-resolution optical mapping. Representative contour plots of electrical conduction properties are shown in Fig. 3-6A and the APD in Fig. 3-6B. The crowding of the isochrome lines in MyBP-C−/− hearts indicates slower electrical conduction, and when quantified, the MyBP-C−/− hearts had slower conduction at 170 msec CL in the transverse direction compared to WT hearts (Fig. 3-6C). Longitudinal conduction velocity and the anisotropic ratio, a ratio of fast (longitudinal) axis to slow
(transverse) axis conduction velocity, showed a trend although did not reach statistical significance (Fig. 3-6D and 3-6E). The APD was significantly longer in MyBP-C−/− hearts compared to WT hearts (Fig. 3-6F).
The expression of the main protein of the connexin complex, Cx43, was evaluated by Western blot, and a representative blot for Cx43 expression in WT and MyBP-C−/− hearts is shown in Fig. 3-5G. P2 and P1 indicate the two phosphorylated Cx43 species, whereas NP indicates the unphosphorylated Cx43. The expression of total Cx43
(P2+P1+NP) was not different between groups (data not shown), however, the phosphorylation of Cx43 (P2+P1/total Cx43) was decreased in MyBP-C−/− hearts compared to WT hearts (Fig. 3-6G).
121 Figure 3-6. Optical Mapping and Protein Expression of Cx43. Representative LV contour plots (A) and tracings of action potential (B) of WT and MyBP-C−/− acquired at 170 ms CL are shown. The transverse conduction velocity (C), longitudinal conduction velocity (D), anisotropic ratio (E), and action potential duration (F) were analyzed using specific custom software. n = 5-6. G: Representative bands of Cx43 and HSC70 by Western blot are shown on the left, with P2 and P1 indicating the two phosphorylated Cx43 species and NP the unphosphorylated Cx43, and the ratio of pCx43 to Cx43 on the right, quantified via densitometry. n = 5-7. * P < 0.05 compared to WT.
122 3.3.8 ECG
To further explore the in vivo mechanisms of MyBP-C deficiency in the heart, we performed continuous ECG recordings in conscious animals using surgically implanted radio telemetry devices. The HR at BL was not different between groups and increased proportionally following DOB challenge (Table 3-3). There were not significant differences between groups in PR segment duration or QRS duration, however, the QT interval (an index of ventricular relaxation) was longer in MyBP-C−/− mice at BL, and the
QT interval corrected for RR interval (QTc), showed a similar trend in MyBP-C−/− mice
(P = 0.06) (Table 3-3).
Table 3-3. ECG at BL and Following -adrenergic Stimulation between WT and MyBP-C−/− Models.
WT MyBP-C−/− BL DOB BL DOB HR (bpm) 704 ± 8 769 ± 12¥ 673 ± 20 732 ± 15¥ PR interval (msec) 32.0 ± 1.0 33.7 ± 4.2 31.2 ± 0.6 30.8 ± 2.7 QRS duration (msec) 20.0 ± 2.2 18.6 ± 1.1 18.3 ± 1.7 20.5 ± 2.8 QT interval (msec) 43.4 ± 2.0 44.3 ± 1.1 50.6 ± 4.0* 48.5 ± 3.2 QTc interval (msec) 47.0 ± 1.9 50.2 ± 1.0 53.4 ± 3.5 53.6 ± 3.3 RR interval (msec) 85.2 ± 0.9 78.1 ± 1.2¥ 89.5 ± 2.7 82.0 ± 1.8¥
Values are means ± SE. BL: baseline; DOB: dobutamine response; HR: heart rate; QTc: HR adjusted QT interval. n = 4-5. ¥ P < 0.05 compared to the individual BL; * P < 0.05 compared to WT BL or DOB.
123 Comparison between WT and MyBP-C+/− models
3.3.9 Myofilament Protein Content and Phosphorylation
To evaluate the molecular changes in myofilament composition, the expression and phosphorylation levels of MyBP-C and other regulatory myofibrillar proteins were quantified in adult animals at age ~6 months. The overall expression of MyBP-C in
MyBP-C+/− hearts was 32% lower than WT hearts (Fig. 3-7B), which was greater than the
25% decline we previously observed in younger MyBP-C+/− mice 2-3 months of age (82).
Further analysis of the pattern of MyBP-C expression in the LV of MyBP-C+/− hearts revealed that MyBP-C expression (normalized to HSC70) was significantly lower in samples isolated from the mid-LV region compared to samples isolated from the apex and base regions (Fig. 3-7C). Although, it is difficult to directly compare MyBP-C expression values derived from a regional LV analysis and a whole LV analysis, the results suggest that MyBP-C haploinsufficiency causes a greater decrease in MyBP-C expression in the mid-LV region compared to the apex and base in MyBP-C+/− hearts.
The phosphorylation levels of the remaining MyBP-C (normalized to MyBP-C expression) at residues Ser273 (Fig. 3-7D), Ser282 (Fig. 3-7E), and Ser302 (Fig. 3-7F) were all decreased in MyBP-C+/− myocardium compared to WT myocardium, by 45%, 32%, and 27%, respectively. Fig. 3-7G shows the relative ratio of MyBP-C phosphorylation at each phosphorylation residue in MyBP-C+/− hearts when compared to its WT counterpart
(which is set to 1). TnI expression (normalized to HSC70) and phosphorylation
(normalized to TnI expression) (Fig. 3-7H) were not different between groups. Consistent with previous studies (82), we observed no differences between groups in the composition of MHC isoforms, or the expression and phosphorylation of TnT or MRLC
124 (data not shown), which were quantified by silver staining and Pro-Q phospho-staining of
SDS-PAGE gels.
125 Figure 3-7. Expression and Phosphorylation of Myofilament Proteins between WT and MyBP-C+/− Models. A: Representative blots of MyBP-C, pMyBP-C273, pMyBP- C282, pMyBP-C302, pTnI, TnI, and the loading control HSC70 by Western blot. n = 6- 10. B: Overall expression of MyBP-C (normalized to HSC70) in MyBP-C+/− whole LV homogenates, and C: Regional differences in MyBP-C expression in LV samples isolated from the apex, mid-LV, and base of MyBP-C+/− hearts. Representative Western blots are shown on the top and quantified protein expression via densitometry on the bottom. n = 5-6. Quantification of MyBP-C and TnI phosphorylation in WT and MyBP-C+/− myocardium: D: pMyBP-C273 (normalized to MyBP-C), E: pMyBP-C282 (normalized to MyBP-C), F: pMyBP-C302 (normalized to MyBP-C). G: Data of MyBP-C phosphorylation at specific phosphorylation residues in the MyBP-C+/− group from D to F are expressed as fold changes to their WT counterparts (set to 1). It can be seen that MyBP-C phosphorylation levels in MyBP-C+/− samples are decreased at each of the phosphorylation residues compared to WT. H: levels of pTnI (normalized to TnI). I: Representative gel images of myofilament proteins prior to and following PKA treatment. Pro-Q staining of phosphorylated proteins is shown on the left and Coomassie-stained total protein expression on the right. Magnified Pro-Q stained MyBP-C bands are shown in the inset. * P < 0.05 compared to WT via Student’s t-test or compared to apex and base LV samples via one-way ANOVA.
To assess the effects of PKA treatment on MyBP-C and TnI phosphorylation, skinned myocardium preparations isolated from WT and MyBP-C+/− hearts were incubated with PKA prior to electrophoresis, and the representative Pro-Q phospho- stained and Coomassie-stained gel images are shown in Fig. 3-7I. PKA treatment resulted in significant and comparable increases in the phosphorylation of MyBP-C and TnI in both WT and MyBP-C+/− myocardium, suggesting that MyBP-C haploinsufficiency does not impair the phosphorylation of the main myofilament targets of PKA.
3.3.10 Steady-state Force and Dynamic Cross-bridge Kinetics Prior to and
Following PKA Treatment
Skinned myocardium was isolated from WT and MyBP-C+/− hearts to determine steady-state force-pCa relationships and for measurements of cross-bridge kinetics using a slack-restretch maneuver and the dynamic response to acute stretch. The minimum and
126 maximum force generation in pCa 9.0 and 4.5 solutions, respectively, were not different between groups (Table 3-4). pCa50 and nH were also not different between groups (Table
3-4). However, incubation of skinned myocardium isolated from MyBP-C+/− hearts in pCa solutions that produced < 15% of maximal force (pCa 6.1 and pCa 6.0) resulted in greater force production compared with WT skinned myocardium (Table 3-4). Following
PKA treatment both WT and MyBP-C+/− skinned myocardium displayed a similar decrease in pCa50 (Table 3-4), without a change in maximal and minimal steady-state force, or nH (Table 3-4).
Table 3-4. Steady-state Mechanical Properties of Skinned Fibers Isolated from WT and MyBP-C+/− Myocardium.
Fmin Fsub Fmax pCa50 nH 2 2 (mN/mm ) (P/Po) (mN/mm ) WT (-PKA) 0.54±0.38 21.89±1.51 5.81±0.02 4.41±0.59 pCa 6.1 0.04±0.01 pCa 6.0 0.08±0.01 WT (+PKA) 0.45±0.39 20.57±1.42 5.70±0.02† 4.24±0.62
MyBPC+/− (-PKA) 0.73±0.29 20.67±1.94 5.83±0.03 4.08±0.64 pCa 6.1 0.08±0.01* pCa 6.0 0.13±0.01* MyBPC+/− (+PKA) 0.57±0.26 19.83±1.79 5.73±0.03† 3.97±0.53
2+ Data are expressed as the means ± SE. Fmin: Ca -independent force at pCa 9.0; Fsub: 2+ 2+ submaximal Ca -activated force at pCa 6.1 and 6.0; Fmax: maximal Ca -activated force at pCa 4.5; pCa50: pCa required for half-maximal force generation; nH: Hill coefficient for total force-pCa relationship. n = 12-20. * P < 0.05 compared to WT, † P < 0.05 compared to untreated (-PKA).
Cross-bridge kinetics measurements in skinned WT and MyBP-C+/− myocardium were performed in pCa solutions that yielded similar isometric forces (P/Po = ~ 0.10 and
0.25) to account for slightly increased Ca2+ sensitivity of force in MyBP-C+/− fibers.
127 +/− Skinned myocardium isolated from MyBP-C hearts exhibited accelerated ktr compared
+/− to WT (Table 3-5). A representative ktr trace recorded from WT and MyBP-C skinned myocardium is presented in Fig. 3-8A. Following PKA treatment both WT and MyBP-
+/− C skinned myocardium displayed accelerated ktr, however, the relative increase was greater in WT myocardium such that basal differences in ktr were no longer apparent following PKA treatment (Table 3-5).
+/− Stretch-activation experiments revealed that kdf was accelerated in MyBP-C skinned myocardium compared to WT, but krel was not different between groups (Table
3-5). The net delayed recruitment of cross-bridges following stretch (P3) was similar between groups at both activation levels, however, the greater decline in the amplitudes
+/− of P2 following stretch in MyBP-C skinned myocardium increased the overall amplitude of the phase 3 force transient compared to WT, as indicated by a larger
+/− increase in Pdf for MyBP-C (Table 3-5). Similar to ktr measurements, PKA treatment of
WT and MyBP-C+/− skinned myocardium resulted in accelerated cross-bridge kinetics indicated by increases in kdf and krel, and there were no differences in kdf between groups following PKA treatment (Table 3-5). Consistent with previous studies (53, 57), PKA treatment significantly decreased the P2 amplitude, thereby resulting in an increase in Pdf for both groups (Table 3-5).
128 - < < 0.05 P † † † † * * * df 20. - P : the rate constant 0.13±0.01 0.09±0.01 rel 0.22±0.01 0.18±0.01 0.21±0.02 0.17±0.02 0.12±0.01 0.16±0.01 k n = 12 : the rate constant of force 3 tr P k 0.12±0.01 0.18±0.02 0.14±0.02 0.16±0.01 0.12±0.01 0.18±0.02 0.14±0.02 0.16±0.01 † † † † * * yocardium. M 2 − P +/ C - 0.03±0.01 0.03±0.01 : maximal force; 0.03±0.02 0.03±0.02 0.04±0.02 0.04±0.02 0.00±0.01 0.01±0.01 o - - - - state state force to the minimum force value at the end of - † † † † state force to the peak value of delayed force of stretch ) - 1 - rel ec k (s 204±22 193±21 241±26 230±24 253±17 247±15 287±18 269±16 stretch stretch steady activated activated force; P - - † † † † * * 2+ stretch steady ) - 1 - df ec k (s solated from WT WT and from MyBP solated I 2.48±0.24 4.96±0.29 PKA). 4.97±0.51 9.02±0.67 5.69±0.54 4.01±0.29 7.37±0.38 10.11±0.79 - between between pre between between pre ibers † † † † * * . The comparisons were made at equivalent levels of activation. : the rate constant of force development during stretch activation; 2 F submaximal Ca ) df 1 k - : the tr 2 P: k ec and and P : the 8±0.33 (s 3 3 kinned P 1.99±0.16 3.59±0.20 3.6 5.90±0.52 4.13±0.35 6.55±0.56 3.05±0.18 4.88±0.23 S ) eans ± SE. between o inetics of inetics K Force (P/P restretch restretch maneuver; - 0.13±0.04 0.25±0.04 0.11±0.02 0.24±0.03 0.15±0.04 0.28±0.04 0.09±0.02 0.22±0.03 : the < 0.05 compared to untreated 0.05 ( < compared df P ng ng stretch activation; P activation protocol; P − − bridge † - - +/ +/ C C - - WT WT PKA) PKA) - - . Cross ( ( (+PKA) (+PKA) 5 - MyBP MyBP 3 phase phase 2 of stretch activation protocol; P to WT, compared Data Data are expressed as the m development after slack of force decay duri Table
129 Time (sec)
Figure 3-8. Rate of Force Development and Fiber Stiffness. A: Representative force traces following a slack and release-restretch protocol showing accelerated rates of force development in MyBP-C+/− skinned myocardium. In this example fibers were activated in solutions containing sufficient Ca2+ to yield in ~ 25% of maximal force. B: The stiffness, as the slope of stretch-tension relationship. The tension was measured following achievement of steady state force at ~half-maximal activation level by application of a series of stretch increases in muscle length (0.25-2.5% of initial muscle length), completed in 0.2 msec. The amplitude of the force transient following stretch (P1) was recorded, and peak force was plotted against the step length. n = 20. The stiffness was significantly (P < 0.05) decreased in MyBP-C+/− fibers compared to WT.
Stepwise rapid stretches of increasing L (0.25%-2.5%) were imposed on skinned myocardium isolated from WT and MyBP-C+/− hearts at steady-state activations of ~
130 50% of maximal activation. The peak amplitude of the force transient following stretch normalized to pre-stretch isometric force (P1 amplitude) was plotted against stretch length
(L) as an estimate of muscle fiber stiffness. The amplitude of P1 was significantly smaller for MyBP-C+/− skinned myocardium at stretch lengths greater than 0.25% of muscle length, and the slope of the relationship between stretch (L) and tension (P1 amplitude) was shallower, indicating a decrease in cross-bridge stiffness (Fig. 3-8B).
3.3.11 Isolated Myocyte Contractile Properties
To evaluate the in vitro contractile properties, we assessed sarcomere shortening in intact ventricular cardiomyocytes isolated from WT and MyBP-C+/− hearts.
Representative tracings of sarcomere shortening are shown in Fig. 3-9A. Diastolic sarcomere length was not different between groups (Fig. 3-9B), however, the % of sarcomere shortening (normalized to diastolic sarcomere length) was depressed in MyBP-
C+/− myocytes compared to WT myocytes (Fig. 3-9C). The rate of sarcomere shortening,
−dL/dt, was also depressed in MyBP-C+/− myocytes compared to WT myocytes (Fig. 3-
9D), but the rate of sarcomere relengthening, +dL/dt, was not different between groups
(WT: 1.21 ± 0.08 m/sec, MyBP-C+/−: 1.16 ± 0.11 m/sec). The time to peak shortening was shorter in MyBP-C+/− myocytes compared to WT myocytes (Fig. 3-9E), however, the time to 50% relaxation (WT: 0.16 ± 0.01 sec, MyBP-C+/−: 0.14 ± 0.01 sec), time to 90% relaxation (WT: 0.23 ± 0.01 sec, MyBP-C+/−: 0.22 ± 0.01 sec), and the relaxation time constant, (Fig. 3-9F), were not different between groups.
131 Figure 3-9. Ventricular Cardiomyocyte Sarcomere Shortening between WT and MyBP-C+/− Models. A: Representative tracings of sarcomere shortening at 2 Hz with 1.8 mM Ca2+ averaged from 8 peaks from the same cell from each group. Diastolic sarcomere length (B), % of sarcomere shortening (C), the rate of sarcomere shortening (−dL/dt) (D), time to peak (E), and the relaxation time constant (F) were measured on fresh isolated intact ventricular cardiomyocytes at 2 Hz with 1.8 mM Ca2+. n = 55-69. * P < 0.05 compared to WT.
3.3.12 Myocyte Ca2+ Handling Properties
Since myofilament function and SR Ca2+ handling can influence and regulate each other, in vitro Ca2+ handling properties were evaluated by measuring the amplitude and kinetics of Ca2+ transients recorded from isolated intact ventricular cardiomyocyte loaded with indo-1. The representative superimposed Ca2+ transient tracings are shown in
Fig. 3-10A. There were no differences between groups in BL Ca2+ levels [(in F405/485 ratio) WT: 1.07 ± 0.06, MyBP-C+/−: 1.22 ± 0.08], Ca2+ transient amplitude as % BL (Fig.
3-10B), time to peak Ca2+ (Fig. 3-10C), time to 50% Ca2+ decay (WT: 150 ± 3 msec,
132 MyBP-C+/−: 147 ± 3 msec), time to 90% Ca2+ decay (WT: 349 ± 4 msec, MyBP-C+/−: 349
± 4 msec), or (Fig. 3-10D).
Figure 3-10. Ventricular Cardiomyocyte Ca2+ Transients between WT and MyBP- C+/− Models. A: Representative superimposed Ca2+ transient tracings at 2 Hz with 1.8 mM Ca2+ averaged from 8 peaks from the same cell from each group. Ca2+ transient amplitude (normalized to BL) (B), time to peak (C), and the relaxation time constant (D) were measured on fresh isolated intact ventricular cardiomyocytes loaded with indo-1 at 2 Hz with 1.8 mM Ca2+. n = 50-68.
3.3.13 Expression and Phosphorylation of Ca2+ Handling Proteins
To determine if differences in myocyte contractile function were related to Ca2+ handling properties, the expression and phosphorylation of several key Ca2+ handling proteins were quantified by Western blot analysis. The expression of Ca2+ handling proteins (all normalized to HSC70) examined in this study were not different between groups, which is consistent with a lack of difference between groups in Ca2+ transient
133 measurements. This includes; RyR2, the Ca2+ releasing channel, (WT: 0.68 ± 0.10,
MyBP-C+/−: 0.60 ± 0.08); SERCA2, the primary protein responsible for sequestering Ca2+ from the cytosol back to the SR, (WT: 1.16 ± 0.60, MyBP-C+/−: 1.07 ± 0.55); PLB, the inhibitor of SERCA2, (WT: 1.86 ± 0.11, MyBP-C+/−: 1.96 ± 0.11), the SERCA2 to PLB ratio (both normalized to HSC70, WT: 0.66 ± 0.35, MyBP-C+/−: 0.59 ± 0.30); NCX1, which contributes to extrusion of Ca2+ into the extracellular space, (WT: 0.27 ± 0.04,
MyBP-C+/−: 0.35 ± 0.05); and pPLB17 (normalized to PLB, WT: 1.63 ± 0.21, MyBP-
C+/−: 1.10 ± 0.31). pPLB16 to PLB ratio was decreased in MyBP-C+/− hearts compared to
WT hearts (WT: 0.78 ± 0.09, MyBP-C+/−: 0.49 ± 0.05, P < 0.05), which would be expected to reduce the inhibition on the activity of SERCA2, however, by itself did not induce a change in Ca2+ transient properties. PP1 protein expression (normalized to
HSC70, WT: 0.61 ± 0.12, MyBP-C+/−: 0.56 ± 0.11) and activity (in AU, WT: 634 ± 43,
MyBP-C+/−: 595 ± 34) were not different between groups, thus cannot account for the decreased pPLB16.
3.3.14 In Vivo Hemodynamic Function
In vivo LV hemodynamic function was assessed in WT and MyBP-C+/− hearts by pressure-volume catheterization at BL and following DOB infusion. The HR was not different between groups (Table 3-6). The end-systolic pressure was not different between groups, however, end-diastolic pressure (EDP) was elevated in MyBP-C+/− hearts at BL (Table 3-6), which could be indicative of diastolic dysfunction. EF was not different between groups, however, there was evidence for systolic dysfunction in MyBP-
+/− C hearts indicated by a decrease in dP/dtmax compared to WT hearts at BL (Table 3-6).
134 Additionally, differences in dP/dtmax between groups were no longer apparent after DOB treatment (Table 3-6). was not different between groups at BL or after DOB treatment
(Table 3-6).
Table 3-6. LV Hemodynamic Function at Baseline and Following -adrenergic Stimulation between WT and MyBP-C+/− Models.
WT MyBP-C+/− BL DOB BL DOB HR (bpm) 410±16 441±16 404±5 442±12 ESP (mmHg) 94.4±4.1 80.2±11.0 93.9±5.2 79.5±8.7 EDP (mmHg) 5.01±0.53 6.04±1.83 7.58±1.08* 4.75±0.81* EF (%) 75.0±4.9 90.2±3.2 65.0±6.9 91.0±4.1
dP/dtmax (mmHg/sec) 7964±273 11072±961 6314±643* 9899±1067 (msec) 9.50±0.87 8.15±0.56 10.6±0.7 9.70±0.99
Data are expressed as the means ± SE. BL: baseline; DOB: dobutamine response at 5 min post injection; bpm: beats per minute; HR: heart rate; ESP: end-systolic pressure; EDP: end-diastolic pressure; EF: ejection fraction; dP/dtmax: peak rate of pressure rise; : relaxation time constant. n = 9. * P < 0.05 compared to WT.
3.3.15 Echocardiography
We also investigated the degree of chamber remodeling and in vivo LV function of the hearts using echocardiography. BW, LV mass to BW ratio, PWTd, ESD, and EDD were all comparable between groups (Table 3-7), indicating that MyBP-C+/− hearts did not display evidence of hypertrophy. Consistent with previous reports (48, 124), we did not observe any functional differences between groups, including EF, FS, ET, and IVRT
(Table 3-7).
135 Table 3-7. LV Morphology and in Vivo Function Measured by Echocardiography between WT and MyBP-C+/− Models.
WT MyBP-C+/− BW (g) 31.1±1.2 30.5±0.8 LV Mass/BW 3.95±0.21 3.84±0.24 PWTd (mm) 0.86±0.03 0.86±0.03 ESD (mm) 2.13±0.20 2.26±0.22 EDD (mm) 3.78±0.07 3.77±0.13 HR (bpm) 427±17 412±1 EF (%) 80.7±4.4 77.2±3.9 FS 0.44±0.04 0.41±0.04 ET (msec) 60.9±2.6 65.1±2.0 IVRT (msec) 22.9±2.2 20.1±2.4
Data are expressed as the means ± SE. BW: body weight; PWTd: posterior wall thickness in diastole; ESD: end systolic dimension; EDD: end diastolic dimension; HR: heart rate; EF: ejection fraction; FS: fractional shortening; ET: ejection time; IVRT: isovolumic relaxation time. n = 7-10.
3.3.16 Histological Assessment of Cardiac Morphology
To provide detailed information on morphology, we performed histological staining studies on cross sections isolated from WT and MyBP-C+/− hearts and the representative images are presented in Fig. 3-11. Masson’s trichrome-stained cross sections at mid-LV level revealed similar LV geometry in both groups, with no obvious chamber remodeling or hypertrophy (Fig. 3-11A). Also, there was no overt fibrosis in either group as very little blue staining was detectable (Fig. 3-11A). Additionally, H&E staining showed no appreciable differences in cardiomyocyte morphology (Fig. 3-11B), as no cellular hypertrophy or disarray was evident in either group.
136 Figure 3-11. Morphology of the Hearts and Cardiomyocytes. A: Representative cross sections of WT and MyBP-C+/− hearts at the mid-LV level, stained with Masson’s trichrome (10X magnification). The viable myocardium is in red and fibrotic area in blue (insignificant). B: Representative magnified cross sections at mid-LV level of WT and MyBP-C+/− hearts stained with H&E (40X magnification). The cardiomyocytes are in pink and their nuclei in blue.
3.3.17 ECG
To further explore the in vivo mechanisms of MyBP-C deficiency in the heart, we performed continuous ECG recordings in conscious animals using surgically implanted radio telemetry devices. The representative ECG waveforms of WT and MyBP-C+/− mice are shown in Fig. 3-12. The RR interval, P wave duration, and PR interval were not
137 different between groups, however, the QRS duration, QT interval, and QTc interval were all prolonged in MyBP-C+/− mice compared to WT mice (Table 3-8).
Figure 3-12. ECG waveforms. Representative ECG waveforms of conscious animals at BL using telemetry implants. The intervals of QRS and QT are indicated with the dotted lines.
Table 3-8. Electrocardiographic Data Acquired by Radio Telemetry between WT and MyBP-C+/− Models.
WT MyBP-C+/− RR interval (msec) 131±4 135±4 P wave duration (msec) 17.5±0.6 18.3±0.3 PR interval (msec) 39.5±1.0 39.0±0.8 QRS duration (msec) 27.3±1.1 30.6±0.8* QT interval (msec) 45.1±1.8 50.4±1.1* QTc interval (msec) 39.2±1.4 44.3±0.9*
Data are expressed as the means ± SE. QTc: HR adjusted QT interval using the Bazett formula: QTc = QT/(RR/100)1/2. n = 8-11. * P < 0.05 compared to WT.
138 Table 3-9. Comparisons between MyBP-C−/− and MyBP-C+/− Models.
MyBP-C−/− MyBP-C+/− Myofilament protein Expression of MyBP-C None ↓ Phosphorylation of MyBP-C None ↓ Myofilament function (322) Fsub ↑ ↑ (322, 324) pCa50 and nH ↔ ↔ (321, 322, 324) ktr and kdf at BL ↑ ↑ (321, 324) krel at BL ↑ ↔ (321, 324) P2 at BL ↓ ↓ (324) ktr and kdf with PKA ↔ ↔ (324) krel with PKA ↔ ↔ (324) P2 with PKA ↔ ↔ Stiffness NA ↓ Myocyte sarcomere shortening Sarcomere length ↓ ↔ % sarcomere shortening ↓ ↓ −dL/dt ↓ ↓ Time to peak ↓ ↓ Time to 50% relaxation ↓ ↔ Myocyte Ca2+ transient measurements Ca2+ transients ↓ ↔ Time to peak ↔ ↔ Time to 50% relaxation & ↓ ↔ Ca2+ handling proteins SERCA2/PLB ↑ ↔ pPLB16/PLB ↓ ↓ pPLB17/PLB ↓ ↔ PP1 expression & activity ↑ ↔ In Vivo hemodynamic function
EDP or Pmin ↑ ↑
139 EF at BL ↓ ↔
dP/dtmax at BL ↔ ↓ at BL ↑ ↔ Contractile reserve ↓ ↔ Echocardiography LV hypertrophy & chamber remodeling yes no EF & FS ↓ ↔ ET ↓ ↔ IVRT ↑ ↔ Optical mapping measurements Conduction velocity ↓ ↔* APD ↑ ↔* Cx43 expression ↔ ↔* Cx43 phosphorylation ↓ ↔* ECG measurements QRS duration ↔ ↑ QT interval ↑ ↑
All measurements were compared to WT. Refer to Fig. 3-2 to 3-12, Table 3-1 to 3-8, and text for details. Data for myofilament function of MyBP-C−/− model were taken from previous studies, and the references are listed in parenthesis. NA: data not available, * data not shown.
3.4 DISCUSSION
The majority of FHC mutations in MyBP-C are predicted to result in MyBP-C haploinsufficiency, however, the link between decreased MyBP-C expression and the development of cardiac dysfunction is not clear. The aim of this study was to examine the in vivo and in vitro functional consequences of decreased MyBP-C expression in MyBP-
C−/− and MyBP-C+/− hearts on myofilament behavior and post translational modifications, and regulatory mechanisms downstream from the myofilament, specifically Ca2+ homeostasis and the electrical activity in the heart. A list of comparisons between WT vs.
140 MyBP-C−/− and WT vs. MyBP-C+/− are summarized in Table 3-9. We found that MyBP-
C deficiency produced altered function at the molecular and whole organ level, which was generally more severe in hearts that were completely devoid of MyBP-C. Complete lack of MyBP-C resulted in reduced sarcomere shortening, −dL/dt, and time to peak shortening in intact myocytes which were accompanied by altered Ca2+ transient kinetics.
Ex vivo optical mapping studies revealed slowed electrical conduction velocity in MyBP-
C−/− hearts which was related to decreased Cx43 phosphorylation, and in vivo ECG measurements in conscious animals also revealed prolonged QT intervals in these animals. Molecular and cellular defects in MyBP-C−/− hearts manifested as severe dysfunction at BL and reduced contractile reserve during stress in in vivo hemodynamic and echocardiographic measurements, as well as profound LV hypertrophy. On the other hand, reduced expression and phosphorylation of MyBP-C in MyBP-C+/− hearts accelerated rates of cross-bridge attachment at low Ca2+ and reduced cross-bridge stiffness in skinned myocardium. Altered myofilament function in turn contributed to reduced sarcomere shortening, –dL/dt, and time-to-peak shortening in intact myocytes isolated from MyBP-C+/− hearts without any alterations in Ca2+ transient kinetics. In vivo hemodynamic systolic and diastolic function in MyBP-C+/− hearts were also impaired as evidenced by decreased dP/dtmax and elevated EDP, respectively, but in the absence of
LV chamber remodeling and hypertrophy. Furthermore, basal differences in cross-bridge behavior and in vivo contractile function between WT and MyBP-C+/− mice were largely diminished following β-adrenergic stimulation by PKA and DOB, respectively. ECG measurements in conscious animals also revealed prolonged QRS, QT, and QTc durations in MyBP-C+/− animals. Collectively, these data demonstrate that complete lack
141 of MyBP-C in the sarcomere result in adaptations in Ca2+ homeostasis and electromechanical coupling, which would be expected to contribute to disease progression in MyBP-C related FHC, and in MyBP-C+/− mouse model which closely mimics the loss of MyBP-C expression in the sarcomere as human patients carrying
MyBP-C mutations, the primary consequence which contributes to impaired in vivo mechanical and electrical function in the heart is abnormal myofilament contractile function. Altered ECG in both models may also predispose them to an increased risk of arrhythmia and SCD, especially during stress.
3.4.1 In Vitro Contractile Function and Ca2+ Handling in MyBP-C−/− Myocytes
Ventricular myocytes isolated from MyBP-C−/− hearts displayed reduced sarcomere length, which can be attributed to the absence of an MyBP-C-induced inhibition on myosin heads (260) which increases their proximity to actin thereby enhances the probability of actomyosin interactions (64). Interestingly, MyBP-C−/− myocytes also displayed impaired sarcomere shortening as evidenced by a decrease in sarcomere shortening (expressed as % from diastolic sarcomere length) and faster time to peak shortening. MyBP-C and its phosphorylation have been shown to be important for maintaining mechanical rigidity of the myofilament lattice and longitudinal stiffness, and prolonging the lifetime of cross-bridge binding (242, 252), thus, the lack of MyBP-C expression and phosphorylation could have contributed to reduced sarcomere shortening in MyBP-C−/− myocytes. A reduction in the time cross-bridges are in strongly-bound states would be expected to result in accelerated cross-bridge turnover to maintain force,
142 and this is consistent with faster time to peak shortening in MyBP-C−/− myocytes and accelerated cross-bridge kinetics recorded in skinned myocardium (82, 324).
Impairments in cardiac contractility can be mediated by disruptions in Ca2+ cycling at the SR, which modulates the availability of Ca2+ to the myofilaments, and thereby force generation. Reduced peak Ca2+ transient amplitude might contribute to in vitro contractile dysfunction in MyBP-C−/− myocytes. Furthermore, their time to 50%
Ca2+ decay and were shortened, which were likely caused by elevated SERCA2 expression, thereby facilitating faster Ca2+ clearance from the cytoplasm into the SR.
Faster Ca2+ transient decay was also observed in myocytes isolated from a knock-in mouse model expressing an FHC missense mutation in MyBP-C that results in decreased
MyBP-C expression and a large increase in Ca2+ sensitivity, and was hypothesized to be a compensatory mechanism to improve diastolic relaxation in the hypercontractile MyBP-
C mutant knock-in hearts (97). However, in the case of the MyBP-C−/− hearts, faster Ca2+ clearing from the SR, which may also be an attempt to accelerate myofilament relaxation in the hypercontractile heart, does not result in accelerated LV relaxation in vivo, likely because the presence of significant LV hypertrophy and fibrosis in MyBP-C hearts markedly reduces diastolic strain and torsion rates (82), which are critical mechanical indices of diastolic function.
It is noteworthy that the phosphorylation of PLB at Ser16 and Ser17 were both significantly reduced in MyBP-C−/− hearts, suggesting downregulated -adrenergic signaling cascade. Consistent with our observations here, a recent study has shown that phosphorylation of TnI and PLB, which are myofilament and Ca2+ handling substrates for
PKA and CaMKII, were both reduced in mouse hearts expressing a TnT FHC mutation
143 that causes myofilament dysfunction (110). Interestingly, both expression and activity of
PP1 were elevated in MyBP-C−/− hearts, counteracting the phosphorylation of PLB.
Upregulated PP1 has been reported in a wide array of cardiac diseases including HF and idiopathic cardiomyopathy and contributing to contractile dysfunction due to its ability to dephosphorylate key myofilament and Ca2+ handling proteins (238), although its role in
MyBP-C−/− hearts remains unclear.
3.4.2 In Vivo Contractile and Hemodynamic Function in MyBP-C−/− Hearts
Consistent with previous studies employing echocardiography to measure cardiac contractility and morphology (124), MyBP-C−/− hearts exhibited severe hypertrophy and enlarged LV chamber size, which could be due to its defective myofilament function since it enhances the vulnerability to the remodeling cascades (370). Similarly, MyBP-
C−/− hearts displayed systolic and diastolic dysfunction evident by depressed EF %, FS, shortened ET, and prolonged IVRT. Hemodynamic assessment by direct LV cannulation
−/− of MyBP-C hearts revealed maintained dP/dtmax compared to WT hearts which has been hypothesized to be due to accelerated rates of cross-bridge formation resulting in a rapid initial pressure rise during systole (250). In contrast, MyBP-C−/− hearts displayed elevated Pmin, and prolonged which are indicative of decreased LV compliance and depressed diastolic relaxation, respectively.
Hemodynamic LV dysfunction in MyBP-C−/− hearts was exacerbated by DOB infusion as evidenced by an inability to maintain Pmax, decreased dP/dtmax, and a blunted increase in EF% compared to WT hearts. Collectively, these data demonstrate that
MyBP-C−/− hearts have a significantly diminished contractile reserve in response to β-
144 adrenergic stimulation and are consistent with previous studies showing that MyBP-C and its phosphorylation are critical for modulation of hemodynamic function in response to increased cardiac workload (235, 280, 281, 342).
3.4.3 Electrical Conduction and ECG Recordings in MyBP-C−/− Hearts
In addition to the contractile dysfunction, decreased MyBP-C expression in
MyBP-C−/− hearts displayed evidence for impaired electrical activity. Ex vivo optical mapping studies revealed that conduction velocity in the transverse direction was significantly slower, and there was a trend towards increased anisotropic ratio in MyBP-
C−/− hearts compared to WT hearts. Slower conduction velocity was accompanied by prolonged APD in MyBP-C−/− hearts. In mice ventricular myocytes, there are small conductance Ca2+-activated K+ channels, which can contribute to longer APD if less
2+ activated, such as with low ICa or [Ca ]i (344). Although ICa is currently unknown, it is possible that faster Ca2+ clearing in MyBP-C−/− ventricular myocytes prolongs APD because of less K+ currents. Decreased conduction velocity is a major contributing factor to decreased contractile function seen in many cardiac diseases (241, 296), and slowed conduction velocity in MyBP-C−/− hearts can be due to increased LV hypertrophy and fibrosis (82, 124), and myocardial disarray and abnormal myofiber architecture (48, 124,
360). However, previous studies show that the degree of hypertrophy or histopathology in the heart do not necessarily correlate with defects in electrical activity in the heart (33,
354, 370), suggesting that additional factors can contribute to slowed conduction in
MyBP-C−/− hearts. In this regard, maintenance of normal propagation patterns of electrophysiological signals between myocytes is also critically dependent on
145 intracellular coupling of gap junctions, which in the LV, are primarily comprised of
Cx43. Decreased expression of Cx43 can be associated with decreased conduction velocity in myocardium [reviewed by (296)]. It is also possible that decreased Cx43 phosphorylation (23, 310) and Cx43 lateralization (moving from the intercalated disc to the lateral edges of the cardiomyocytes, often as a result of dephosphorylation) (283) can contribute to slowed electrical conduction in MyBP-C−/− hearts, and accordingly we observed significant reductions in Cx43 phosphorylation levels in MyBP-C−/− hearts.
ECG recording in conscious ambulatory animals also revealed abnormalities in electrical activity in MyBP-C−/− hearts as evidenced by prolonged QT intervals, which is consistent with results from our optical mapping studies which demonstrate slowed conduction velocity and from human studies showing prolonged QT intervals in patients carrying
MyBP-C mutations (155). Together with LV hypertrophy, fibrosis, and myocyte disarray, these factors may predispose MyBP-C deficient hearts to re-entry arrhythmias.
3.4.4 Myofilament Contractile Function in MyBP-C+/− Hearts
We have previously shown that hearts of young MyBP-C+/− mice (i.e., 8-10 weeks of age) display impairments in in vivo mechanical function, which is related to altered myofilament function (82). In this study, we observed a significant increase in ktr and kdf at low levels of Ca2+-activation in MyBP-C+/− skinned myocardium, the latter being consistent with results from our previous study (82). Assuming that ktr is the sum of the f and g rate constants of the transitions between force-generating and non-force generating
+/− cross-bridge states, the lack of acceleration in krel in MyBP-C skinned myocardium suggests that accelerated ktr was mostly due to accelerated cross-bridge recruitment and
146 transitions to force-generating states (i.e., greater f). It has previously been shown that complete ablation of MyBP-C in the sarcomere of MyBP-C−/− mice radially displaces the heads of myosin cross-bridges closer to actin and increases the probability of actomyosin interactions, thereby accelerating rates of cross-bridge recruitment and force generation
(65). Force generation at low Ca2+-activation is highly cooperative in that strong binding of cross-bridges to actin can promote further cross-bridge recruitment within the thin filament. At low levels of Ca2+-activation cooperative cross-bridge recruitment slows the rate of force development as strongly-bound cross-bridges gradually recruit additional cross-bridges into strongly-bound force-generating states. It is possible that although the decrease in MyBP-C expression in the MyBP-C+/− sarcomere is substantially smaller than the MyBP-C−/− ones, it may be sufficient to enhance the probability of actomyosin interactions in regions of the sarcomere where MyBP-C is absent (82, 338) by accelerating rates of cross-bridge binding throughout the sarcomere, thereby reducing the time course of cooperative cross-bridge recruitment and accelerating force development.
At higher levels of Ca2+-activation, force generation becomes less dependent on cross- bridge induced cooperative recruitment of cross-bridges as increased binding of Ca2+ to
TnC would recruit the majority of available cross-bridges to interact with open actin sites.
Cross-bridge kinetics were not different between WT and MyBP-C+/− skinned myocardium at Ca2+-activations yielding forces greater than ~30% of maximal (data not shown), suggesting that decreased MyBP-C expression accelerates cooperative recruitment of cross-bridges.
Accelerated rates of cross-bridge kinetics at low Ca2+-activation in MyBP-C+/− skinned myocardium were also accompanied by an increase in steady-state force
147 generation. An increase in steady state force at low Ca2+-activations was also previously noted in MyBP-C−/− skinned myocardium (322), which can be attributed to increased actomyosin interactions due to the closer juxtaposition of actin and myosin in the absence of MyBP-C (65). Thus, it is possible that a decrease in MyBP-C content in MyBP-C+/− skinned myocardium may also contribute to increased force generation by a similar mechanism as MyBP-C−/− skinned myocardium. Moreover, because we observed an
+/− increase in kdf but not krel in MyBP-C myocardium, it is possible that increased force generation at low Ca2+-activations may reflect an increase in the number of cross-bridges bound at a given time without a decrease in the amount of time cross-bridges are in strongly-bound states (i.e., no change in rates of cross-bridge detachment). Furthermore, because MyBP-C phosphorylation has been proposed to decrease force generation at submaximal Ca2+-activations (56) presumably by decreasing the time that cross-bridges are in strongly-bound states (67), the overall decrease in MyBP-C phosphorylation in
MyBP-C+/− hearts could also have contributed to increased cross-bridge dwell time, and thereby force generation.
Basal differences in cross-bridge kinetics between WT and MyBP-C+/− skinned myocardium were abolished following treatment with PKA, as both groups displayed accelerations in ktr and stretch activation kinetics. The overall response to PKA was smaller in MyBP-C+/− compared to WT skinned myocardium due to a reduction in the amount of MyBP-C in the sarcomere available to be phosphorylated. However, the data suggest that reduced MyBP-C expression in MyBP-C+/− myocardium does not impair the ability to accelerate cross-bridge kinetics in response to PKA phosphorylation. The decrease in Ca2+-sensitivity following PKA phosphorylation was also similar in WT and
148 MyBP-C+/− skinned myocardium suggesting that decreased MyBP-C expression also does not impair PKA-dependent TnI phosphorylation.
The presence of MyBP-C in the sarcomere and its phosphorylation have been shown to be important for maintaining mechanical rigidity of the myofilament lattice which is an important determinant of the properties of cross-bridge binding to actin (242,
252), and a decrease in both MyBP-C content and MyBP-C phosphorylation in the sarcomeres have been shown to significantly reduce radial and longitudinal lattice stiffness (252). Consistent with this notion, we found that skinned myocardium isolated from MyBP-C+/− hearts displayed reduced cross-bridge stiffness compared to WT skinned myocardium when subjected to step-wise acute stretches at half-maximal Ca2+- activations. Furthermore, we observed a greater decline in the minimum amplitude of the
+/− 2+ force transient (P2) following stretch in MyBP-C skinned myocardium at low Ca - activations, corresponding to greater cross-bridge detachment in response to stretch, which may suggest increased cross-bridge compliance (321, 324). The precise mechanisms for decreased cross-bridge stiffness in MyBP-C+/− myocardium in response to acute stretch are unclear, but are unlikely to be due to differences in the number of strongly-bound cross-bridges prior to stretch because steady-state force at sub-maximal activation was adjusted to be similar between WT and MyBP-C+/− skinned myocardium.
Rather, it is possible that individual MyBP-C+/− cross-bridges are inherently more compliant and detach more easily when subjected to significant strain such as acute stretch. It has been proposed that MyBP-C normally provides a significant elastic and viscous load which is important for storing elastic recoil energy to facilitate subsequent relaxation function (252). Thus, a decrease in cross-bridge stiffness resulting in more
149 compliant myofilaments in MyBP-C+/− hearts may impair LV mechanical relaxation in vivo, as we have previously observed (82).
A few studies have examined the mechanical properties of skinned myocardium isolated from human hearts expressing MyBP-C mutations (133, 349, 350). These investigations have consistently shown decreased MyBP-C expression, MyBP-C phosphorylation, and TnI phosphorylation, which together result in large deficits in maximal force generation and increases in submaximal force generation (when expressed relative to maximal force). Although the decrease in MyBP-C expression in the hearts of the MyBP-C+/− mice studied here (32%) mirrors the loss of MyBP-C in myectomy samples from hearts of human patients expressing MyBP-C truncation mutations (33%)
(349, 350), we did not observe any changes in maximal force generation between MyBP-
C+/− and WT skinned myocardium. Furthermore, the increases in submaximal force generation in MyBP-C+/− skinned myocardium were small and limited to very low activation levels (< 15% of maximal). Altered steady-state force generation in skinned myocardium isolated from human MyBP-C truncation mutation carriers might be related to large decreases in TnI phosphorylation (349, 350). However, the finding that myectomy samples isolated from human FHC hearts that did not express MyBP-C mutations displayed similar force generation properties suggests that these changes are general features related to FHC and not decreased MyBP-C expression per se (350).
Thus, our results suggest that in the absence of LV remodeling and activation of compensatory cascades, the initial response to MyBP-C insufficiency is an acceleration in the rates of cross-bridge attachment at low Ca2+-activation and increased steady-state
150 force generation, which in conjunction with decreased MyBP-C phosphorylation result in decreased cross-bridge stiffness.
3.4.5 In Vitro Contractile Function and Ca2+ Handling in MyBP-C+/− Myocytes
Impairments in cardiac contractility can be mediated by disruptions in Ca2+ cycling at the SR, which modulates the availability of Ca2+ to the myofilaments, and thereby force generation. We investigated whether MyBP-C insufficiency in MyBP-C+/− sarcomeres results in altered Ca2+-handling properties in intact myocytes. Indeed, MyBP-
C+/− myocytes displayed abnormal in vitro contractile properties as evidenced by reduced sarcomere shortening, −dL/dt, and a reduced time to peak shortening. However, altered
MyBP-C+/− myocyte contractility was not related to changes in BL Ca2+ levels, Ca2+ release kinetics, peak Ca2+ transient amplitude, and Ca2+ transient clearance/decay. The expression and phosphorylation of Ca2+ handling proteins responsible for the individual components of Ca2+ transient measurements were not different between groups with the exception of decreased pPLB16 in MyBP-C+/− hearts. However, we did not observe differences in the rates of the Ca2+ transient decay, therefore it is unlikely that in this case, decreased pPLB16 significantly affected SERCA2 function. The increases in Ca2+- sensitivity of force generation observed here in MyBP-C+/− skinned myocardium may not have been substantial enough to invoke a compensatory response in Ca2+ clearance, suggesting that depressed MyBP-C+/− myocyte contractile behavior was due to impaired myofilament function rather than altered Ca2+-handling properties.
151 3.4.6 In Vivo Contractile and Hemodynamic Function in MyBP-C+/− Hearts
Consistent with previous investigations employing echocardiography to study contractile function in heterozygous null MyBP-C hearts (48, 124) we did not find differences in LV chamber wall dimensions or indices of cardiac function compared to
WT hearts. However, invasive pressure-volume catheterization studies revealed that despite relatively preserved EF, MyBP-C+/− hearts displayed significantly depressed dP/dtmax compared to WT hearts, indicative of systolic dysfunction. This finding differs from a previous study that found no significant changes in contractile function between
WT and MyBP-C+/− hearts at nine months of age (48). The causes for the differences between studies is unclear but may reflect differences in the genetic backgrounds of the
MyBP-C+/− animals employed, and perhaps due to the greater decrease in cardiac MyBP-
C expression in the mice used here (~32%) compared to Carrier et al. (48) (~25%), and/or
MyBP-C phosphorylation which was not reported in (48), all of which can contribute to the more pronounced contractile impairments observed here. Our finding that dP/dtmax was not different between WT and MyBP-C+/− hearts following DOB treatment suggests that decreased MyBP-C phosphorylation may have contributed to basal systolic impairment in MyBP-C+/− hearts and underscores the importance of MyBP-C phosphorylation in maintaining normal cardiac function.
Impaired systolic function in MyBP-C+/− hearts is consistent with measurements in intact myocytes showing reduced sarcomere shortening and rates of sarcomere shortening (−dL/dt). In addition to systolic dysfunction, MyBP-C+/− hearts also display evidence for diastolic dysfunction as evidenced by increased EDP compared to WT hearts. Although the increase in EDP in MyBP-C+/− hearts is modest, it could be
152 indicative of the early phases of diastolic dysfunction that could further impair systolic function. Elevated EDP can be associated with decreased LV compliance (114), which in this case could be due to enhanced cross-bridge binding and accelerated rates of force generation in skinned myocardium, and reduced time-to-peak shortening in intact myocytes at low cytosolic Ca2+ concentrations. Ultimately, this hypercontractile state prevents the myofilaments from relaxing completely, thereby impairing ventricular filling during diastole (97, 260).
Our finding here that in vivo systolic function is impaired in MyBP-C+/− hearts is consistent with previous data showing depressed systolic torsion and circumferential strain in MyBP-C+/− hearts compared to WT hearts (82). Although, accelerated rates of cooperative cross-bridge activation and force generation at low Ca2+-activations in
MyBP-C+/− skinned myocardium would be expected to enhance systolic function in vivo,
+/− MyBP-C hearts displayed depressed dP/dtmax. The precise cause for systolic dysfunction in MyBP-C+/− hearts is unclear but could be related to a disruption in the timing of fiber shortening in early and late-activated regions of the heart during the early isovolumic contraction phase due to accelerated cross-bridge kinetics at submaximal workloads. Depressed mechanical function could also be due to regional inhomogeneity in rates of force development due to variable MyBP-C expression within sarcomeres such that regions with less MyBP-C content may exhibit accelerated cross-bridge kinetics compared to regions with higher MyBP-C content, which ultimately results in uncoordinated sarcomere shortening and stretching. We found that MyBP-C expression in MyBP-C+/− hearts was more depressed in the mid-LV region compared to the apex and base. The cause of the differences in regional MyBP-C expression in MyBP-C+/− hearts is
153 unclear but a greater decrease in MyBP-C expression in the mid-LV is consistent with our previous observations in younger MyBP-C+/− mice which show that the mid-LV region displays greater mechanical dysfunction than the apex and base as evidenced by greater abnormalities in measurements of radial and circumferential strains (82).
Furthermore, LV rotational mechanics during systole were particularly depressed in the regions between the mid-LV and base in MyBP-C+/− mice, resulting in a decrease in overall LV twist which compromises systolic function and efficiency (82).
3.4.7 ECG Recordings in MyBP-C+/− Hearts
Arrhythmia induced SCD is the most common cause of death in FHC patients
(208) and can affect young and asymptomatic individuals. LV hypertrophy is considered an independent risk factor for SCD (316), however, in many cases arrhythmia and SCD occur in the absence of overt LV hypertrophy (5, 206, 207, 354, 362). In this study we recorded electrical activity in un-anaesthetized ambulatory MyBP-C+/− and WT mice, and despite a lack of LV hypertrophy, MyBP-C+/− hearts displayed abnormal ECG patterns associated with increased risk of arrhythmia and cardiac dysfunction. Specifically,
MyBP-C+/− mice had prolonged QT intervals compared to WT mice, perhaps indicative of prolonged APD and ventricular repolarization (284, 286), a feature that has also been documented in patients carrying MyBP-C mutations (155). Furthermore, we also observed a prolongation of ventricular depolarization evidenced by an increase in QRS duration in MyBP-C+/− mice compared to WT mice, which is associated with impaired ventricular conductance (284, 286). The precise causes that underlie the abnormal ECG patterns in MyBP-C+/− animals are unclear, however, enhanced myofilament Ca2+-
154 sensitivity and dys-synchronous mechanical function have been implicated as potential triggers for abnormal cardiac electrical activity (139, 170, 333), and both of these characteristics have been observed in MyBP-C+/− hearts [present study and (82)].
Regardless of the mechanisms, our ECG data suggest that MyBP-C+/− animals are at an increased risk for developing arrhythmias especially in conditions of increased cardiac stress, a known trigger for LV hypertrophy in these animals (289).
Collectively, our data show that decreased expression of MyBP-C in MyBP-C−/− hearts results in complex remodeling cascades that impact in vivo and in vitro contractile function, Ca2+ handling, electrical activity, and chamber remodeling of the heart. On the other hand, MyBP-C+/− animal provides a closer model for FHC since they posses similar decrease in MyBP-C expression and phosphorylation as FHC patients with MyBP-C mutations. Decreased MyBP-C expression in MyBP-C+/− hearts and concomitant down regulation of MyBP-C phosphorylation in the sarcomere results in altered cross-bridge function at the myofilament level and contributes to in vivo contractile dysfunction and
ECG abnormalities. Myofilament dysfunction in MyBP-C+/− hearts was not accompanied by compensatory adaptations in Ca2+ transient kinetics, and was independent of changes in LV chamber remodeling and hypertrophy, which further indicates that myofilament dysfunction is the primary insult associated with MyBP-C abnormalities and chamber remodeling is the secondary compensatory response. Because decreased MyBP-C expression is a common feature in MyBP-C mediated FHC, increasing MyBP-C expression in the sarcomeres in MyBP-C-deficient hearts may normalize cross-bridge function, thereby providing a therapeutic application to delay the emergence of FHC or reverse the pathological course of the disease (224).
155 Chapter 4
Summary and Future Directions
4.1 SUMMARY AND FUTURE DIRECTIONS OF “CHANGES IN
MYOFILAMENT PROTEINS, BUT NOT CALCIUM REGULATIONS, ARE
ASSOCIATED WITH A HIGH FAT DIET-INDUCED INPROVEMENT IN
CONTRACTILE FUNCTION IN HEART FAILURE”
We report that SAT diet feeding after the induction of mild-moderate HF/LV dysfunction can improve cellular contractility (–dL/dt), and this change was associated with decreased MHC content. Furthermore, changes in isolated ventricular myocyte systolic and diastolic function correspond with improvements in in vivo myocardial contractile function and reductions in myocyte hypertrophy without alterations in Ca2+ handling. These results suggest that the “cardio-protective” effect attributed to SAT feeding in HF may result from changes in the cardiac myocyte that lead to changes in myofilament function without affecting SR Ca2+ regulatory properties.
4.1.1 Potential Factors Contributing to MHC Changes
Although MHC utilizes less energy to produce work and has a steeper Frank-
Starling relationship compared to MHC (121), a switch from MHC to MHC in HF
156 ultimately leads to further LV dysfunction (111). The major question left unanswered in this study is how MHC isoform is impacted by SAT diet. MHC expression is known to be affected by the metabolic status of the heart. Decreased glycolytic pathway and increased FFA oxidation favor MHC expression in diabetic animals. Conversely, sensitizing insulin in diabetic animals promotes MHC expression (111). It is unclear whether this is also the case in HF, however, MHC was decreased in our HFSAT animals (58) which had insulin resistance (60) and increased FFA oxidation (32, 60).
Although SAT diet does not have a known direct effect on MHC isoform expression, it could have impacts on other factors that do, as ones listed in Table 4-1. In this regard, we had investigated endothelin 1 (ET-1) and IGF-1, both of which upregulate
MHC. ET-1 production has been shown to be mediated by leptin (7), the product of the ob gene mainly secreted by adipose tissue. Leptin increases in response to a high-fat diet in a circadian fashion and contributes to decreased food intake and increased energy expenditure, and it is chronically elevated in obesity (123, 355). Indeed, both SHSAT and
HFSAT animals had increased serum leptin (270), although HFSAT animals did not have significant weight gain compared to HFNC animals and animals in all four groups remained isocaloric during this study. It is unknown whether increased leptin would cause increases in ET-1 and whether increased ET-1 would upregulate MHC expression in pathological state such as HF. Our preliminary results showed that tissue ET-1 was not different between HFNC and HFSAT animals (data not shown), thus ET-1 might not be responsible for changes in MHCin our animal model.
157 Table 4-1. A List of Pathophysiological Stimuli Controlling mRNA and/or Protein Expression of Cardiac MHC Isoforms.
Stimuli MHC MHC Hormones and Cytokines Hyperthyroid ↑ ↓ Hypothyroid ↓ ↑ Diabetes NA ↑ Gonadectomy ↓ ↑ Basic fibroblast growth factor (FGF) ↓ ↑ Endothelin 1 (ET-1) ↑ ↑ Insulin-like growth factor 1 (IGF-1) NA ↑ Interleukin 1(IL-1) ↓ ↑/↔ Transforming growth factor (TGF) ↓ ↑ Adrenergic drive Sympathectomy ↓ NA Dobutamine (DOB) ↑ NA Isoproterenol (ISO) NA ↑ Forskolin ↑ NA Norepinephrine NA ↑ Phenylephrine ↓/↔ ↑ Metabolism High-fructose diet ↑ NA -oxidation inhibition ↑ NA Semi-starvation NA ↑ Hypoxia NA ↑ Function Exercise ↑ NA Hemodynamic/Pressure overload ↓ ↑ Chromatin remodeling Trichostatin A ↑ NA NA: data not available. [The content of this table is taken from (126) and (111).]
On the other hand, IGF-1 is produced by many tissues including the heart in response to growth hormone (GH) stimulation, and it is known to promote physiological
158 hypertrophy through activation of phosphoinositide 3 kinase (219). GH secretion can be inhibited by somatostatin, a major regulator of pituitary function, which is upregulated by nutrients including FFA along with many other factors (26, 104). Conversely, leptin negatively regulates both somatostatin (255) and GH (104), therefore it is unclear whether somatostatin level is increased in our SAT fed animals. Although it has been shown that in high-fat fed healthy animals, leptin was increased while IGF-1 was decreased compared to animals fed NC (50), our preliminary results showed that plasma
IGF-1 level was not different between groups in our HF model (data not shown).
However, the IGF-1 level in tissue remains undetermined and a candidate for the observed MHC changes. The tissue IGF-1 concentration can be assessed by commercial enzyme-linked immunoassay kits using perfused LV tissue homogenate (122).
There are many other candidates on the list that we have not investigated. Thyroid hormone (T3) is perhaps the most potent and well-understood regulator of MHC expression. When T3 binds to thyroid hormone receptors, they travel to the nucleus where they can bind to the thyroid hormone response elements in the promoter region of the effecter genes. When bound to the promoter, they upregulate MHC but downregulate MHC transcription (157). Thyroid hormone signaling is downregulated in failing hearts due to lower level of both T3 and thyroid hormone receptors (83). On the other hand, leptin stimulates thyrotropin-releasing hormone (256) which stimulates the release of thyroid-stimulating hormone (102), resulting in increased T3 production. Since our SAT fed animals had increased leptin (270), and hyperleptinemia is associated with increased circulating T3 (76, 292), it is likely that T3 is increased in our HFSAT animals compared to HFNC. T3 level in our animal model is currently unknown and an attractive
159 candidate for decreased MHC expression in HFSAT hearts. If T3 is indeed elevated in
HFSAT animals, it would also conveniently explain their improved LV function as T3 stimulates the rate and force of systolic function as well as the rate of relaxation in HF through alterations of contractile and Ca2+ handling proteins (83, 157). Serum T3 level can be measured by commercial enzyme-linked immunoassay kits, and tissue T3 level can be measured by tandem high-performance liquid chromatography (HPLC) mass spectrometry (365).
4.1.2 Other Potential Mechanisms for Improved Contractility in HFSAT Animals
Apart from the MHC isoform switch, there are other possibilities that might explain the improved contractility associated with SAT diet in HF animals. Apoptosis, also known as programmed cell death, occurs commonly in CVD and is a major contributor to deterioration of LV function especially in the transition from compensatory remodeling to HF (186). Although the apoptosis rate is very low in myocardium (0.01-
0.001% in normal hearts and 0.12-0.7% in human HF of New York Heart Association class III-IV), a 0.1% apoptosis rate is estimated to result in ~37% loss of cardiomyocytes in a year due to limited proliferation capacity of matured cardiomyocytes (203). Such a loss in contractile mass would exacerbate LV dysfunction in HF. Furthermore, apoptosis can play a causal role in HF progression and inhibiting it can cease the development of contractile dysfunction (367). The intrinsic pathway of apoptosis is mediated by mitochondria, which function can be disrupted by ROS species (307). ROS oxidizes mitochondrial pores and leads to releasing of apoptotic signaling molecules. ROS generation increases under lipotoxic condition, however, there was no alterations in H2O2
160 (a ROS species) production in our animal model with HF or SAT feeding (271). Whether there are other ROS species that are upregulated in our model is unknown. On the other hand, among the signaling pathways that promote cell survival, Akt activation plays a vital role and can be stimulated by growth factors such as IGF-1 (304). Akt activation was decreased in SHSAT and HFNC compared to SHNC but not different between
HFSAT and HFNC animals (60). Thus, it is unclear whether the level of apoptosis is altered in our animal model with HF or SAT diet. Preliminary results showed that the degree of apoptosis measured by terminal deoxynucleotidyl transferase dUTP nick end labeling assay was not different between groups (data not shown). However, a detailed examination is needed to rule out apoptosis as a potential mechanism. Several apoptotic protein markers including caspase 3, cleaved caspase 3, Bax, and Bcl-2 can be quantified using western blot (254), and the activity of calpain and caspases involved in apoptotic pathways can be measured through fluorescence-based biochemical assays (191).
Leptin signaling has been a major area of interest given its central satiety hence weight-control function in the hypothalamus and more recently, a role in regulating cardiac pathophysiology (3, 378). Obese subjects have elevated leptin which fails to decrease appetite and increase energy expenditure, a state of leptin resistance. The mechanisms for leptin resistance is thought to be saturated and/or dysfunctional leptin transport system in the blood-brain barrier and altered leptin downstream signaling including leptin-induced negative feedback (204, 378). Although both our SAT fed animals had increased serum leptin, they were not obese and their food intake remained isocaloric during the study as mentioned above. Therefore, it is not likely that their leptin levels are in the hyperleptinemia range nor they had hypothalamic leptin resistance.
161 Peripheral leptin is mainly secreted by adipocytes and increases proportionally to the volume/mass of fat tissue. However, cardiac tissue is also capable of leptin secretion, and there are leptin receptors in the heart, therefore the heart is also a site of leptin action (3,
378). In ob/ob mice which are leptin-deficient, increasing leptin within the physiological range reversed LV hypertrophy (19), improved cardiomyocyte contraction and Ca2+ homeostasis (268), restored -adrenergic responsiveness (225), and decreased apoptosis
(20). Also, after coronary artery ligation or ischemia-reperfusion in normal mice, administration of leptin has been shown to reduce infarct size (84, 309) and improve LV function (2). Furthermore, leptin increases fatty acid uptake (325), which might be beneficial if the heart is capable of increasing -oxidation in response (see below).
Therefore, although hyperleptinemia is associated with increased risk of CVD (3, 378), increasing leptin signaling within the physiological range in the disease state seems to be beneficial. However, MI and HF are associated with increased serum leptin independent of obesity in patients (221, 291) and mice (217). McGaffin KR et al. reported intact and upregulated leptin signaling in mice after MI but it did not prevent/improve LV dysfunction (217). Since the endogenous leptin level is unknown in the studies demonstrating beneficial effects of leptin after cardiac injury (2, 84, 309), it is not clear whether leptin signaling activation occurs in all cases, and whether the protective effects of leptin are concentration dependent. The current notion is that too little (leptin- deficient) or too much (hyperleptinemia) leptin both contribute to cardiac pathophysiology (3, 204, 325). In humans, leptin treatments for weight control are largely ineffective due to hypothalamic leptin resistance, however, leptin resistance is tissue specific/selective and it is debatable whether cardiac leptin resistance exists (204, 325).
162 Thus, it is unclear whether the heart can benefit from extra leptin in the disease state.
Although the serum leptin was elevated with SAT diet in our animal model, it was not elevated with HF (271). It remains unestablished whether leptin in the cardiac tissue is different and whether cardiac leptin signaling is altered in our model. The expression of leptin, leptin receptor, and phosphorylated (and hence activated) leptin receptor in LV tissue as well as its downstream signaling molecules including signal transducer and activator of transcription 3 and its phosphorylated forms can be assessed by western blot in conjunction with immunoprecipitation (187).
Interestingly, as described previously, although obesity increases the risk of CVD, increased BMI is associated with decreased mortality compared to low BMI once the disease is established. This obesity paradox is observed in patients with coronary artery disease and/or HF (1, 74, 87, 109, 113, 117, 132, 162, 163, 182, 183, 240, 245, 262), however, this association is lost if the patients also have diabetes (6) or are severely obese
(BMI ≥ 35 kg/m2) in some cases (1, 87, 117). [In addition to this U-shape association between BMI and survival rate, there are also concerns about the body fat composition/distribution, muscle mass/fitness, and water content that are masked or misrepresented by the use of BMI, malnutrition in lean patients being the true underlying cause of mortality, and potential selection bias between groups (e.g. age, smoking habit, the “healthy survivor” effect, and the severity of disease) (12, 159, 214).] There is no definitive answer for the obesity paradox so far, but it is tempting to speculate leptin signaling as the underlying cause. Intact cardiac leptin signaling might play a positive role in HF up to the point where leptin resistance is apparent and/or the detrimental effects of diabetes and obesity weigh in.
163 In keeping with the obesity paradox, there is increasing evidence suggesting that elevated FFA in the setting of cardiac injury could be beneficial. Carbohydrate oxidation uses less oxygen to produce ATP compared to fatty acids, thus the metabolic shift from
FFA to glucose in HF has been assumed protective especially in conditions with limited oxygen supply such as MI (173). Insulin resistance is a risk factor for HF, and inhibition of fatty acid utilization has been proposed to ameliorate cardiac dysfunction with hypoxic condition (e.g. ischemia) or dyslipidemia (e.g. obesity) (196). However, high-fat feeding in the diseased state does not necessarily exacerbate LV dysfunction and can actually have beneficial effects (as discussed previously), while upregulating insulin signaling and promoting glucose utilization have been linked to deteriorated cardiac function and increased cardiac events and mortality (108, 303), challenging the idea that the metabolic switch is protective. Insulin resistance in SAT fed HF animals actually provides a condition for the diseased heart to switch back to the normal metabolic profile, utilizing fatty acids as the primary energy source (32, 60). Together with increased FFA oxidation induced by leptin, promoting normal metabolic profile can be a potential mechanism for the improved function in HFSAT animals. While peripheral insulin resistance predicts and correlates with the severity of HF (144, 372), cardiac insulin resistance might promote normal substrate utilization profile and improve cardiac function in the diseased state (32, 60). The beneficial effects of SAT in the diseased state, however, need to be evaluated in long-term studies as well as with more severe MI/HF since MI and HF usually progress further with time.
164 4.1.3 Limitations
We proposed that SAT diet improves cardiac function in a MI induced mild LV dysfunction rat model by restoring its metabolic profiles, however, it remains unclear whether this beneficial effect is limited to rodents and whether SAT diet would have similar beneficial effect if the LV dysfunction is more severe. Cautious interpretation is warranted because of limitations described below. We have an unique model of HF with
SAT intervention. MI induced only mild LV dysfunction with no Ca2+ handling defects and no deterioration or progression into HF, even though the coronary artery ligation was severe enough to cause ~20% surgery-related death. SAT feeding produced no obesity or compromised satiety control and no lipotoxicity, although the food was provided ad libitum. While it allows us to study the specific metabolic changes, such a model may not be clinically relevant. Furthermore, the SAT feeding was designed to begin after ligation surgery due to previous experience of extremely high surgical mortality rate when the animals were pre-fed with SAT (271). The design was strategically inevitable but also clinically irrelevant because most patients have MI as a result of atherosclerosis which is induced by endothelial cell dysfunction, lipid oxidation, and lipid accumulation, often by overconsumption of SAT (190, 343). The potential consequence of increased atherosclerosis is ischemia on a more global scale, whereas in our HF animals the ischemia might be more restricted to the scar and border zone, and hence there is a larger proportion of non-ischemic/functional tissue left in HFSAT hearts that can benefit from increased FFA oxidation. Increased FFA oxidation is otherwise known to be detrimental in ischemic condition (194).
165 In addition to the problems with the study design, there are also major differences between species (rat vs. human) that might undermine the potential metabolic benefits of
SAT diet, especially differences in cholesterol homeostasis. Cholesterol is synthesized partly from fatty acids and travels in the blood stream bound to lipoproteins, which are classified into high-density lipoproteins (HDL), low-density lipoproteins (LDL), intermediate-density lipoproteins, very-low-density lipoproteins, and chylomicrons based on their density (in a high-to-low order) (27, 343). Diets rich in saturated fatty acids increase LDL in humans and promote atherosclerosis, and reducing LDL has been the main goal of coronary artery disease managements (21, 190, 222, 343). On the other hand, HDL mobilizes and clears atherosclerotic plaques and has anti-inflammatory functions. There are profound differences in the relative content of these lipoproteins between rodents and humans. LDL is the main lipoprotein in humans, whereas HDL is predominant in rodents (21, 62, 116) due to their lack of cholesteryl ester transfer protein
(CETP) (21, 115, 180, 331). CETP mediates the exchange of phospholipids from HDL to other lipoproteins, and increasing CETP activity is positively correlated with atherosclerosis (21). Contrary to the clinical population, rodents are naturally resistant to diet-induced atherosclerosis because of the absence of CETP activity (21). The relative levels of LDL and HDL have not been established in our animal model, but since there is no difference in SVR between groups, it is likely that their LDL is low and HDL is high even with SAT diet. Therefore, the beneficial effects associated with SAT feeding are potentially limited to the setting of diet-insensitive low LDL.
166 4.1.4 Fat vs. Carbohydrate
The traditional diet-heart hypothesis describes the relation between high intake of cholesterol and saturated fatty acids and the resulting atherosclerosis and cardiac contractile dysfunction. This is supported by several clinical studies as well as in certain animal models (135, 136, 196). To date, no clinical trial has shown that a saturated-fat diet after MI could be beneficial. Quite the contrary, unsaturated fatty acids are beneficial
(see below) and a low-fat plant-based diet has been shown to reverse severe coronary artery disease to normal with no further cardiac events or complications (89-91).
Together with life style changes, a low-fat plant-based diet reversed severe coronary artery disease and improved LV function during exercise, whereas patients receiving usual care continued to progress (77, 107, 169, 246). The same intervention also improved LV function in patients with ischemic heart disease (247) and stage B HF
(258). Even if the patients were genetically predisposed, a low-fat diet has been shown to decrease the risk of MI and CVD by nearly 2 fold (85). Also, a low-fat plant-based diet has been indicated as the most efficient treatment for type 2 diabetes (371), a major risk factor for CVD. Although an obesity paradox exists, Lavie CJ et al. stated that “the constellation of data still supports purposeful weight reduction in the prevention and treatment of CVD” (183).
However, the issue with fat is still not settled. There is substantial controversy in the literature regarding whether fat is bad for the heart (both directly through cardiac- specific effects and indirectly through weight management) based on the observation that although total fat intake as % calorie has decreased, the prevalence of obesity and type 2 diabetes has increased (228), the incidence of CVD remains high (275), and the
167 correlation between “total” fat intake and CVD is poor (137, 164). Furthermore, there has not been any convincing evidence that reducing total fat intake can sustain weight lose in the long run (369), and poly-unsaturated fat is more effective at lowering cholesterol and
CVD risk than low-fat with high refined-carbohydrate diets (16, 135). Since the introduction of the “low-fat movement” as a result of the diet-heart hypothesis, significant amount of research has been done on the different types of dietary fat, and we now have better understanding of their distinct effects. Generally speaking, saturated and trans fatty acids are atherogenic and can induce or worsen CVD, while -3, mono- and poly-unsaturated fat are inversely correlated with cholesterol and CVD [reviewed in
(135)]. However, different types of fatty acids within the same category can also have different actions on plasma cholesterol level. Specifically considering the three main fatty acids used in our SAT diet, palmitic acid (16:0) increases TG and LDL and also HDL to a minor degree, stearic acid (18:0) decreases TG and LDL and also HDL to a minor degree, and oleic acid (18:1) decreases TG and LDL but increases HDL (176). This complicates the interpretation of our study because the total fat content in the SAT diet consisted of
25% of palmitic acid but 66% stearic and oleic acids combined. The atherogenic potency of this diet is therefore unclear. Nonetheless, -3 and poly-unsaturated fatty acids are increasingly recognized as beneficial to the heart, while lowering “total fat” is not a justifiable recommendation (135).
On the flip side of the story, increasing carbohydrate —especially refined carbohydrates— intake plays an even more significant role in obesity and CVD than increasing fat consumption. In fact, a low-carbohydrate diet high in vegetable fat and vegetable protein was reported to significantly lower risk of CHD while a low-fat and
168 high-carbohydrate diet did not (119). Refined carbohydrates are digested and absorbed more easily than complex carbohydrates due to smaller particle size and lower soluble fiber content (from grinding and other food processing) and hence have a higher glycemic index (higher glycemic responses after digestion) and are positively correlated with TG and negatively correlated with HDL (135). Multiple clinical trials reported the association between diets high in refined carbohydrate and type 2 diabetes and CVD
(135), perhaps due to the pro-inflammatory and pro-thrombotic properties of a hyperglycemic state (75, 196). This correlation is more significant in obese individuals
(135), and because refined carbohydrates are known to trigger hunger and overeating
(272), diets high in refined carbohydrates worsen the correlation through weight gain and if severe, the resulting insulin resistance. While the total fat consumption has decreased over the years, the intake of carbohydrate has increased (often as a consequence of intentional fat intake reduction), especially refined carbohydrates such as added sugar in soft drinks (202). This provides an explanation of the increased obesity and type 2 diabetes epidemic albeit with less total fat intake.
More studies are clearly needed to determine if there are beneficial effects associated with SAT diet or any particular category of nutrients in the context of MI. As discussed previously, using animal models may not be ideal, and hence clinical trials are preferred. It is relatively easy to carry out clinical trials with diet intervention. However, it is difficult to pin point a specific nutrient (e.g. palmitic acid) using such intervention due to shared food sources and potential troubles with accuracy and adherence to the diet, and other nutrients (e.g. proteins and vitamins) in the diet can complicate the interpretation of results. Also, there are potential differences in the digestive system
169 among individuals that can influence the plasma concentration of the nutrients supplied in the diet. To bypass all these difficulties, direct infusion of nutrients into the blood stream has been used in clinical studies, including fatty acids, glucose, as well as vitamins and other substances such as natriuretic peptides and insulin (75, 174, 274, 318). To parallel our current study on rats fed SAT after coronary artery ligation surgery, direct infusion of palmitic acid, or stearic acid, or oleic acid on post-MI patients can be done in comparison. Techniques such as echocardiography, in vivo hemodynamic measurements through catheterization, contrast-enhanced MRI, and electrocardiography can be used to assess end-points such as contractile function, infarct size (314), and arrhythmicity. The timing, duration, and dosage of infusions can be evaluated in correlation to cardiac function and mortality rate. Most importantly, it would allow us to distinguish the various types of fatty acid. Studies like this would help us gain further understanding of nutrients’ impacts on MI and CVD pathophysiology.
4.2 SUMMARY AND FUTURE DIRECTIONS OF “IMPAIRED CONTRACTILE
FUNCTION DUE TO DECREASED CARDIAC MYOSIN BINDING PROTEIN C
CONTENT IN THE SARCOMERE”
Our data show that decreased expression of MyBP-C in MyBP-C−/− hearts results in complex remodeling cascades that impact in vivo and in vitro contractile function, Ca2+ handling, electrical activity, and chamber remodeling of the heart. On the other hand, decreased MyBP-C expression in MyBP-C+/− hearts and concomitant down regulation of
MyBP-C phosphorylation in the sarcomere result in altered cross-bridge function at the myofilament level and contributes to in vivo contractile dysfunction and ECG
170 abnormalities in the absence of adaptations in Ca2+ transient kinetics or LV chamber remodeling and hypertrophy. Normalization of contractile function in MyBP-C+/− myofilaments and intact hearts following β-adrenergic stimulation suggests that basal contractile dysfunction in MyBP-C+/− hearts is at least partly mediated by impaired
MyBP-C phosphorylation.
4.2.1 Regional Differences in Expression of MyBP-C
One potential mechanism of decreased contractile function in MyBP-C+/− hearts is inhomogeneous expression and/or phosphorylation of MyBP-C. It is now known that there are intrinsic differences in different regions of the LV muscle, giving it the ability to contract in an orchestrated and sequential manner. Epicardium contracts faster than endocardium due to shorter action potential and Ca2+ transients as well as greater
2+ phosphorylation of MRLC, which contributes to greater force at sub-maximal [Ca ]i (46,
78). It is not known whether MyBP-C protein expression and/or phosphorylation gradient exist in a similar fashion. However, it has been shown that the incorporation of the remaining functional MyBP-C is not uniform in FHC patients (338), and a MRLC mutation which renders it non-phosphorylatable and hence disrupts the MRLC phosphorylation gradient in the myocardium can cause FHC (46). Furthermore, LV remodeling pattern partially depends on mechanogenic stimuli. Since in FHC patients hypertrophy is found to be asymmetric and more profound in the septum (165), fibrosis is more pronounced in anteroseptal area (80), and myocyte disarray is located excessively in interventricular junction (152, 178), these area likely suffer from more stress than other area in the myocardium and could be due to more disruption at the molecular level.
171 Inhomogeneous expression of the remaining functional MyBP-C in MyBP-C+/− hearts could cause regional disruption of sarcomere pattern and cross-bridge assembly, which can lead to unsynchronized contraction/relaxation and hence decreased efficiency as observed using MRI (82).
We have attempted to locate the site of inhomogeneous expression through multiple approaches, including Western blot on different regions of LV as shown in
Chapter 3 and immunofluorescent microscopy on both tissue and myofilament levels.
From bird’s-eye view on whole LV tissue sections of MyBP-C+/− mice, there were no discernible blank spots/patches of missing MyBP-C (preliminary data not shown). At the myofilament level however, we were able to observe some sites with decreased staining of MyBP-C in MyBP-C+/− samples (Fig. 4-1). The sites missing MyBP-C appear in a patchy pattern with some sites missing one out of the two MyBP-C strips in one sarcomere and some others missing both of them. This finding however, is not conclusive because it is possible that the blank spots were simply incomplete segments of myofilaments, a result of mechanical injury by the polytron during the isolation process.
The low appearance rate (< 1 in 100 myofilaments, preliminary) of myofilaments possessing these blank spots also makes them less likely to be influential. Since there is no enzymatic or other non-mechanical method for myofilament isolation, we could not reassess this finding and therefore cannot provide concrete evidence of inhomogeneous incorporation of MyBP-C in MyBP-C+/− samples at this point.
172 Figure 4-1. Regional Differences in Expression of MyBP-C. Isolated LV myofibrils from WT and MyBP-C+/− hearts were stained with specific fluorescent antibodies recognizing -actinin (shown in green) and MyBP-C (shown in red). Preparations of multiple myofibrils are shown on the left and single/double myofibrils on the right. MyBP-C showed up as classic doublets in WT samples but in a patchy pattern in MyBP- C+/− samples (pointed out with white arrows).
There is another possibility, which is at the sarcomere level. It is thought that the physiological ratio of MyBP-C to myosin is 1:3 in the C-zone of the sarcomere (72, 278).
If the ratio stays the same in some but significantly less than 1:3 in some other MyBP-
C+/− sarcomeres, the inhomogeneous pattern could affect myofilament function as well.
Techniques such as negative staining and electron microscopy can be used to assess this
(298) and will also help us determine if the distance between thin and thick myofilaments
173 is closer in MyBP-C+/− sarcomeres as observed in MyBP-C−/− model, which could explain their increased cross-bridge attachment rates.
4.2.2 Cardiac Energetic Alterations
There is emerging evidence suggesting that alterations in cardiac energetics play an important role in FHC disease progression (15). The FHC causing mutations in myofilament proteins often lead to an increase in energy cost of force production, which has been shown in mice carrying TnI (150, 366), TnT (229), and MHC mutations (315), rats with a TnT mutation (99, 198), a patient with a MHC mutation (25), a patient with both MyBP-C and MHC mutations (161), and in silico studies with TnI mutations (160,
373). On the other hand, both FHC patients (346) and mice with MHC or TnT mutation
(197) exhibited mitochondrial abnormalities and dysfunction, and there is a metabolic switch from fatty acids to glucose in hypertrophied hearts (171), which together can result in lower ATP production. Inefficient utilization of ATP due to the myofilament protein abnormalities together with the impairments in ATP synthesis leads to an energy supply/demand imbalance in FHC hearts, which can compromise key functions such as ion homeostasis (145). For example, decreased Ca2+ reuptake can activate hypertrophy signaling. Also, an elevation of ADP hence lower chemical driving force as a result of increased energy cost of contraction has been shown to slow the cross-bridge kinetics and contribute to diastolic dysfunction as seen in FHC (145). Through magnetic resonance spectroscopy, it has been shown that PCr to ATP ratio is markedly decreased in FHC patients with or without the manifestation of symptoms (73, 156, 306), suggesting that the bioenergetic deficit precedes and can be the leading cause of the LV dysfunction.
174 Furthermore, the metabolic drug perhexiline has been shown to ameliorate diastolic dysfunction and improve exercise capacity in FHC patients, which might due to its ability to substantially increase the PCr/ATP ratio, albeit inconclusive (4, 131). Mutations in several metabolic genes including AMP-activated protein kinase also have been reported to cause FHC-like phenotypes, further indicating that alterations in cardiac energetics can be a mechanism for FHC (100). Our preliminary data showed that there was no difference in ATP/ADP ratio by luciferase assay and HPLC (data not shown). However, these assays might not be ideal for heart tissue since the endpoints include the myosin- bound ADP which is not bioenergetically available. The large amount of myosin-bound
ADP in muscle tissue could mask the real ATP/ADP differences and contribute to false negative outcomes. Also, it remains unknown whether the PCr level and ATP hydrolysis rate are different in our model. Magnetic resonance spectroscopy, considered as the gold standard for measuring metabolic changes, can be used to measure PCr, ATP, ADP,
AMP, as well as many other metabolites in living beating hearts (34). The ATP hydrolysis rate can then be calculated based on mathematical equations (51).
4.2.3 Autonomic Regulations
Impaired autonomic function, specifically decreased parasympathetic activity is a common feature in FHC patients (70, 94, 200, 305, 377) and has also been observed in a mouse model of FHC (151). We investigated the possibility of altered autonomic regulations in conscious WT, MyBP-C−/−, and MyBP-C+/− animals through pharmacological approaches (discussed in section 1.2.5) by telemetered electrocardiogram. However, we were not able to detect any difference between groups in
175 sympathetic or vagal tone, as the BL HR (Fig. 4-2A), DOB response (Fig. 4-2B), atropine response (Fig. 4-2C), and propranolol response (Fig. 4-2D) were all comparable between groups.
Figure 4-2. Sympathetic and Parasympathetic Tone. ECG data were collected on conscious animals (n = 8-19) with telemetry implants and used for HR analysis. HR was recorded for 2 hrs after an initial 30 min settling period. DOB (10 g/g BW), atropine (1 g/g BW), or propranolol (1 g/g BW) was then injected intraperitoneally, and the HR response was recorded for 2 hrs. BL was determined by the lowest HR segment (~8 min) before the injection without any motion based on the activity channel of the recordings. HR response typically peaked ~5-10 min post-injection and remained stable for at least 15 min. A ~8 min segment of the peak HR response was selected for analysis. Only segments with clear and stable tracings were used. BL HR (A), differences between peak DOB response and BL HR (B), differences between peak atropine response and BL HR (C), and differences between peak propranolol response and BL HR (D) between groups were analyzed using one-way ANOVA, and no difference was found.
176 Although there were no discernible alterations in autonomic function in these animals, prolonged QT intervals and slower electrical conduction in conjunction with down regulation of phosphorylation of myofilament and Ca2+ handling proteins in
MyBP-C−/− animals would be expected to significantly increase their risk of cardiac arrhythmia and SCD, especially in conditions of increased peripheral metabolic demand, such as exercise. Indeed, increased -adrenergic stimulation by DOB in MyBP-C−/− animals resulted in 38% mortality from severe heart block, while WT and MyBP-C+/− animals did not have any death within the 2 hrs post-injection recording. However, there were no differences at BL, and other symptoms of arrhythmia such as tachycardia and premature ventricular contraction were not different between groups with or without
DOB. Increased heart block but not tachycardia in MyBP-C−/− animals indicates that the dysfunction is more likely to be in the electrical conduction system (sinoatrial node, atrioventricular node, and His-Purkinje fiber) and not coupling between ventricular cardiomyocytes. Examining the function of the electrical conduction system [e.g. measuring the main connexin involved in nodal (connexin 45) and His-Purkinje connexin
(connexin 40) by western blot (297)] might help elucidate the conduction defects and arrhythmogenesis in MyBP-C−/− animals.
Alterations in autonomic regulations can also be assessed by HRV, a more sensitive analysis looking at fluctuations between intervals of consecutive heart beats.
The concept of HRV analysis is that in cardiovascular system, the sympathetic and vagal inputs create a non-stationary balance, and because vagal inputs are more rapid, healthy hearts have higher overall HRV. In contrast, diseased hearts would have lower HRV due to sympathetic dominance (which can be due to either increased sympathetic inputs or
177 decreased vagal inputs). Moreover, there are parameters in HRV analysis that are more specific to the individual components of nervous regulation (375). For example, the square root of the mean of the sum of the squares of differences between adjacent normal-to-normal R-R intervals is a measure of vagal activity, whereas standard deviation of all normal-to-normal R-R intervals if less than 100 msec (in human) is an index of abnormal sympathetic outflow. HRV has correlations with multiple CVD and
CVD risk factors including diabetes, hypertension, hyperlipidemia, ischemic heart disease, atherosclerosis, HF, SCD, among others (375), and we suspect that both MyBP-
C−/− and MyBP-C+/− animals would also have abnormalities in HRV due to observed
SCD in MyBP-C−/− animals and abnormal ECG parameters in both MyBP-C−/− and
MyBP-C+/− animals. HRV analysis can be done with the aid of commercial software packages such as A.R.T. and Ponemah (Data Sciences International) (261).
4.2.4 Beyond Mutations?
One of the key questions left in this study that cannot be answered by mutations is what causes decreased MyBP-C phosphorylation in MyBP-C+/− hearts. Decreased MyBP-
C phosphorylation is a common feature of diseased hearts and not limited to MyBP-C mutation related FHC. Our protein study showed that MyBP-C expression is less in
MyBP-C+/− myocardium but the remaining MyBP-C can be phosphorylated just as well as the ones in WT when subjected to PKA, and the mechanical function is similar between MyBP-C+/− and WT skinned myocardium treated with PKA, suggesting that haploinsufficiency does not limit MyBP-C from phosphorylation and PKA-mediated phosphorylation is important for proper myofilament function. Additionally, when given
178 the same amount of DOB, the in vivo hemodynamic function is also similar between
MyBP-C+/− and WT hearts, suggesting that the downstream -adrenergic signaling pathway is intact in MyBP-C+/− hearts. The decreased MyBP-C phosphorylation under basal condition suggests that there might be alterations upstream of ARs and/or unchecked phosphotase activity. However, PP1 expression and activity were not elevated in MyBP-C+/− hearts as mentioned in Chapter 3 and other phosphatases have limited ability to dephosphorylate MyBP-C (179). Therefore, the culprit is more likely to be changes that affect kinase activity although not identified in this study. Physiological activators of ARs such as epinephrine, norepinephrine, and dopamine can be measured either with plasma or urine samples by HPLC with electrochemical detection (129).
Interestingly, the expression of MyBP-C in ~6 month old MyBP-C+/− mice is 32% less than WT mice, which is a greater decline than the 25% decrease observed in 2-3 month old MyBP-C+/− mice. It is unclear if the decrease in MyBP-C progresses with age and what causes the progressive decline. However, base on the evidence that MyBP-C phosphorylation is decreased in the disease state and decreased phosphorylation promotes degradation of MyBP-C, one can hypothesize that the decreased phosphorylation contributes to the decline of MyBP-C expression. It is unknown if the decreased phosphorylation progresses with age also and if it correlates with the decline of expression. While phosphorylation of MyBP-C has several important functions, several of them depend on the MHC composition and are quite different in a predominantly
MHC background [reviewed in (18)]. Since human LV consists of > 94% of MHCin healthy state and > 98% of it in failing state (267), understanding the role of MyBP-C in the MHCbackground should be made priority. Studying myofilaments from patients
179 carrying MyBP-C truncation mutations who are in early FHC stage (and hence without profound secondary adaptations) would be ideal.
The most intriguing aspect of MyBP-C induced FHC is perhaps the fact that the mutations alone do not necessarily predict the outcome, as the disease penetrance, age of disease onset, and degree/prevalence of hypertrophy and other symptoms were all extremely variable both within and between families that share the same mutations (248).
Specifically, the disease onset ranged from 5 to 80 years old, the prognosis ranged from
SCD in childhood to no symptom at an advanced age, and all patterns of hypertrophy were observed (severe obstructive asymmetrical septal hypertrophy, apical, concentric, and biventricular hypertrophy with mid-cavity obstruction, dilated-phase FHC, and FHC with restrictive physiology). [In addition to FHC, mutations in MyBP-C also causes dilated cardiomyopathy (357).] This indicates that the disease modifying factors are just
(if not more) as important and powerful as the mutation itself. The mechanisms might include chromosomal or hormonal specific factors (201), disease modifying genes and gender specific single nucleotide polymorphisms (205), dietary pattern (85), physical stress (i.e. exercise) (98, 147, 209), emotional stress (181), and potentially other environmental or behavioral factors. Since symptoms are among the most important determinants of quality of life of FHC patients (59, 71, 231), understanding the mechanisms for these secondary factors that either trigger or prevent the development of symptoms would be helpful for disease management.
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