The Role of ShcA Phosphotyrosine Signaling in the Myocardium€¦ · The Role of ShcA...
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The Role of ShcA Phosphotyrosine Signaling in the Myocardium by Rachel Vanderlaan A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Molecular Genetics University of Toronto © Copyright by Rachel Vanderlaan, 2011
Transcript of The Role of ShcA Phosphotyrosine Signaling in the Myocardium€¦ · The Role of ShcA...
The Role of ShcA Phosphotyrosine Signaling in the Myocardium
by
Rachel Vanderlaan
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Molecular Genetics
University of Toronto
© Copyright by Rachel Vanderlaan, 2011
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The Role of ShcA Phosphotyrosine Signaling in the Myocardium
Rachel Vanderlaan
Doctor of Philosophy
Graduate Department of Molecular Genetics
University of Toronto
2011
Abstract
Tyrosine kinases (TK) are important for cardiac function, but their downstream targets in the
adult heart have yet to be established. The ShcA docking protein binds specific phosphotyrosine
(pTyr) sites on activated TKs through its N-terminal PTB and C-terminal SH2 domains and
stimulates downstream pathways through motifs such as pTyr sites in its central CH1 region. To
explore the role of this TK scaffold in the adult heart, we generated a myocardial-specific
knockout of murine ShcA (ShcA CKO). Such mice developed a dilated cardiomyopathy
phenotype involving impaired systolic function with enhanced cardiomyocyte contractility. This
uncoupling of global heart and intrinsic myocyte functions was associated with altered
perimysial collagen and extracellular matrix complicance properties, suggesting disruption of
mechanical coupling. In vivo dissection of ShcA signaling properties revealed that selective
inactivation of the PTB domain in the myocardium had effects resembling those seen in ShcA
CKO mice, while disruption of the SH2 domain caused a less severe cardiac phenotype.
Downstream signaling through the CH1 pTyr sites was dispensable for baseline cardiac function,
but necessary to prevent adverse remodeling after hemodynamic overload. Therefore, ShcA
mediates pTyr signaling in the adult heart through multiple distinct signaling elements that
control myocardial functions and response to stresses.
Acknowledgments
My PhD has been a great experience and has taught me many things about science, life and
myself. Countless people have enriched my experience as a graduate student and I thank them
for their support.
My supervisor Dr.Tony Pawson has taught me much about science and research. His enthusiasm
and continual pursuit of understanding signaling at its fundamental level has made him a great
mentor. Likewise, my co-supervisor Dr.Peter Backx has provided the opportunity to learn
cardiovascular physiology and has challenged me to be independent. I also thank my committee
members Dr. Jim Woodgett and Dr. Ian Scott for their thoughtfulness and guidance. I also thank
Dr. Norm Rosenblum and Sandy McGugan from the MD/PhD program for their continual
support.
I would like to thank all the members of the Pawson and Backx laboratories for their knowledge,
kindness and friendship. They have enriched the past 6 years and while, we all pursue different
paths, I hope for continued years of friendship.
Most importantly, I would like to thank my family. I thank my husband for never
complaining about long hours or scheduling our life around the mice. Without his support, my
degree would be meaningless. I thank our oldest son, Owen, whose enthusiasm and love is
infectious. His constant questions and marvel of all things ‘science’ has always given me a fresh
perspective and reminds me of why science is fun. Along with Owen, I thank our youngest son,
Elliott, for reminding me daily that being a mom is the best part of who I am.
I have not failed. I've just found 10,000 ways that won't work.
Thomas A. Edison
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Table of Contents
Acknowledgments.......................................................................................................................... iii
Table of Contents........................................................................................................................... iv
List of Tables ............................................................................................................................... viii
List of Figures ................................................................................................................................ xi
Chapter 1 General Introduction ...................................................................................................... 1
1 Introduction ................................................................................................................................ 2
1.1 Congestive Heart Failure and Hypertrophy ........................................................................ 2
1.2 Signal Transduction: Cardiac Signaling in Hypertrophy and HF....................................... 5
1.2.1 Calcium Kinetics..................................................................................................... 7
1.2.2 Mitogen Activated Protein Kinases ........................................................................ 8
1.2.3 JAK/STAT Family................................................................................................ 10
1.2.4 Protein Kinase C ................................................................................................... 11
1.2.5 PI3K/GSK 3 Signaling.......................................................................................... 11
1.3 Membrane Signaling in Cardiac Maintenance and Hypertrophy ..................................... 13
1.3.1 Integrins and Sarcolemmal Proteins ..................................................................... 14
1.3.2 G-Protein Coupled Receptors ............................................................................... 14
1.3.3 Tyrosine Kinases................................................................................................... 15
1.4 Tyrosine Kinase Signaling................................................................................................ 16
1.4.1 Tyrosine Kinase Signaling in the Heart: Mouse Models ...................................... 17
1.4.2 Tyrosine Kinase Signaling: Clinical Evidence ..................................................... 25
1.5 ShcA, an Adaptor for Tyrosine Kinases ........................................................................... 27
1.5.1 ShcA Isoforms and Signaling Domain Architecture ............................................ 27
1.5.2 Biological Role of ShcA Signaling....................................................................... 39
1.5.3 The Role of ShcA in the Myocardium.................................................................. 41
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1.5.4 Strategies for Condition Gene Targeting in the Myocardium .............................. 41
1.6 Rationale and Objectives .................................................................................................. 43
Chapter 2....................................................................................................................................... 45
2 Phenotypic Analysis of ShcA Allele Series ............................................................................. 46
2.1 Mouse Models................................................................................................................... 46
2.2 Cardiac Phenotyping......................................................................................................... 46
2.2.1 Global Cardiac Function Methods ........................................................................ 47
2.2.2 Tissue and Cellular Physiology Methods ............................................................. 49
2.3 Cellular Biology and Signal Transduction Methods......................................................... 57
2.3.1 Histological Analysis and Microscopy ................................................................. 57
2.3.2 Electron Microscopy............................................................................................. 58
2.3.3 Western Blotting and Immunoprecipitation.......................................................... 58
2.3.4 Zymography.......................................................................................................... 59
2.3.5 RNA Isolation and Real Time RT PCR................................................................ 59
2.3.6 Preparation of Tamoxifen ..................................................................................... 59
Chapter 3....................................................................................................................................... 61
3 ShcA is required for Cardiac Structure and Function .............................................................. 62
3.1 Introduction....................................................................................................................... 62
3.2 Results............................................................................................................................... 63
3.2.1 Generation of ventricular cardiomyocyte - specific ShcA null mice.................... 63
3.2.2 ShcA is required for the maintenance of cardiac structure and function.............. 66
3.3 Discussion ......................................................................................................................... 73
Chapter 4....................................................................................................................................... 75
4 Loss of ShcA in the myocardium uncouples single myocyte function and global heart function .................................................................................................................................... 76
4.1 Introduction....................................................................................................................... 76
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4.2 Results............................................................................................................................... 83
4.2.1 The loss of ShcA signaling results in enhanced single myocyte contractility...... 83
4.2.2 The loss of ShcA leads to deregulation of the ECM components in the heart ..... 86
4.2.3 ShcA CKO mice undergo an eccentric remodeling response with transverse aortic constriction.................................................................................................. 89
4.3 Discussion ......................................................................................................................... 94
Chapter 5....................................................................................................................................... 97
5 ShcA uses distinct signaling mechanisms to regulate myocyte contractility and global heart function ........................................................................................................................... 98
5.1 Introduction....................................................................................................................... 98
5.2 Results............................................................................................................................... 99
5.2.1 Generation of mice with ventricular specific ShcA point mutations.................... 99
5.2.2 The PTB domain of ShcA couples to upstream TKs to maintain cardiac structure and function ......................................................................................... 103
5.2.3 ShcA phosphotyrosine derived signaling is required in hemodynamic overload106
5.2.4 Myocyte contractility requires ShcA phosphotyrosine-based signaling............. 112
5.3 Discussion ....................................................................................................................... 112
Chapter 6..................................................................................................................................... 114
6 Summary and Future Directions ............................................................................................ 117
6.1 Summary ......................................................................................................................... 117
6.2 Future Directions ............................................................................................................ 119
6.2.1 What are the specific TK intereactions of the PTB and SH2 domains of ShcA within the myocardium? ..................................................................................... 119
6.2.2 What is the effect of ErbB2-ShcA signaling in the myocardium?...................... 120
6.2.3 What are the clinical correlates to the loss of ShcA or TK signaling in the myocardium?....................................................................................................... 120
6.2.4 How does the loss of ShcA regulate myofilament homeostasis? ....................... 121
6.2.5 What are the downstream signaling consequences attributed the loss of ShcA in cardiomyocytes? ............................................................................................. 121
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6.2.6 What is the molecular mechanism by which the loss of ShcA affects the mechanical integrity of the myocardium?........................................................... 122
6.2.7 What is the role of Grb2 signaling in the myocardium?..................................... 122
6.3 Concluding Remarks....................................................................................................... 123
References .............................................................................................................................. 124
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Abbreviations and Symbols
α Alpha
β Beta
γ Gamma
δ Delta
φ any hydrophobic region
A Alanine
Ant wall Anterior wall
BSA Bovine serum albumin
Bpm beats per minute
CH1 Collagen homology region-1
CH2 Collagen homology region 2
CO2 Carbon dioxide
CML Chronic myeloid leukemia
Cre Cre recombinase
DNA Deoxyribonucleic acid
dP/dT first derivative of pressure development
dSl/dT first derivative of sarcomere shortening
EGFR Epidermal growth factor receptor
ERK1/2 Extracellular-signal-regulated kinase ½
F Phenylalanine
% FS percent fractional shortening
F-actin Filamentous actin
Grb2 growth factor receptor bound protein-2
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G-actin Globular actin
GSK-3 Glycogen synthases kinase-3
HF Heart failure
HR Heart rate
HW/BW Heart weight-to-body weight ratio
K Lysine
LungW/BW Lung weight-to-body weight ratio
LVEDD Left ventricle end diastolic dimension
LVESD Left ventricle end systolic dimension
MMP Matrix metalloproteinase
PI3K Phosphoinositide 3-kinase
Post Wall Posterior wall
PTB Phosphotyrosine binding
pTyr Phosphotyrosine
Q Glutamate
R Arginine
RTK receptor tyrosine kinase
SDS PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SOS Son of sevenless
SH2 Src homology 2
SH3 Src homology 3
TAC Transverse aortic constriction
TR Time to relaxation
Y tyrosine
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List of Tables
Table 1.1 ShcA PTB domain interaction partners ........................................................................ 34
Table 1.2. ShcA SH2 domain interaction partners ....................................................................... 35
Table 1.3. ShcA interactions by conserved signaling motifs........................................................ 37
Table 1.4. Cardiomyocyte specific Cre transgenic mouse models ............................................... 42
Table 3.1. Echocardiography timecourse data......................................................................... 69
Table 4.1. ECM components that contribute to mechancal integrity of the myocardium... 81
Table 4.2. Single myocyte assays ................................................................................................. 85
Table 4.3. Echocardiography and cardiac catheterization data for TAC studies ................ 92
Table 4.4. Echocardiography data of TAC study for acute model of ShcA excision................... 93
Table 4.5. Gravimetric data for ShcA CKO and control mice after TAC .................................... 94
Table 5.1. Echocardiography data for 1 year timecourse ........................................................... 109
Table 5.2. Echocardiography and cardiac catheterization for TAC study ......................... 111
Table 5.3. Contractility and calcium transient assays for ShcA mutant domain KI mice .......... 113
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List of Figures
Figure 1.1 Adaptive and maladaptive remodeling pathways in the heart....................................... 3
Figure 1.2. Signal transduction pathways in cardiac hypertrophy................................................. 6
Figure 1.3 MAPK signaling in the heart ....................................................................................... 10
Figure 1.4 PI3K/AKT signaling in the heart................................................................................. 13
Figure 1.5 Domain structure of tyrosine kinases important in the cardiovascular system ........... 19
Figure 1.6 ShcA locus and domain architecture. .......................................................................... 30
Figure 2.1 Papilary muscle force-length apparatus....................................................................... 51
Figure 2.2 Configuration of single cardiomyocyte apparatus....................................................... 55
Figure 3.1 Ventricular cardiomyocyte-specific deletion of ShcA. ............................................... 64
Figure 3.2 ShcA is required for the maintenance of cardiac function. ......................................... 67
Figure 3.3 ShcA CKO mice undergo eccentric remodeling with preserved cyto-ultrastructure and
minimal fibrosis. ........................................................................................................................... 71
Figure 4.1 Excitation Contraction Coupling in Cardiomyocytes.................................................. 77
Figure 4.2 The loss of ShcA leads to deregulation of the ECM components in the heart. ........... 87
Figure 4.3 ShcA CKO mice undergo an eccentric remodeling response with TAC. ................... 90
Figure 5.1 Schematic of ShcA allele strategy to generate ventricle cardiomyocyte specific ShcA
KI mutants................................................................................................................................... 101
Figure 5.2 TK-ShcA signaling is required to maintain cardiac structure and function, while
downstream signaling from the CH1 pTyr sites is dispensable. ................................................. 104
Figure 5.3 TK-ShcA signaling is required to maintain cardiac structure and function, while
downstream signaling from the CH1 pTyr sites is necessary after hemodynamic overload. ..... 107
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Figure 6.1 Model of ShcA signaling in the myocardium............................................................ 118
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Chapter 1 General Introduction
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1 Introduction
1.1 Congestive Heart Failure and Hypertrophy
Heart failure (HF) is a leading cause of death in the western world and also contributes
to considerable morbidity and health care costs. HF currently affects over half a million
Canadians and sees over 50,000 new diagnoses per year (Liu et al. 2001). Despite a decline in
acute cardiovascular prognoses, as with myocardial infarction, HF, the most common of all
cardiovascular diseases for hospital admissions, has not seen improvement in its mortality rate
(Ho et al. 1993; Ho et al. 1993; O'Connell et al. 1994). Indeed, the 5 year survival rate after
diagnosis of HF is 45%, worse than most cancer prognosis (McMurray et al. 2000; Jessup et al.
2003). As 4% of all Canadians over the age of 70 live with HF, which incurs over 1 billion
dollars for inpatient care alone, the aging population of Canada will further burden the public
health care system in the coming years (Ross et al. 2006; Russell et al. 2008). In the pediatric
population, HF results in multiple admissions, long term pharmacological management and,
ultimately, cardiac transplantation (Boucek Jr et al. 2002). Understanding the pathogenic
processes that contribute to HF is therefore paramount to impacting patient prognosis.
HF is operationally defined as the inability of the heart to meet the systemic metabolic
needs of the body, and results in a clinical syndrome of fatigue, dyspnea, edema, and left
ventricle dysfunction. HF can be caused by primary disease of the heart tissue (genetic
cardiomyopathies) or secondary to complex polygenic disease such as hypertension, coronary
artery disease or myocardial infarction (Figure 1.1). In the former case, cardiomyopathies can
impact directly on the myocardium decreasing cardiac function at an early age of onset. In the
latter case, various conditions, such as hypertension, can increase ventricular wall stress and
hence initiate a pathological hypertrophic remodeling program. Under chronic conditions, these
changes become maladaptive resulting in ventricle remodeling and/or dilation that leads to HF
(Chien 1999; Hunter et al. 1999; Liew et al. 2004). Given the significant morbidity and mortality
associated with HF, understanding the adaptive and maladaptive molecular mechanisms the heart
employs to maintain cardiovascular function is clearly important.
Hypertrophy is the main protective response the heart employs to match pump function
with enhanced systemic needs. Hypertrophy can be either physiological, as with pregnancy and
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exercise, or pathological. The pathological hypertrophy response is the primary mechanism the
heart employs to maintain pump function in the face of disease. This is a complex
Physiological hypertrophy
Pathological HypertrophyDilated Cardiomyopathy
Heart Failure
Normal Heart
ExercisePregnancy
GeneticPolygenicPressure overload
GeneticValvular insufficency
Volume overload
Concentric Hypertrophy
parallel addition of sarcomerescardiomyocyte thickening
Eccentric Hypertrophy
serial addition of sarcomerescardiomyocyte lengthening
Sarcomere
Figure 1.1 Adaptive and maladaptive remodeling pathways in the heart.
Physiological hypertrophy results in hypertrophy of the myocardium to accommodate increased
blood volume resulting in myocytes lengthening (eccentric remodeling); however, no fibrosis or
dysfunction is noted. Pathological hypertrophy is the disporportionate hypertrophy of the
myocardium wall at the expense of the chamber volume, with dysfunction and fibrosis being
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evident. Dilation of the ventricles occurs with eccentric remodelling whereby the cardiomyocytes
lengthen in response to increased volume or mechanical instability. The syndrome of heart
failure is the end consequence of chronic and maladaptive remodeling.
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process that, at the level of the myocardium, involves neurohumoral activation and/ or
mechanoreceptors such as integrins to stimulate signaling pathways that ultimately effect gene
transcription (MacLellan et al. 2000; Heineke et al. 2006). Hallmarks of the process are early
expression of proto-oncogenes such as c-fos, c-jun and c-myc (Mulvagh et al. 1987; Komuro et
al. 1988; Izumo et al. 1998), coincident upregulation of fetal isoforms for various sarcomeric
proteins such as β myosin and skeletal actin (Lompre et al. 1979; Schwartz et al. 1986; Schwartz
et al. 1993), and expression of the biomarkers atrial naturetic peptide (ANP) and brain naturetic
peptide (BNP) (Mercadier et al. 1989; de Bold et al. 2005) Ultimately, there is an increase in the
number of contractile units of the myocyte by parallel addition of sarcomeres (concentric
hypertrophy). Neurohumoral contributions such as adrenergic inputs, the renin –angiotensin
system and cytokines also help to make the initial hypertrophy response compensatory, so that
the heart is able to maintain cardiac homeostasis (Kan et al. 2001; Port et al. 2001). In chronic
disease, where the initiating stress is persistent, the heart moves into a decompensated state,
where fibrosis, cardiac enlargement and decreased systolic function evolve into symptomatic
HF(Chien 1999). While current therapies attempt to relieve the symptoms of HF, understanding
the complex signaling cascades that are involved in maintenance of normal cardiac function and
the cascades involved in transitioning the heart into overt HF will potentially contribute to novel
therapeutic strategies.
1.2 Signal Transduction: Cardiac Signaling in Hypertrophy and HF
From initiating signals at the cardiomyocyte membrane, a host of signal transduction events
occur in a complex and integrated manner to evoke adaptive or maladaptive responses. Research
has highlighted multiple signaling networks that receive converging inputs from various
upstream molecules to impact on hypertrophy and the maintenance of cardiac function. While
neither comprehensive nor inclusive, I will highlight signaling pathways important in
hypertrophy and/or heart failure signaling, such as calcium kinetics, the mitogen- activated
protein kinase (MAPK) pathways, the janus kinase/stat family, PKC signaling and the PI3K-
GSK pathways. Important upstream membrane receptors that trigger these cascades are integrins
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and sarcolemmal proteins, G-protein coupled receptors and tyrosine kinases (TK) (Section
1.3)(Figure 1.2).
Figure 1.2. Signal transduction pathways in cardiac hypertrophy
Proximal membrane inputs activate downstream signal transduction cascades that ultimately
impact on hypertrophy. Important mediators of hypertrophy signaling are outlined in Section 1.2
and 1.3.
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1.2.1 Calcium Kinetics
Calcium signaling is paramount to cardiomyocyte function as a rise in intracellular calcium can
initiate both cardiomyocyte contraction and calcium triggered signal transduction pathways.
With respect to cardiac contraction, calcium entry from the voltage gated L type calcium channel
(Ca+2 L type) triggers the ryanodine receptor (RyR) mediated sarcoplasmic reticulum calcium
release. Relaxation of myocyte contraction involves reuptake of calcium by the sarcoplasmic
reticulum ATPase pump ( SERCA2a) and its negative regulatory protein phospholamban (PLN)
(Bers 2002) (Chapter 4). Indeed, downregulation of SERCA2a gene expression is found in
human and mouse models of hypertrophy and heart failure (Nagai et al. 1989; Anger et al. 1998;
Frank et al. 2002), and mutations that prevent PKA mediated phosphorylation of PLN decrease
SERCA2a mediated decay of the calcium transient, resulting in a dilated cardiomyopathy
(Schmitt et al. 2003).
Within discrete microdomains, calcium can also act as a potent signal transduction
molecule and can lead to modulation of key calcium activated pathways, termed excitation-
transcription coupling (ETC); ETC is coordinated by the multifunction calcium sensor,
calmodulin (CaM). CaM binds to the phosphatase, calcineurin, to activate the transcription factor
NFAT and to Calmodulin kinase II (CaMKII) to impact on hypertrophy signaling. While both
pathways can impact on excitation- contraction coupling (ECC), it is the modulation of
transcription factors that impacts on hypertrophy signaling(Bers et al. 2005). Overexpression of
CaM results in severe cardiac hypertrophy(Gruver et al. 1993), solidifying its role as a pivotal
molecule in cardiac signaling. One arm of CaM signaling is through its interactions with
calcineurin, a serine/threonine protein phosphatase that exists as a heterotrimer. Activation of
calcineurin by sustained calcium levels (Dolmetsch et al. 1997) leads to dephosphorylation of
NFAT, allowing coordination of hypertrophy genes in the nucleus with the transcription factor
GATA4 (Frey et al. 2000). Indeed, overexpression of calcineurin and NFAT c4 leads to robust
hypertrophy (Molkentin et al. 1998), while deficiency of calcineurin decreases the response to
hypertrophy agonists (Wilkins et al. 2002). The CaM-CaMKII axis is activated by high calcium
spikes (De Koninck et al. 1998) and can impact on hypertrophy through phosphorylation of
CREB (cAMP binding response element) transcription factor(Sun et al. 1996; Deisseroth et al.
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1998; Hook et al. 2001) and histone deacetylase (HDAC), which liberates Mef2, thus impacting
on transcription (Frey et al. 2000; Zhang et al. 2002; Berger et al. 2003). In vivo models support
these associations as increased activity of Mef2 leads to exaggerated hypertrophy in mouse
hearts (Zhang et al. 2002).
1.2.2 Mitogen Activated Protein Kinases
MAPKs are serine/threonine kinases that are primarily activated by tyrosine /threonine
phosphorylation. Activated MAPKs phosphorylate downstream kinases and nuclear substrates
such as c-myc, c-jun and ATF-2. The MAPK cascades include the stress activated protein kinases
(SAPKs) and the 3 subfamilies include the extracellular regulated kinases (ERKs), the c- jun N-
terminal kinases (JNKs) and the p38 MAPKs (Figure 1.3). These subfamilies are organized as
hierarchal cascades with MAPK being the terminal kinase activated. The initiation of the kinase
cascades is by activation of serine/threonine MAPK/ERK kinase kinases (MEKKs) which,
through dual phosphorylation of a Ser-XXX-Ser/Thr motif (with x denoting an amino acid),
activate MAPK/ERK kinases (MEK/MKK). These in turn activate MAPK by dual
phosphorylation of Thr-X-Tyr motifs (Garrington et al. 1999).
In the heart, the 3 subfamilies are activated by varying stimuli, each contributing to
different facets of hypertrophy signaling. The ERK pathway consists of 6 kinases, with ERK
1(44kDa) and ERK2 (42kDa) being the best characterized. The ERK cascade has the hierarchical
order of: Raf kinase /MEKK1→MEK1 / MEK2→ERKs, with substrates including Elk-1, c-jun
and 90 kDa S6 kinase (Garrington et al. 1999). In vivo data support a role for ERK signaling in
hypertrophy, as overexpression of MEK1 in the myocardium of mice causes concentric
hypertrophy that is not accompanied by fibrosis or premature death (Bueno et al. 2000).
Overexpression of Ras, an upstream activator of Raf, also elicits robust hypertrophy, however
due to pleiotropic effects, the mice die suddenly and have pathological remodeling (Hunter et al.
1995). Disruption of c-raf-1 in the myocardium leads to systolic dysfunction and chamber
dilation, associated with an increased number of apoptotic cells (Yamaguchi et al. 2004). The
clinical relevance of this pathway is reiterated as gain of function Raf mutations result in Noonan
and LEOPARD syndromes with hypertrophic cardiomyopathies (Pandit et al. 2007). Therefore,
proper levels of ERK signaling are important for maintaining normal cardiac function.
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The JNK pathway is encoded by 3 genes that yield the 3 isoforms: JNK1/SAPKγ, JNK2
/SAPK α and JNK 3/SAPK β. In the heart, the upstream kinases in the JNK pathway are ill
defined, but appears to be MEKK1, MEKK2, MEKK3 and MEKK5, which activate MKK4 and
MKK7 (Yan et al. 1994; Tournier et al. 1997; Wang 2007). JNKs activate transcription factors
such as c-jun and ATF2 (Wang 2007). While the role of JNK signaling in hypertrophy is
debated, loss of MEKK1 results in chamber dilation, systolic dysfunction and premature death
after pressure overload (Sadoshima et al. 2002), while cardiac specific loss of MKK4 results in
an exaggerated pathological hypertrophy response with apoptosis (Liu et al. 2009).
The p38 MAPK pathway is activated by MEKK5 which activates MKK3 and MKK6.
These MKKs activated the isoforms p38 MAPK α, β, δ and γ leading to phosphorylation of
MAPK-activated protein kinase 2 (MK2) which activates heat shock protein 25 and 27 and ATF
2 (Ichijo et al. 1997; Zechner et al. 1997; New et al. 1998; Kerkela et al. 2006). The main
isoform expressed in the heart is p38α (Liao et al. 2002), and cardiac specific expression of
upstream activators of p38, MKK3 and MKK6, show divergent signaling roles. Expression of
activated MKK3 leads to chamber dilation with reduced wall thickness and fibrosis, while
expression of activated MKK6 results in mild hypertrophy with preserved chamber dimensions
(Liao et al. 2002). Myocardial expressed dominant negative p38α results in a hypertrophic
response to pressure overload, with little fibrosis(Zhang et al. 2003). This suggests p38 is a
critical player in ventricular remodeling.
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Figure 1.3 MAPK signaling in the heart
1.2.3 JAK/STAT Family
Another family of signal transduction implicated in hypertrophy is the janus kinase / signal
transducers and activator of transcription pathway (JAK/STAT). The JAK family are protein
tyrosine kinases which consists of JAK 1, 2, and 3 and TYK2 (Ihle et al. 1995), while the STAT
family are transcription factors activated by tyrosine phosphorylation and consist of STAT1,
2,3,4,5a, 5b and 6 (Schindler et al. 1995; Horvath et al. 1997; Boengler et al. 2008). Initiation of
the JAK pathway is through ligand binding to the cytokine receptor, such as cardiotrophin (CT-
1) binding to the glycoprotein 130 receptor in the heart. This induces activation of the receptor-
JAK complex and recruitment of STAT3 which migrate to the nucleus, regulating gene
transcription (Horvath et al. 1997; Robledo et al. 1997). Indeed, loss of gp130 in the myocardium
results in a dilated cardiomyopathy accompanied by apoptosis of myocytes (Hirota et al. 1999),
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while constitutive expression of gp130 leads to cardiac hypertrophy (Hirota et al. 1995).
Clinically, cytokines have been shown to be involved in the transition into overt heart failure and
are important biomarkers for disease prognosis (Wollert et al. 1997; Deswal et al. 2001; Fischer
et al. 2007).
1.2.4 Protein Kinase C
Protein kinase C (PKC) is a serine/threonine kinase that has 3 subcategories for its isoenzymes:
1) classical PKC (α/β/γ) regulated by DAG, phosphatidylserine and calcium, 2) novel PKC (δ, ε,
η,Ω, μ) not regulated by Ca+2 and 3) atypical PKC (ζ and λ) with ill defined regulation (Puceat
et al. 1996; Newton 1997). PKC signaling can activate the ERK pathway through Raf, the Iκβ
pathway, and modulated intracellular calcium all of which can initate the hypertrophy response
in cardiomyocytes(Kolch et al. 1993; Buchner 1995; Ho et al. 1998). In vivo, the role of PKC is
difficult to decipher due to the overlapping function of multiple isoforms; however, with
hemodynamic stress, PKCα has been shown to regulate hypertrophy and contractility, as the loss
of PKCα in the myocardium leads to enhanced cardiomyocyte function and protection against
heart failure (Braz et al. 2004).
1.2.5 PI3K/GSK 3 Signaling
The phosphoinositide-3’ kinase (PI3K) signaling pathway (Figure 1.4) involves the
phosphorylation of phosphatidylinositols (PtdIns), with the phosphorylation pattern dictated by
the class of PI3K. The two PI3K classes are delineated by their catalytic and accessory subunits,
and in the heart, class 1, and its subclasses 1A and 1B, are distinguished by their effects on
hypertrophy signaling(Oudit et al. 2009). Physiological hypertrophy is coupled to PI3K subclass
1A, 110 kDa lipid kinase (p110α) downstream of IGF-1 and other TKs (Lupu et al. 2001).
Cardiac-specific constitutively active p110α does not cause transition to maladaptive
hypertrophy (Shioi et al. 2000), while a dominant-negative mutant impairs physiological
hypertrophy in response to exercise (McMullen et al. 2003). PI3K subgroup IB, which utilizes the
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catalytic p110γ subunit, is recruited to the membrane by the G-protein subunits Gβγ of Gq/11
receptor associated proteins, and is required for pressure overload induced hypertrophy
(Crackower et al. 2002; Patrucco et al. 2004), while also regulating cAMP levels in
cardiomyocytes (Crackower et al. 2002). These pathways converge on the protein kinase AKT,
which is critical in trophic growth. AKT1 is the most important AKT gene in the heart, and
germline deletion leads to reduction in organ size including the heart (Cho et al. 2001), while
overexpression of AKT in the heart results in hypertrophy with depressed contractility (Matsui et
al. 2002; Shioi et al. 2002). Other key molecules in this pathway are the negative regulator of
PI3K, PTEN (phosphatase and tensin homolog on chromosome 10), and the upstream activator
of AKT, phosphoinositide-dependant kinase-1 (PDK1). Cardiac specific loss of PTEN results in
cardiac physiological hypertrophy (Schwartzbauer et al. 2001; Crackower et al. 2002);
conversely, cardiac ablation of PDK-1 leads to reduced hypertrophy and subsequent
cardiomyopathy (Mora et al. 2003).
The PI3K-AKT axis can activate mammalian target of rapamycin (mTOR) to effect
protein synthesis, but can also negatively regulate the praline-directed serine/threonine protein
kinase, GSK-3 (Fiol et al. 1987; Plyte et al. 1992). Of the 2 isoforms of this kinase, GSK-3β is
the most studied in the heart . GSK-3β receives converging inputs from multiple protein kinases
such as PKC and PKA and phosphorylation of Serine 9 of GSK-3β leads to release of negative
inhibition to impact on hypertrophy (Hardt et al. 2002). The phospho-null mutant GSK-3βS9A,
when overexpressed in the myocardium, inhibits hypertrophy after pressure overload and
isoproterenol stimulation (Antos et al. 2002). While loss of GSK-3β downstream of the Gq
activator, endothelin-1, results in hypertrophy in neonatal rat cardiomyocytes (Haq et al. 2000).
As GSK-3β is a multifunctional protein, its ability to regulate hypertrophy can be through a
variety of mechanisms. These could include regulation of important transcription factors
involved in hypertrophy, such as GATA4 or through stabilization of β-catenin, the main
consequence of Wnt signaling (Toyofuku et al. 2000; Morisco et al. 2001; Woodgett 2001).
Recently, the GSK-3α isoform has been shown to regulate beta-adrenergic responsiveness and
hypertrophy signaling in response to hemodynamic overload, therefore expanding our
understanding of GSK signaling in the heart (Zhou et al.).
13
Figure 1.4 PI3K/AKT signaling in the heart
1.3 Membrane Signaling in Cardiac Maintenance and Hypertrophy
Proximal inputs implicated in the maintenance of the myocardium and the initiation of
hypertrophy includes integrins and associated cytoskeletal proteins, G protein coupled receptors
14
and tyrosine kinases. Engagement of these membrane proteins activates various signal
transduction pathways mentioned above to impact on hypertrophy and cardiac function.
1.3.1 Integrins and Sarcolemmal Proteins
Maintenance of cardiac function and the initiation of the hypertrophy signal are sensed by
integrins and/or sarcolemma proteins, particularly resulting from mechanical stimuli such as
hypertension. Indeed, many of the genetic cardiomyopathies result from mutations in sarcomeric
proteins such as cardiac α-actin, desmin, troponins, cardiac myosin heavy chain, dystrophin and
sarcoglycan (Fatkin et al. 2002; Liew et al. 2004). Likewise, integrin signaling has been shown
to initiate hypertrophy in cell culture in the absence of neurohumoral inputs, and the β1 integrin
knockout mouse develops a dilated cardiomyopathy (Ross et al. 1998; Shai et al. 2002).
Molecules downstream of integrins such as Melusin have also shown to be critical in
hypertrophy signaling after hemodynamic overload (Brancaccio et al. 2003).
1.3.2 G-Protein Coupled Receptors
Currently, the main targets for therapeutics and the focus of much cardiac research are G protein-
coupled receptors (GPCR) in cardiac tissue. GPCRs are ligand activated seven transmembrane
receptors that are coupled to heterotrimeric guanine-nucleotide regulatory proteins. Over 200
GPCRs exist in the heart with the adrenergic, angiotensin, endothelin and muscarinic receptors
being most notably implicated in heart failure(Salazar et al. 2007). In the treatment of heart
failure, adrenergic and angiotensin GPCR therapeutics account for the majority of prescribed
therapies (Tang et al. 2004).
Adrenergic signaling in the heart consists of α and β adrenergic receptors (AR), with β
ARs being predominant: αARs are one tenth the number of βARs; within the βARs, there is an
80:20 expression ratio of β1ARs to β2ARs (Rockman et al. 2002). In general, the adrenergic
receptors couple to either G stimulatory (Gs) or G inhibitory (Gi) proteins to modulate adenylyl
cyclase and its second messengers cAMP to impact on PKA regulation. Clinically, in heart
failure, when catecholamine levels are elevated for a sustained duration, desensitization of β
15
adrenergic receptors leads to loss of cardiac reserve (the inability to increase SNS mediated
effects on heart function) and compromised ventricular function (Tilley et al. 2006). Therefore,
βAR antagonists are used as a symptomatic therapeutic intervention. The β1ARs have a positive
ionotropic (enhanced contraction) and lustropic (enhanced relaxation) effect in that they
modulate calcium fluxes through phosphorylation of the L-type calcium channel, ryanodine
receptor , troponin I and phospholamban , the inhibitory regulator of SERCA2a, while β2ARs
are thought to exert their effect on vasodilation of the vasculature . Loss of β1AR leads to
dampened iontropic and chronotropic response to isoproterenol (Rohrer et al. 1996) and loss of
β2 ARs results in exercise-induced hypertension(Chruscinski et al. 1999). Conversely,
overexpression of β1AR results in hypertrophy and fibrosis which transitions into heart failure
(Engelhardt et al. 1999; Bisognano et al. 2000), while overexpression of β2ARs leads to
enhanced cardiac function without progression into heart failure (Milano et al. 1994; Liggett et
al. 2000). αARs are also important in mediating the hypertrophy response and the transition into
heart failure. Double knockouts of α1A/C and α1B fail to develop physiological hypertrophy, and
fail to initiate an adaptive hypertrophy response after pressure overload (O'Connell et al. 2003).
The Angiotensin receptors are another important class of GPCR implicated in
hypertrophy and heart failure and mainly signal through the peptide ligand Ang II which
activates Gq coupled AT1R (Lambert et al. 1995; Touyz et al. 2000). Overexpression of AT1aRs
result in exaggerated hypertrophy response and fibrosis (Paradis et al. 2000), while loss of
At1aRs diminishes these responses (Bridgman et al. 2005), thereby implicating Ang II signaling
in hypertrophy and extracellular matrix production.
1.3.3 Tyrosine Kinases
Individually, many tyrosine kinases have been shown to have an important role in cardiac
structure and function. Not only do they initiate discrete signaling pathways, but also serve as
nodes for other proximal signals. Indeed, integrins utilize FAK and Src, two non receptor TKs, to
impact on cardiac signaling (Plopper et al. 1995), while GPCRs, such as endothelin, use TKs to
activate growth pathways (Chung et al. 2007). Clinically, GPCRs have dominated the research
and treatment of heart failure, but tyrosine kinases have also been shown to be integral in cardiac
16
homeostasis. With research directed at elucidating the signaling networks from various tyrosine
kinases perhaps novel and efficacious treatment strategies will evolve.
1.4 Tyrosine Kinase Signaling
Tyrosine phosphorylation, by protein TKs, is a key covalent modification that involves the
transfer of the γ phosphate of ATP to hydroxyl groups of tyrosines of target proteins thereby
propagating signals within a cell (Hunter 1998). TKs exist as 2 main classes, the transmembrane
receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases (nRTK). Typically, RTKs
have a common structure of a glycosylated extracellular domain which binds ligand, a
transmembrane helix and the cytoplasmic tail containing a protein tyrosine kinase core and
regulatory sites for covalent motifications (Hubbard et al. 2000; Schlessinger 2000). Signaling
through RTKs involves ligand-induced oligimerization of the receptor to induce a
conformational change with subsequent trans autophosphorylation in the activation loop of the
cytoplasmic domain (Ullrich et al. 1990; Heldin 1995). nRTK can be activated by
phosphorylation in the activation loop, either in trans or by a different nRTK, to increase
tyrosine kinase activity (Superti-Furga 1995). Negative regulation of TK signaling can be
through protein tyrosine phosphatases (PTP), autoinhibitory phosphorylation, receptor-mediated
endocytosis, or ubiquitin-directed proteolysis (Hubbard et al. 2000; Schlessinger 2000). Tyrosine
kinase signaling plays a critical role in development, differentiation, proliferation, and migration.
In the case of RTKs, tyrosine phosphorylation of consensus sites in the cytoplasmic
domain creates docking sites to lend specificity and complexity to ligand induced activation.
Autophosphorylation targets of PTKs are typically located outside the catalytic unit of the
receptor and hence serve as binding sites for modular phosphotyrosine recognition proteins, such
as those containing SH2 (Src homology 2) or PTB (phosphotyrosine binding) domains. Protein
signaling/interaction domains are usually 3-120 amino acids in length and allow proteins to bind
to covalently modified sites on target proteins (Pawson et al. 2000). Common examples of
interaction domains include SH2 domains, which bind sequences pY-X-X-θ generally, PTB
domains, which bind NP-X-pY motifs and SH3 domains which bind proline rich sequences
defined by the P-X-X-P motifs (where θ is any hydrophobic amino acids, X is any amino acid).
17
The modular nature of proteins allows for the various domains to impact on signaling
through diverse ways. First, domain binding to an activated kinases increases the probability of
that protein being phosphorylated and hence propagation and amplification of the signal. Second,
domains can be used as a means to localize signaling molecules to discrete microdomains, as
seen with RTK mediated Ras activation, whereby recruitment of Grb2-SOS to the EGFR allows
SOS to be in proximity to its substrate, Ras (Pawson et al. 1993). Finally, in the case of domain
containing proteins with catalytic activity, binding to substrates can relieve intramolecular
regulation allowing for subsequent activation, as in the case of Src (Superti-Furga et al. 1993).
1.4.1 Tyrosine Kinase Signaling in the Heart: Mouse Models
TK signaling has been shown to be critical in oncogenic transformation and in many
developmental processes. Over the years, evidence has also demonstrated the importance of TK
signaling in the heart, through use of mouse knockout models. Indeed, TKs have an under-
appreciated role in both the maintenance of cardiac function and the complex signaling programs
that are initiated with disease. As more conditional TK alleles emerge, the role of individual TKs
within the myocardium will be elucidated. Additonally, the literature has demonstrated the
importance of TK reciprocal signaling in the myocardium. Indeed, the paracrine induction of
coronary vasculature by VEGF-A secreted from myoyctes and NRG-1- Erbb2 interplay between
the endothelium and the myocytes, suggests TK signaling is critical in maintaining tissue
physiology. Therefore, I will highlight some of the heart specific findings for a variety of TK
receptors (Figure 1.5).
1.4.1.1 Epidermal Growth Factor Receptor Family
The epidermal growth factor receptor family consists of 4 main receptors, the epidermal growth
factor receptor (EGFR/ErbB1), ErbB2, ErbB3 and ErbB4. These RTKs exist in monomeric form
and upon ligand binding, can form homo or heterodimers to initiate autophosphorylation events.
EGF signaling ligands include EGF, transforming growth factor alpha, amphiregulin, epiregulin,
neuregulins, heparin binding EGF-like growth factor and betacellulin. Complexity of signaling is
18
achieved by homo and heterodimer preferences, ligand binding preference and signaling
recruitment by non ligand affiliated ErbB2 (Fuller et al. 2008) In the heart, EGF family
members are required for heart development and for the maintenance of cardiac structure and
function in the postnatal heart. Despite having no known ligand, ErbB2 facilitates dimerization
and coordination of signaling networks and is critical in heart function (Horan et al. 1995; Graus-
Porta et al. 1997). Mice null for ErbB2, ErbB4 and their endothelium-derived ligand NRG are
embryonic lethal at E10.5 with poor trabeculation and peripheral nervous system defects
(Gassmann et al. 1995; Lee et al. 1995; Meyer et al. 1995). Conditional loss of ErbB2 in the
myocardium results in severely dilated ventricles, with potential defects in apoptosis (Crone et
al. 2002; Ozcelik et al. 2002), while cardiomyocyte specific loss of ErbB4 results in dilated
ventricles and electrical disturbances (Garcia-Rivello et al. 2005). ErbB3, though not expressed
in cardiomyocytes, is a key player in valve morphogenesis as germline deletion results in
defective cardiac cushion formation, thus resulting in valve abnormalities and lethality at E13.5
(Erickson et al. 1997; Riethmacher et al. 1997). HB-EGF KO mice have perinatal/postnatal
lethality and demonstrate dilated cardiac chambers and cardiac valve enlargement (Iwamoto et
al. 2003; Jackson et al. 2003). EGFR knockout mice have varying severity of phenotypes
depending on the genetic background, and surviving KO mice have semilunar valve enlargement
(Chen et al. 2000). As the NRG/HB-EGF-ErbB2/ErbB4 axis is required for cardiac development
and maintenance of cardiac function, understanding the signal transduction downstream of the
autophosphorylation events is of interest. Phosphorylated consensus sites on the activated
receptor allow many different adaptor and scaffold molecules to bind, thereby enhancing the
complexity of receptor-ligand interactions; in the case of ErbB2, ShcA is the preferred adaptor,
while Grb2 is preferred by ErbB4 (Schulze et al. 2005). Physiological processes regulated by
NRG-ErB2 signaling are apoptosis (Crone et al. 2002) and myocyte-myocyte/matrix interactions
(Kuramochi et al. 2006); while the molecular mechanisms of NRG signaling are still emerging,
the use of NRG-1 as a restorative agent for diseased myocardium, suggests it may be a clinically
relevant therapy (Pentassuglia et al. 2009), especially as the NRG-1-ErbB4 axis has been shown
to regenerate cardiomyocytes within the myocardium (Bersell et al. 2009).
19
Figure 1.5 Domain structure of tyrosine kinases important in the cardiovascular system
Tyrosine kinase signaling has been shown to be critical in cardiovascular development and
myocardial function. The structure of various TKs outlined in Section 1.4 are shown (adapted
from (Hubbard et al. 2000)).
20
Figure 1.5. Domain structure of tyrosine kinases important in the cardiovascular system
21
1.4.1.2 Platelet-Derived Growth Factor Signaling
The platelet derived growth factor (PDGF) family consists of 4 ligands: PDGF-A, B, C and D,
with the latter 2 being secreted latent factors. These ligands bind to PDGFRα and β and have a
general mitogenic response on mesenchymal cells (Simm et al. 1998; Heldin et al. 1999). PDGF-
B and PDFGR-β are important for development of vascular support cells, while cardiac
overexpression of PDGF- C and D results in cardiac fibrosis and vascular defects (Edelberg et al.
2003; Ponten et al. 2003; Ponten et al. 2005). Conditional alleles, used for cardiac ablation
studies, will allow evaluation of the importance of PDGF paracrine signaling in supporting
vascular integrity within the myocardium.
1.4.1.3 Vascular Endothelial Growth Factor Family
The vascular endothelial growth factor (VEGF) family of receptors consists of VEGF receptors
1, 2,and 3 which can bind the ligands VEGF-A, VEGF-B, VEGF-C, VEGF-D and placental
growth factor (PIGF) (Tammela et al. 2005). While VEGF -C and D bind VEGFR3 to stimulate
lymphangiogenesis, VEGF-A is the primary ligand for hypoxia induced angiogenesis (Tammela
et al. 2005). VEGF-A can form homo or heterodimers with other VEGF ligands and binds
VEGFR1 and 2 in the vascular endothelium to stimulate the formation of new blood vessels.
Germline ablation of VEGF-A results in early embryonic lethality resulting from defects in
endothelial cell development, blood island formation and vascular formation (Ferrara 1999).
Loss of specific VEGF-A isoforms in mice results in impaired myocardial angiogenesis and
ischemic cardiomyopathy (Carmeliet et al. 1999). In the heart, paracrine signaling is paramount
for maintaining vascular-myocyte function. Conditional loss of VEGF-A in the myocardium
showed that the paracrine release of VEGF from myocytes is required for heart function, as loss
of Vegf-A resulted in thin, dilated ventricles due to coronary hypovascularization (Giordano et
al. 2001). The clear role for VEGF signaling in hypoxic induced angiogenesis has lead to
22
therapies designed to increase collateral vessel formation in ischemic heart disease (Carmeliet et
al. 2000; Freedman et al. 2002; Yla-Herttuala 2003).
1.4.1.4 Integrins/FAK/Src Signaling
Integrins are membrane associated receptors that are comprised of heterodimers of α and
β subunits. As 8 β and 18 α subunits consisting of a large extracellular domain connected to a
shorter cytoplasmic domain can dimerize, signal diversity is generated by combinations of
heterodimers and ligand preference. Extracellular matrix proteins such as fibronectin, laminin
and collagen are examples of common ligands (Barczyk et al.; Hynes 2002). In the adult heart,
laminin is bound predominately by α7β1D heterodimer, and signaling is directed by the
consensus motifs in the cytoplasmic domain (Brancaccio et al. 1998; Ross et al. 2001). The β1D
integrin isoform is neccessary for cardiac structure and function as loss of β1D in the postnatal
period results in a dilated cardiomyopathy with enhanced fibrosis deposition (Ross et al. 1998).
Signaling downstream from integrins involves many cytoskeletal proteins such as talin and α
actinin, but of particular importance are the cytoplasmic tyrosine kinase FAK and Src (Manso et
al. 2009). FAK is a FERM domain containing tyrosine kinase, that has a C-terminal Focal
adhesion targeting domain to localize it to costomeres (focal adhesion complexes) in
cardiomyocytes (Mitra et al. 2005). Recruitment to clustered integrins at the costomeres leads to
autophosphorylation of Tyr-397 to allow for the SH2 domain docking of ShcA, Src or p85
subunit of PI3K (Mitra et al. 2005). FAK has been implicated in the hypertrophic response of
cardiomyocytes, particularly upon mechanical stretch (Franchini et al. 2000; Domingos et al.
2002; Torsoni et al. 2003), while selective loss of FAK in the myocardium results in heart
failure after pressure overload (DiMichele et al. 2006; Peng et al. 2006), as well as eccentric
remodeling associated with age (Peng et al. 2006). Downstream effectors of FAK signaling
include ERK 1 / 2 and AKT, thereby impacting both on hypertrophy effectors and apoptosis
(Heidkamp et al. 2002; Brancaccio et al. 2006).
1.4.1.5 Ephrin Family
23
The largest subfamily of tyrosine kinases is Eph receptors, and, with their surface-
associated ligands, ephrins, they specialize in cell-cell communication. Eph receptors initiate
forward signaling in the receptor bearing cell, while reverse signaling occurs in the ephrin
expressing cell (Holland et al. 1996; Kullander et al. 2002). Eph receptors are broken into 2
subclasses where generally EphA (A1-A10) receptors bind ephrin A (A1-A6) ligands and EphB
(B1-B6) receptors bind ephrinB (B1-B3) ligands. Eph receptors are critical in blood vessel
formation as ephrin A1 is expressed in developing vasculature (McBride et al. 1998) and EphB2,
EphB3 and EphB4 along with their ligands ephrin B2 and B1 are important in directing
circulatory system development (Wang et al. 1998; Gerety et al. 1999; Foo et al. 2006; Himanen
et al. 2007). Eph signaling has also been shown to be important in heart morphogenesis and
cardiac function. EphA3 null mice have defects in atrial septa and atrioventricular endocardial
cushions, possibly from impaired epithelial to mesenchymal transformation (Stephen et al.
2007). Cardiac trabeculation during ventricular chamber morphogenesis is critical for embryonic
viability, and recent studies have demonstrated that Notch controls the expression of ephrin B2
to regulate cardiomyocyte differentiation (Grego-Bessa et al. 2007). To date, no conditional
deletion of any ephrin or Eph receptors has been reported in the myocardium; however, given
their importance in development and general cell-cell communication, they are likely to play a
role in maintaining the adult myocardium.
1.4.1.6 Trk Family
Signaling by neurotrophins (NT1-5) is mediated by their binding to one of 3 Trk receptors
(TrkA, B, and C), and is well known for its effects on axon guidance and neuronal development
(Huang et al. 2003). The NT3-TrkC axis has been shown to have a role in embryonic heart
formation, as loss of either NT3 or TrkC results cardiac abnormalities consistent with defects in
cardiac neural crest migration, such as atrial and ventricular septal defects and valvular
abnormalities (Donovan et al. 1996; Tessarollo et al. 1997). While no known role in the adult
myocardium has been reported, the observation that TrkC is expressed in adult cardiomyocytes
suggests it could play a role in a post mitotic myocardium (Kawaguchi-Manabe et al. 2007).
24
1.4.1.7 Insulin Growth Factor Family
The insulin family consists of multiple peptides, such as insulin and insulin growth factors 1 and
2 (IGF1and IGF2). The peptide ligands bind 3 main receptors, the insulin receptor, IGF-1
receptor (IGF1R) and the non tyrosine kinase IGF2-receptor (IGF2R), that exist on the cell
surface as homodimers or heterodimers (Nakae et al. 2001). While loss of insulin signaling
results in the metabolic syndrome of diabetes and obesity (Accili et al. 2001), IGF-1 signaling is
implicated in physiological cardiac growth upstream of AKT (McMullen et al. 2004). Indeed,
loss of the IGF-1R in cardiomyocytes resulted in a blunted hypertrophy response to exercise
(Kim et al. 2008).
1.4.1.8 Fibroblast Growth Factor Family
The fibroblast growth factor (FGF) family consists of 23 related polypeptide growth factors that
bind to receptors FGF receptor 1 and 2 (FGFR1 and FGFR2) (Turner et al.). Fibroblast growth
factor 2 (FGF2) is the most well known in the myocardium and is expressed in multiple cell
types and binds to mainly to FGF receptor 1 (FGFR-1) (Detillieux et al. 2003). FGF-2 has a
protective role in ischemia/reperfusion as mice overexpression FGF-2 show limited injury
following an ischemic event (184). Solidifying its role in hypertrophy signaling, mice deficient
in FGF-2 have a reduced hypertrophy response in a pressure overload model (Schultz et al. 1999)
and a dilated cardiomyopathy phenotype with Angiotensin II stimulus (Pellieux et al. 2001). The
ability of FGF-2 to act in many different cell types gives rise to pleiotropic effects on fibrosis
and inflammation in addition to ischemic injury and hypertrophy signaling (Detillieux et al.
2003).
1.4.1.9 Discoidin Domain Receptor Family
Discoidin Domain Receptors (DDR) 1 and 2 bind collagen and are critical in cell-matrix
interactions (Vogel et al. 2006). While no cardiomyocyte specific knock out exists, the germline
25
deletion of DDR1 results in altered remodeling at atherosclerotic lesions (188) while preliminary
studies suggest a role in ventricular remodeling (Unpublished Results, Vanderlaan, RD; Vogel,
WF; Backx, PH).
1.4.1.10 Ror Family
Ror family receptor (Ror) TKs consist of Ror1 and Ror2 receptors, and in the mouse, Ror1 null
mice display no obvious phenotype, while Ror2 displays skeletal and cardiac defects in
embryonic development (189-192). Mutations in human Ror genes result in Robinow syndrome,
comprised of bone abnormalities with some cardiac findings, and brachydactyly type B, a
syndrome affecting skeletagenesis. While little is known regarding the signal transduction
cascades involved, the Ror family of receptors is important in the pathogenesis of specific hand-
heart syndromes.
1.4.1.11 Abl Kinase
Recently, the nonRTK, c-Abl has been implicated in cardiac function due to the cardiotoxicity
seen with Gleevec in oncology patients (Section 1.4.2) (193). In mice, c-Abl was found to be
important in heart development in a strain-dependant manner. Loss of c-Abl results in perinatal
lethality with dilation of both ventricles and atria that is also accompanied with increased
proliferation of cardiomyocytes and abnormal mitochondria (Qiu et al.). This study provides
insight into the mechanism by which TK inhibitors impact on cardiac function.
1.4.2 Tyrosine Kinase Signaling: Clinical Evidence
Research has shown the importance of tyrosine kinases in oncogenic processes. Mutations in
tyrosine kinases such as the BCR-Abl fusion protein in chronic myleloid leukemia (CML)
(Burke et al.) and over-amplification of the ErbB2 receptor in some breast cancers (Tagliabue et
26
al.) are examples of the pivotal role that TKs can play in the initiation and progression of
oncogenic events. As targeted tyrosine kinase inhibitors (TKI) have been developed, either as
humanized monoclonal antibodies or small molecule inhibitors, the efficacy of these treatments
have been proven; however, a side effect of cardiac disease has been reported in a subset of
patients treated with TKIs. In particular, two TKIs known to cause cardiac side effects are
Herceptin/Trastuzumab and Gleevec/Imatinib, highlighting the central role of TKs in the
maintenance in cardiac function.
1.4.2.1 Herceptin Mediated Cardiotoxicity
TKI cardiac effects were first reported for Herceptin/Ttrastuzumab, a humanized monoclonal
antibody against the ErbB2 receptor. Clinical trials demonstrated left ventricle dysfunction with
an incidence of 4-7% when Herceptin is used alone. When Herceptin is used in conjuction with
chemotherapy, such as anthracyclines, the incidence of ventricular dysfunction increased to 27%
*. Herceptin binds ErbB2-ErbB3 heterodimers (De Keulenaer et al. 2010), and while the direct
mechanism by which Herceptin mediates the cardiotoxicity is unknown, clinical trials with
Pertuzumab, a humanized monoclonal antibody against the dimerization domain of ErbB2, and
Lapatinib, a small molecular inhibitor against the kinase activity of ErbB1and ErbB2, will help
to understand ErbB2 signaling in the myocardium *. Basic science research focused on
understanding ErbB2 signaling networks in the heart will perhaps lead to selective targeting of
ErbB2 signaling to minimize cardiotoxicity.
1.4.2.2 Gleevec Mediated Cardiotoxicity
Recently, the cardiotoxicity seen in Gleevec/Imatinib patients, has further focused the
importance of TKs in cardiac maintenance. Imatinib is an Abl kinase ATP competitive small
molecule inhibitor used in the treatment of CML. Other tyrosine kinases inhibited are Abl-related
27
gene (ARG), the PDGF receptors α and β and KIT (Cheng et al.). While the overall incidence of
cardiotoxicity reported for Imatinib is low (1%) (Force et al. 2007), many feel this is an
underestimation due to short nature of monitoring in clinical trials, the lack of specific cardiac
end points and the sample bias of patients entering clinical trials (Force et al. 2007). Although
the exact mechanism by which Imatinib mediates cardiotoxicity is poorly understood, one study
has shown that cultured myocytes treated with Imatinib have significant mitochondrial
dysfunction. In mice receiving Imatinib, mitochondrial dysfunction can be recapitulated, and
clinically, heart biopsies taken from 2 patients administered Imatinib demonstrated non-specific
abnormalities in mitochondria while taking Imatinib (Kerkela et al. 2006). While the incidence
of HF remains low in current TKI clinical trials, the potential for retrospective reports of
cardiotoxicity is high, especially if multikinase TKIs are utilized (Force et al. 2007). Therefore,
understanding downstream signaling from TKs in the myocardium will help to design
therapeutics that minimizes side effects and potential cardiotoxicities.
1.5 ShcA, an Adaptor for Tyrosine Kinases
Adaptor proteins are critical signaling molecules downstream of TKs, as their recruitment
localizes and coordinates signal transduction networks. Adaptor proteins are non-enzymatic
signaling molecules that are modular in nature, in that they contain multiple protein domains and
regulatory sites for binding other proteins and phospholipids (Pawson et al. 1997). Examples of
adaptor proteins include Grb2, Shc, Nck and Crk. Of particular interest in this thesis is the
broadly expressed adaptor/scaffold molecule ShcA, Src homology 2 containing transforming
protein 1(Shc1), and how its two phospho-tyrosine recognition domains mediate downstream
signaling from TKs in the myocardium.
1.5.1 ShcA Isoforms and Signaling Domain Architecture
ShcA is a member of the Shc family that contains ShcB, ShcC and ShcD. While ShcB, C and D
are expressed in neuronal tissue, with ShcD also expressed in the neural crest lineage, ShcA is
found in most tissues (Ravichandran 2001). ShcA has 3 isoforms: p66, p52 and p46. The p52/46
28
isoforms result from translation initiation sites within the same transcript, while the p66 isoform
arises from induction at a distant promoter site (Migliaccio et al. 1997) (Figure1.6). All 3
isoforms contain an N terminal phospho-tyrosine binding (PTB) domain (truncated in p46) and C
terminal Src homology 2 (SH2) domain with an intervening collagen homology region (CH1);
the p66 isoform is unique in that it has an additional N terminal collagen homology 2 region
(CH2) (Figure 4). Given the modular architecture of ShcA, many different signaling partners and
downstream pathways can be utilized, in a context-dependant manner. In the myocardium, the in
vivo role of ShcA remains largely unexplored; therefore, to begin to understand the biological
significance of ShcA in the myocardium, one must first consider its domain architecture.
1.5.1.1 The PTB domain of ShcA
PTB domains are 100-170 amino acid sequences that typically bind phosphopeptides with the
NP-X-pY motif (X is any amino acid). Additional specificity is conferred by hydrophobic
residues 5 amino acids C terminal to the pTyr (Ravichandran 2001). The first structure of the
PTB domain of ShcA was completed in 1995 in complex with the TrkA phosphopeptide (Zhou
et al. 1995). This enabled the tertiary structure of a β sandwich comprised of 2 nearly
orthonganol β sheets and 3 α helices to be described. The phosphotyrosine (pTyr) binding site is
formed by a cleft of β5 and the C terminal of α3. Zhou and colleagues showed that the
phophopeptide forms a β turn to fit precisely into the binding pocket, establishing interactions
with Arg 67, Ser 151, Lys 169 and Arg 175 (Zhou et al. 1995). Of these residues, Arg 175 was
found to be required for pTyr binding as site directed mutagenesis of Arg 175 to glycine or
lysine resulted in loss of phosphopeptide binding. This is recapitulated in Drosophila studies
where a mutation in the site analogous to R175 inactivates ShcA function (Ravichandran 2001).
Despite sharing little sequence similarity, the topology of the PTB domain closely
resembles that of a pleckstrin homology domain, which binds acidic phospholipids
(Ravichandran 2001). Phospholipid-binding by the ShcA PTB domain could allow membrane-
protein interactions, perhaps in the absence of phosphopeptide binding, and could explain the
localization of ShcA at membranes in unstimulated cells (Ravichandran 2001). While initial
observations for ShcA binding was shown for TrkA and middle T antigen, ShcA was also shown
29
to bind the EGFR, ErbB3, cytokine receptors for GM CSF and IL2, ErbB2 and ErbB4, amongst
others (Ravichandran 2001) (See Table 1.1 for complete list of interactions).
30
.
Figure 1.6 ShcA locus and domain architecture.
(A) ShcA locus and exon arrangement in ShcA transcripts. P66ShcA transcript is formed by
exons 2-13, while p52/p46 transcripts are formed by exons 1-2a and 3-13 through alternative
splicing. ShcA isoforms have a common domain architecture containing a PTB and SH2 domain
separated by an intervening CH1 region. P66ShcA has an additional CH2 domain, while p46 has
a truncated PTB domain. (B) ShcA pTyr signaling. The phosphotyrosine recognition motifs bind
to consensus motifs on activated TKs, while subsequent phosphorylation leads to activation of
downstream signaling pathways
31
Figure 1.6. ShcA locus and domain architecture.
32
1.5.1.2 The SH2 domain of ShcA
SH2 domains are ~100 amino acid elements that bind to phosphotyrosine motifs and are
divided into 2 broad groups: group I have consensus sites pY-φ-θ-φ (φ denoting hydrophobic
residues and θ as hydrophilic residues) and group II SH2 domains which prefer pY-φ-x-φ
(Marengere et al. 1994). SH2 domains typically have a central antiparallel β sheet flanked by 2 α
helicies, and SH2 domains bind the pTyr containing sidechain into their pTyr binding pocket by
coordinating the negatively charged phosphate with the positive charge on the sidechain of Arg
βB5 (Marengere et al. 1994), which is part of the signature FLVR sequence found in most SH2
domains (Blaikie et al. 1994). Loss of this critical anchor through point mutation prevents SH2
recognition of phosphoproteins (Mayer et al. 1993). The SH2 domain of ShcA is located at the
C-terminus, and preferentially binds pY -X- X- Ile/Leu (Songyang et al. 1993; Songyang et al.
1994). While sharing much homology with other SH2 domains, the SH2 domain of ShcA has a
leucine at βD5, where βD5 residues influences phosphopeptide binding specificity (Zhou et al.
1995). The SH2 domain of ShcA has relatively low binding affinity for phosphopeptides, and
may use its N terminal phosphotyrosine binding domain to help augment recruitment of ShcA to
signaling networks (Ravichandran 2001). Initially, the SH2 domain of ShcA was shown to bind
EGF, PDGF and the ζ chain of the T-cell receptor, but subsequently, many other binding partners
have been demonstrated (Ravichandran 2001) (See Table 1.2 for a complete list). Of interest, the
VEGF family, EGF family, and PDGF family of receptors have the potential to influence cardiac
structure and function.
1.5.1.3 The CH1 Region of ShcA
The collagen homology region is a 52-167 amino acid region that like its namesake
collagen, is highly enriched in prolines and glycines. Within the CH1 region of ShcA, there are
critical tyrosine residues for mediating downstream phosphotryosine signaling, primarily through
recruitment of Grb2, a SH3-SH2-SH3 adaptor protein. These tyrosine residues, Y239/Y240 and
Y313 (denoting mouse nomenclature), may represent a mechanism that has evolved in multi-
cellular organisms to increase the complexity of signaling (Luzi et al. 2000). While all three
33
tyrosines are present in higher mammals, Y239/240 are present in Drosophila but absent in
nematodes, and Y313 is generally absent in “lower” metazoans (Luzi et al. 2000). Given the
requirement for CH1 phosphotyrosine signaling in only some morphogenic processes (Hardy et
al. 2007), the evolutionary advantage conferred by pTyr sites in the CH1 region could be through
the introduction of specificity to signal transduction downstream of activated TKs.
Y239/240 and Y313 are phosphorylated by a variety of receptor kinases, and mutagenesis
studies suggest that these are essential for ShcA mitogenic signaling leading to Ras activation.
Upon receptor tyrosine kinase activation, phosphorylation of tyrosines in their cytoplasmic tail
creates docking sites for either the PTB or SH2 domain of ShcA. ShcA is subsequently
phosphorylated in its CH1 region to create consenus motifs (pY-X-Asn) for Grb2-SH2 domain
binding. The Ras guanine nucleotide exchange factor SOS (Son of Sevenless) is recruited by the
SH3 domain of Grb2, and localized to the plasma membrane (Ravichandran 2001). The TK-
ShcA-Grb2-SOS complex thus allows for Ras activation by conversion of GDP bound Ras to the
active GTP bound state. ShcA has also been shown to activate PI3K signaling via Grb2-Gab
associations, whereby phosphorylation of the pTyr sites in the CH1 region can potentially lead to
the recruitment of Grb2-Gab complexes to activate AKT signaling (Ravichandran 2001).
While Grb2-MAPK activation is the predominant signaling pathway from the CH1
region, other studies have shown that pY239/240 can lead to c-Myc activation (Gotoh et al.
1997). A diverse array of phosphopeptides can also bind to the pTyr sites in the CH1 region of
ShcA, however the functional relevance of these is still unknown(van der Geer et al. 1996). In
addition to SH2 binding motifs, the CH1 region also has several P-X-X-P motifs for SH3 binding
(Weng et al. 1994) and a documented adaptin binding site (Okabayashi et al. 1996). Recent
studies suggest that IQGAP links ShcA to the actin cytoskeleton through a non-canonical
interaction with the PTB domain (Smith et al.) (see Table 1.3). Therefore, ShcA is able to
coordinate multiple different signal transduction networks when recruited to upstream TKs. The
importance of these, in the context of the myocardium, is largely unexplored.
34
Table 1.1 ShcA PTB domain interaction partners
PTB domain interaction partner Reference Cardiovascular phenotype
APP (amyloid β (A4) precursor protein) (Zambrano et al. 1997; Tarr et al.
2002)
Tetraspanin CD81 (Carloni et al. 2004)
CSF2RB (β subunit of IL-3 R) (Bone et al. 2000)
DDR1 (Discoidin domain receptor) (Vogel et al. 1997; Foehr et al. 2000)
Section 1.4.1.9
EGFR (O'Bryan et al. 1996; Sakaguchi et
al. 1998)
Section 1.4.1.1
EphA2 (Pratt et al. 2002)
ERBB2 (Olayioye et al. 1998)
Section 1.4.1.1
ERBB3 (Vijapurkar et al. 1998)
Section 1.4.1.1
ESR1 (ERα) (Song et al. 2002) Associated with Coronary artery disease(Kunnas et al.)
IGF-I receptor (Dey et al. 1996) Section 1.4.1.7
IL-2R β chain (Ravichandran et al. 1996)
IL-4Rα (Ikizawa et al. 2000)
Insulin Receptor (Wolf et al. 1995; Sasaoka et al. 2000)
Section 1.4.1.7
β 3 integrin (Cowan et al. 2000) Section 1.4.1.4 and (Ren et al. 2007)
β 4 integrin (Dans et al. 2001)
SHIP (SH2 containing inositol phosphatase)
(Damen et al. 1996; Lamkin et al. 1997)
35
LDL receptor-related protein 1 (Barnes et al. 2003)
Mpl (myeloproliferative leukemia virus oncogene)
(Drachman et al. 1997)
TrkA (Dikic et al. 1995; Obermeier et al.
1996)
Section 1.4.1.6
TrkC (Guiton et al. 1995) Section 1.4.1.6
PP2A (Ugi et al. 2002) Dephosphorylates myofilament cTnI and MyBP-C (Solaro et al.
2002)
PTP-PEST (PTPN12) (Habib et al. 1994; Faisal et al. 2002)
T-cell protein tyrosine phosphatase (PTPN2)
(Tiganis et al. 1998)
ZAP-70 (Lck, Syk) (Pacini et al. 1998; Walk et al. 1998)
Table 1.2. ShcA SH2 domain interaction partners
SH2 domain interaction partner Reference Cardiovascular Phenotype
CEACAM1 (carcinoembryonic antigen-related cell adhesion molecule 1)
(Poy et al. 2002)
AP2A1/2 (Adaptin) (Okabayashi et al. 1996)
AXL tyrosine kinase (O'Bryan et al. 1996)
Cbl (Fukazawa et al. 1996; Park et al.
1999)
Noonan-like syndrome (Martinelli et al.)
CD22 (Poe et al. 2000)
CD3E (CD3ε) (Guirado et al. 2002)
36
CD3Z (CD3ζ) (Labadia et al. 1996)
CDH5 (cadherin 5, type 2) (Zanetti et al. 2002)
CSF1R (colony stimulating factor 1 receptor)
(Lioubin et al. 1994)
CSF2Rβ (β subunit of IL-3 R) (Bone et al. 2000)
CSF3R (G-CSF receptor) (Ward et al. 1998)
DAG1 (β-Dystroglycan; dystroglycan 1) (Sotgia et al. 2001) Cardiomyopathy/Muscular dystrophy (Lapidos et al. 2004)
DDR2 (discoidin domain receptor) (Ikeda et al. 2002) Section 1.4.1.9
EphA2 (Pratt et al. 2002)
Erythropoetin receptor (Damen et al. 1993)
ESR1 (ERα) (Song et al. 2002)
Fc γRIIb (Koncz et al. 1999)
FcγRIII (CD16) (Galandrini et al. 1997)
Flt1 (Warner et al. 2000)
Section 1.4.1.3
Flt4 (Fournier et al. 1995)
Section 1.4.1.3
Gab1 (Ingham et al. 1998)
Growth hormone receptor (VanderKuur et al. 1995;
Moutoussamy et al. 1998)
β4 integrin (Dans et al. 2001) Section 1.4.1.4
Jak2 (VanderKuur et al. 1995)
Section 1.2.3
Lyn (Ptasznik et al. 1995)
37
HGF receptor (Pelicci et al. 1995)
STK/RON tyrosine kinase (Iwama et al. 1996)
PDGF β-receptor (Yokote et al. 1994) Section 1.4.1.2
Fak (Hecker et al. 2002) Section 1.4.1.4
Pyk2 (Lev et al. 1995)
Ret (Borrello et al. 1994)
Tie2 (Audero et al. 2004)
Section 1.4.1.3
PAL (Protein expressed in Activated Lymphocytes
(Schmandt et al. 1999)
SLP-76 (Chu et al. 1998)
Table 1.3. ShcA interactions by conserved signaling motifs
Interaction partner Interaction Region/motif References Cardiovascular phenotype
Crk CH1 pTyr sites for SH2 domain binding
(Matsuda et al. 1994; Gesbert et al. 1998)
Embryonic cardiac dilation and thin ventricles (Park et al. 2006)
CrkL CH1 pTyr sites for SH2 domain binding
(Chin et al. 1997)
Eps8 PXXP motif for SH3 domain binding
(Matoskova et al. 1995)
Candidate Noonan syndrome gene (Ion et al. 2000)
38
Src, lyn, fyn PXXP motif for SH3 domain binding;
(Weng et al. 1994; Sato et al. 2002)
Section 1.4.1.4
Grb7 CH1 pTyr sites for SH2 domain binding
(Yokote et al. 1996)
Inhibitory function on cardiac K channels (Ureche et al. 2009)
Grb2 CH1 pTyr sites for SH2 domain binding
(Nicholson et al. 2001)
Section 1.2.2
Shp-2 (PTPN11) Binds pTyr sites on ShcA with its SH2 domains
(Eck et al. 1996) Noonan and LEOPARD syndromes (Neel et al. 2003)
Grap Binds pTyr sites on ShcA with its SH2 domains
(Trub et al. 1997)
Gads Binds pTyr sites on ShcA with its SH2 domains
(Liu et al. 1998)
MAPKAP kinase 2, (MK2)
Outside of pTyr binding motif on the PTB domain
(Yannoni et al. 2004)
Section 1.2.2 (Streicher et al.)
IQGAP1 Outside of pTyr binding motif on the PTB domain
(Smith et al.)
1.5.1.4 The CH2 region of ShcA
The second collagen homology region is found only in p66 ShcA at its N terminus and
also in the long forms of ShcB and C (205). It is thought to be recently acquired in vertebrates, as
it is lacking in Drosophila Shc protein (Luzi et al. 2000). The CH2 region is linked to the
oxidative stress pathway, as selective loss of the p66 isoform results in mice that are long lived
with reduced levels of intracellular free radicals (Migliaccio et al. 1999; Pinton et al. 2008). It
has been demonstrated that p66ShcA is phosphorylated at serine 36 in response to exposure of
H2O2, PMA or UV irradiation and is implicated in apoptosis signaling through p53 mediated
39
increase in intracellular reactive oxygen species (ROS) (Trinei et al. 2002; Pacini et al. 2004).
P66ShcA also participates in the oxidative stress response by inhibiting FOXO transcription
factors (Nemoto et al. 2002) and localizing to the mitochondria membrane to aid in
depolarization (Orsini et al. 2004; Giorgio et al. 2005). Overall, the loss of p66 ShcA confers
protection from conditions related to oxidative stress damage (Purdom et al. 2003; Cosentino et
al. 2008).
1.5.2 Biological Role of ShcA Signaling
The first biological role for ShcA was shown by Lai and Pawson in 2000 (Lai et al. 2000). Mice
homozygous for the ShcA null allele are embryonic lethal at E12.5 resulting from defects in the
cardiovascular system. ShcA null mice demonstrated dilated ventricles and poorly developed
vasculature; the developing myocardium displayed decreased trabeculation in the ventricles at
E10.5 and hypoplastic endocardial cushions resulting in poor contractility and cardiac
congestion. Whole mount RNA in situ hybridization demonstrated ShcA is highly expressed in
the developing cardiovascular system, specifically in the endothelium and mesenchymal cells.
The vasculature in these mice was found to have decreased complexity and aberrant cell-cell
contacts, owing to defects in late angiogenic remodeling. As ShcA is important for MAPK
activation, Lai and Pawson demonstrated decreased MAPK activation in the developing
cardiovascular system, while in embryonic fibroblasts, ShcA sensitized cells to growth factor
stimulation.
To circumvent the embryonic lethality and to address more specific roles for ShcA in
vivo, a ShcA allele series was generated that allowed for dissection of pTyr requirements of
ShcA through mutant Knock-in (KI) models, as well as conditional excision of ShcA by use of
loxP /Cre recombinase technology (Hardy et al. 2007). This allows tissue and temporal
specificity of ShcA signaling to be addressed.
The ShcA KI models contained individual point mutations that precluded 1) pTyr binding
of the PTB domain to upstream TKs by mutating arginine 175 to glutamate (δPTB), 2) pTyr
binding of the SH2 domain by mutation of arginine 397 to lysine (δSH2) and 3) downstream
signaling from the tyrosines in the CH1 region by mutating tyrosines 239/240/313 to
40
phenylalanine (1F (Y313F), 2F (Y239/240F) and 3F (Y239/240/313F). Mice homozygous for
the PTB mutation phenocopied the ShcA null mouse, demonstrating a requirement for TK
signaling in heart development, which is myocardial-specific (Hardy et al. 2007). Surprisingly,
mice homozygous for the 3F allele showed normal heart development, suggesting an alternative
downstream signaling mechanism is required for heart development.
Mice homozygous for the ShcA 3F and SH2 alleles were viable, but postnatal
phenotyping revealed severe coordination defects. ShcA 3F/3Fmice failed to develop normal
muscle spindles, while ShcA SH2/SH2 mice had defects in spindle morphogenesis. Both
genotypes had altered synaptic conductivity of group Ia sensory neurons. Using the conditional
allele (ShcA flx/flx) in conjuction with the muscle specific Mlc1f Cre, Hardy et al demonstrated
that ShcA functions in the stretch reflex circuit and requires signaling from both the PTB domain
and protein- protein interactions downstream of the pTyr sites in the CH1 region using a novel
genetic strategy (Hardy et al. 2007). These studies not only highlight the diversity of ShcA
signaling requirements for various morphogenic processes in different tissues, but also the
potential for a ShcA signaling independent of Grb2.
The emerging theme that CH1 signaling is context dependant was solidified by
experiments demonstrating differential signaling from the CH1 region in an ErbB2 breast cancer
model (Ursini-Siegel et al. 2008). By using the phospho-deficient 3F ShcA alleles, Ursini-Siegel
et al showed differential pTyr signaling requirements in early stages of mammary tumour
progression: Y313 is critical for tumor cell survival, while Y239/240 augments tumour
vascularization. In vitro studies also have demonstrated differential signaling from the pTyr
residues based on receptor type (Gotoh et al. 1997; Pratt et al. 1999). These studies demonstrate
that ShcA is critical in coordinating morphogenic processes in multiple tissues. They also
highlight the complexity of signaling as ShcA can recruit downstream effectors either through
pTyr sites in the CH1 region or other signaling motifs.
These studies also demonstrated the importance of reciprocal signaling in tissue
physiology. Hardy and collegues demonstrated that the loss of SH2 signaling in the muscle
compartment acts to limit paracrine signaling required in proper spindle development. Ursini-
Siegel and collegues also showed that loss of Y313 signaling in the stroma can limit tumour
progression by disrupting cellular communication between the endothelial and myoepithelial
41
compartments. ShcA is therefore an important mediator of TK signaling that maintains cell
autonomous and non-cell autonomous effects.
1.5.3 The Role of ShcA in the Myocardium
The importance of ShcA signaling in the developing cardiovascular system and the strong
requirement for TK signaling in the myocardium suggests that ShcA could play a role in the
postnatal myocardium. While no in vivo data have been reported for the conditional loss of ShcA
in the functioning myocardium, there have been several reported roles for ShcA in the
cardiovascular system. One of the most explored roles for ShcA is that of p66ShcA in
cardiovascular diseases associated with free radical damage. The discovery that serine36
phosphorylation of p66 ShcA leads to its translocation into the mitochondria to induce ROS
generation and apoptosis (Pinton et al. 2007) has offered mechanistic insight into the protection
incurred by germline ablated p66 ShcA mice in conditions of ROS damage, such as diabetic
cardiomyopathies (Rota et al. 2006), endothelial dysfunction (Camici et al. 2007), atherosclerosis
(Napoli et al. 2003), ischemia-reperfusion (Zaccagnini et al. 2004) and AngII-mediated
myocardial remodeling (Graiani et al. 2005). In vitro data have placed ShcA downstream of
thrombin activation of the GPCR PAR-1 (Obreztchikova et al. 2006), while a role for ShcA in
integrin /FAK signaling has also been described in feline pressure overload studies (Barberis et
al. 2000; Laser et al. 2000). These studies point to an important role for ShcA in the
myocardium, however, little is known regarding the signaling mechanisms by which ShcA
mediates its effects or the physiological implications within the myocardium. Therefore, to
address this, a conditional gene targeting strategy must be employed to define tissue specificity
and circumvent embryonic lethality.
1.5.4 Strategies for Condition Gene Targeting in the Myocardium
As germline deletion of key signaling molecules can result in embryonic lethality or phenotypic
ambiguity due to cellular heterogeneity within the tissue of interest, conditional gene targeting
approaches are preferred. In the heart, choice of Cre transgenic mouse models allow for spatial
42
regulation and the use of ligand inducible systems refine excision, allowing for cardiomyocyte
specific excision by Cre/loxP recombination. Techniques for inducing point mutations are
currently evolving (Bayascas et al. 2006) and new recombination methods await rigorous in vivo
testing (Feil 2007).
The main system to achieve conditional allele activation is through LoxP/Cre
recombinase technology. Cre is a bacteriophage P1 gene that recognizes a 34 bp site designated
LoxP and catalyzes the reciprocal DNA recombination between 2 loxP sites (Sauer 1998). The
directionality and lack of requirement for host factors to mediate efficient recombination make it
suitable for eukaryotic cells (Sauer 1998). LoxP/Cre recombination can be used to ‘turn on’
genes by excision of a STOP cassette between a promoter and its transgene or ‘turn off’ genes by
excision of targeted alleles. Conditional targeting of genes of interest in the myocardium has
allowed for regulation, as the choice of the promoter used to drive Cre expression determines the
developmental window for the phenotype and specific cell lineage. To circumvent any
developmental deficiencies, one can use a ligand inducible Cre system such that Cre expression
is acutely turned on by drug administration (Table 4).
Table 1.4. Cardiomyocyte specific Cre transgenic mouse models
Heart Specific Cre Promoters
Temporal Expression Pattern Tissue Expression Pattern
Reference
Isl1 Early Embryonic 1st and 2nd heart field (Ma et al. 2008)
Nkx2.5 E9.5-adulthood Cardiomyocyte (Moses et al. 2001)
β-MHC E8.5-birth/perinatal
Reactivated in disease states
Cardiomyocyte
Fetal: V>A
(Lyons et al. 1990; Parsons et al. 2004)
α-MHC
E8.5 to midgestation
Reactivated at birth
Downregulated with disease
Cardiomyocyte
Fetal: A>V
Adult: V and A
(Agah et al. 1997; Wang et al. 2005)
MLC2v Fetal ( E10-E15) to adulthood Cardiomyocyte (Chen et al.
43
(Ventricular) 1998)
Cardiac Troponin I
Fetal <neonate<adult
Cardiomyocytes
V and A
(Opherk et al. 2007)
Inducible Systems
Temporal Expression Pattern Tissue Expression Pattern
Reference
α- MHC CrePR1
Post RU486 administration
Cardiomyocyte
(Minamino et al. 2001)
α-MHC-tTA Post Doxycycline administration
Cardiomyocyte (Yu et al. 1996)
α-MHC MerCreMer
4 days post Tamoxifen administration
Cardiomyocyte (Sohal et al. 2001)
TnT-iCre Post Doxycycline administration
Cardiomyoyctye (Wu et al.)
A, atrial cells; V,ventricular cells; MHC, myosin heavy chain; MLC, myosin light chain; Isl, Islet ; TnT, troponin T
1.6 Rationale and Objectives
The germline deletion of ShcA demonstrated a requirement for ShcA in the developing
cardiovascular system, but due to the embryonic lethality, the defined roles of ShcA in the
postnatal myocardium have remained elusive. Therefore, conditional ablation of ShcA in the
myocardium is required to fully understand the role of ShcA in cardiac structure and function.
Genetic analysis of embryonic heart development demonstrated the CH1 pTyr sites of ShcA are
not required for heart morphogenesis, therefore, generation of cardiac-specific mutant ShcA KI
mice using the ShcA allelic series would allow for evaluation of the PTB and SH2 domains of
ShcA in coupling to upstream TKs and the importance of the pTyr sites in the CH1 region in
cardiac structure and function. As ShcA has the potential to signal downstream of multiple TKs
in the myocardium, and can activate Erk- MAPK and PI3K pathways, these studies can further
our understanding of pTyr signaling networks in the myocardium. Of clinical importance, the
44
ability of ShcA to be recruited to activated ErbB2 suggests it could be relevant to Herceptin-
mediated cardiotoxicity, thereby furthering our understanding of the effects of TKIs in the
myocardium. Therefore, the objectives of this thesis are: 1) to understand the role of ShcA in the
postnatal myocardium by evaluating its effects on cardiac structure and function, 2) to explore
the molecular mechanism by which ShcA effects cardiac structure and function, and lastly 3) to
understand how ShcA coordinates its effects on cardiac structure and function through its various
signaling domains and motifs. To this end, ShcA was conditionally excised in ventricular
cardiomyocytes and the ShcA allelic series (Hardy et al. 2007), which is comprised of ShcA KI
alleles each containing discrete point mutations that inactivate specific domains or motifs, were
restricted to ventricular cardiomyocytes only and evaluated for effects on cardiac structure and
function.
Our studies demonstrate that the loss of ShcA in ventricular cardiomyocytes leads to a
dilated cardiomyopathy characterized by elevated cardiomyocyte contractility and impaired
myocyte-matrix interactions. In vivo dissection of ShcA signaling properties revealed that
selective inactivation of the PTB domain in the myocardium had effects resembling those seen in
ShcA CKO mice, while disruption of the SH2 domain caused a less severe cardiac phenotype.
Downstream signaling through the CH1 pTyr sites was dispensable for baseline cardiac function,
but necessary to prevent adverse remodeling after hemodynamic overload. Therefore, ShcA
mediates pTyr signaling in the adult heart through multiple distinct signaling elements that
control myocardial functions and response to stresses.
45
Chapter 2
Phenotypic Analysis of the ShcA Allele Series
46
2 Phenotypic Analysis of ShcA Allele Series
This section will highlight all the methods used to do the experiments in the subsequent data
chapters.
2.1 Mouse Models
All mice were housed in a pathogen-free facility (Department of Comparative Medicine,
University of Toronto) and handled using standard protocols in accordance with animal welfare
regulations. All study protocols were approved by the Animal Care Committee at the University
of Toronto.
The ShcA allele series was generated by W.R Hardy in the Pawson lab and targeting was
described elsewhere (Hardy et al. 2007). The ShcA allele series consists of the ShcA floxed
allele and mutant domain KI alleles of ShcA (Hardy et al. 2007). The Mlc2v Cre KI mouse
(Chen et al. 1998) and α MHC- MerCreMer transgenic mouse (Sohal et al. 2001) were used to
conditionally excise ShcA in the myocardium through Cre/loxP strategies. Targeting and
recombination strategies were previously described (Hardy et al. 2007) and outlined in results
section. All mouse lines were bred in a mixed background and used appropriate littermate
controls.
Mouse tails were used for PCR amplification of the various genotypes. In short, mouse
tails were digested in SDS tail buffer (50Mm Tris (pH 8.5), 0.5mM EDTA, 0.1% SDS, 0.1M
NaCl with 100ug/ml Proteinase K) diluted and deactivated at 100οC for 15 minutes. Genotyping
primers distinguishing the KI and wild type alleles and the various Cre recombinase lines were
as previously described (Chen et al. 1998; Sohal et al. 2001; Hardy et al. 2007). DNA
sequencing from tail samples confirmed the individual mutations as previously reported (Hardy
et al. 2007). The excision of the ShcA floxed allele was confirmed using genomic DNA as
previously described (Hardy et al. 2007).
2.2 Cardiac Phenotyping
47
Cardiac function is determined by various levels of regulation. Depending on the context, cardiac
contractility can have many layers of compensation built in to maintain cardiac homeostasis.
Systemic regulation, external to the heart itself, involves autonomic nervous system modulation
and volume regulation at the level of the kidneys, while effective contraction at the tissue level
requires proper electrical and mechanical coupling. Electrical propagation ensures synchronous
and progressive waves of depolarization and repolarization with each heart beat, while effective
mechanical force transmission requires intact extracellular matrix that is continuous along the
myocytes. At the most fundamental level, cardiomyocyte contractility, governed by calcium
handling and myofilament interactions, determines the ability of the individual myocyte to
contract. Therefore, to understand heart function in various mouse models, one must look at each
level of regulation to understand the various contributions, which ultimately lead to the
phenotype.
2.2.1 Global Cardiac Function Methods
At the organ level, the heart must function to meet the systemic needs of the body, while
maintaining beat to beat regulation. As an organ, global cardiac function is determined by the
heterogeneous cellular composition and is also subject to regulation extrinsic to the myocardium
itself. For example, in the face of disease, systemic regulation (sympathetic stimulation) can
augment poor cardiomyocyte function to maintain cardiac homeostasis. To ascertain the global
cardiac function in the mouse, echocardiography, cardiac catheterization and transverse aortic
constriction were employed. These methods look at characterization of left ventricle function, the
main systolic pumping chamber, to infer global heart mechanics.
2.2.1.1 Echocardiography of the Mouse Heart
Transthoracic echocardiography allows for in vivo assessment of cardiac function under
physiological conditions and also the serial evaluation of disease states. Echocardiography
visualizes the left ventricle dimensions during the cardiac cycle , thereby assessing dimensions
48
during the ejection phase (systole) and during the relaxation phase (diastole). Trans thoracic
echocardiography was done as previously described (Crackower et al. 2002). In brief, mice were
anaesthetized with 2% isoflurane, shaved in the pericardial region and then brought to a semi-
conscious state with 0.6% isoflurane. Mice were maintained at homeostatic conditions with the
use of a heating pad and low isoflurane with a target heart rate between 450 to 650 beats per
minute( bpm). Imaging was recorded on the Sequoia C256 ultrasound machine (15L8 transducer,
Acuson). For visualization and assessment of left ventricular systolic function, as measured by
the degree of reduction of chamber dimension, mice were placed in a lateral dicubitus position
and the transducer was placed to first visualize wall motion in the M-mode view at the mid
papillary level and then positioned to read the aortic velocities by Doppler in the 2 dimensional
plane using the parasternal short axis view. 3 independent views were obtained for each position.
Analyses of 3 consecutive cardiac contractions were averaged using the Sequoia platform and
standard parameters of cardiac structure and function were assessed. Parameters included left
ventricular end diastolic and systolic dimensions ( LVEDD and LVESD, respectively), anterior
and posterior wall thickness and % fractional shortening (LVEDD-LVESD)/LVEDD*100), heart
rate (beats per minute) and velocity of circumferential shortening (VCF= %FS/AoV). Doppler
imaging evaluates the aortic velocities (AoV) and time to peak ejection amplitude (ET) for
subsequent estimations of ejection performance. Parameters recorded on tables are: LVEDD,
left ventricular end diastolic dimension; LVESD, left end systolic dimension; %FS, % fractional
shortening; VCF, velocity of circumferential shortening; Ant, anterior; post, posterior.
2.2.1.2 Cardiac Catheterization
Cardiac Catheterization allows for detection of pressure development within the aortic and left
ventricular compartments.A pressure transducer is introduced by a wire catheter to measure the
hydrostatic pressure responsible for blood ejection within the left ventricle. These surgical
procedures were done by Dr. M.G. Kabir at the Heart and Stroke Richard Lewar Centre of
Excellence at the University of Toronto as described previously (Crackower et al. 2002). In
brief, mice for the various time points were anesthetized with inhaled isoflurane (3% induction,
1% maintenance) placed in a supine position and a small midline incision from the peristernal
notch toward the mandible was made to expose the right common carotid artery and a 1.4 French
49
pressure transducer catheter (Millar Instruments) was inserted in a 1mm incision. Retrograde
advancement of the catheter into the ascending aorta and subsequently through the aortic valve
into the left ventricle allowed for 3 independent recordings of aortic and left ventricle pressures
to be collected. Measurements were recorded at a stable internal temperature of 37ºC with a
target heart rate above 350 bpm. Both aortic and left ventricle parameters were recorded.
Dobutamine challenge (3.0mg/kg body weight) was injected in the peritoneal cavity and
measurements were recorded at baseline and 20 minutes after injection. Parameters recorded on
tables are: LVESP, Left ventricular end systolic pressure; LVEDP, Left ventricle end diastolic
pressure; dP/dT max, first derivative of maximal pressure development in left ventricle; dP/dT min,
first derivative of minimal pressure in left ventricle.
2.2.1.3 Transverse Aortic Constriction Model
To analyze the effect of biomechanical stress on the remodeling processes of the left ventricle in
the various genetic models, a minimally invasive transverse aortic constriction procedure was
employed. These surgical procedures were done by Dr. M.G. Kabir at the Heart and Stroke
Richard Lewar Centre of Excellence at the University of Toronto as described previously
(Crackower et al. 2002). Briefly, 25-30g male mice were anesthetized (3% isoflurane) and
intubated. Once placed on a respirator and held at a stable internal temperature of 37οC, a small
incision above the suprasternal notch was made to expose the aortic arch. A 6.0 mm silk suture
was positioned under the aorta with a blunted 27 gauge needle to set the diameter of the ligation.
Sham animals underwent every aspect of the surgery with the exception of the ligation
procedure. The animal was then sutured closed and allowed to recover in a heated environment.
2.2.2 Tissue and Cellular Physiology Methods
To isolate various physiological parameters of cardiac muscle, tissue physiology and isolated
myocyte function were investigated. This allowed for tissue mechanics and cardiomyocyte
contractility to be evaluated independent of integrated systemic compensation responses.
50
2.2.2.1 Isolated Ventricular Muscle Preparation Methods
The heart possesses sturcutes that have myocardium arranged into linear strips. One example is
the papillary muscule which assists in holding the valves in place during cardiac ejection.
Trabeculae are small ultrathin myocardial strips that prevent inversion of the mitral and tricuspid
valve during sytole. The force length relationship was investigated in papillary and trabeculae
muscles to evaluate the contribution of extracellular matrix to the passive tension. The force
length relationship in tissue preparations, allows for isolation of the mechanical components of
the Frank Starling principle, which states an increase in end diastolic volume will necessitate an
increase in pressure generation. At the tissue level, this translates to increased force as sarcomere
length is increased.
Adult mice (10-12wks) were killed by cervical dislocation. The hearts were rapidly
excised and cannulated and perfused with a cardioplegic Krebs solution (112.5mM NaCl, 4.7mM
KCl, 1.2mM MgCl2, 1.2mM KH2PO4, 25mM NaHCO3, 12mM Glucose, containing1mM CaCl2
and 15mM KCl, pH 7.4). The right ventricle was opened from the pulmonary artery to the apex
and the papillary muscle (septal side) or trabeculae (right ventricle free wall) was dissected by
cutting a portion of the valve leaflet above and a small portion of the ventricular wall at the base
of the muscle. Only muscles preparations free from the wall and cylindrical in shape were
selected, while muscle preparations with spontaneous electrical activity were discarded. Muscles
were measured manually using a stereoscopic microscope for their thickness and width to allow
for calculation of cross sectional area to normalize the force measurements.
The muscle was then placed in the bath and mounted on the force transducer (Aurora
Scientific Canada, 5mN) (Figure 2.1). Muscles preparations were mounted horizontally with the
ventricular wall piece mounted on a small wire basket connected to the force transducer by a
glass tube while the valve leaflet is connected to a hook on the motor server arm allowing for
incremental length changes. The perfusion set up allowed for constant perfusion rate and
thermostatic conditions at 27οC. The preparation was field-stimulated by platinum electrodes
allowing for pulse durations of 2ms, at 1 Hz with voltages a minimum of 20% over threshold.
The preparation was gradually stretched to 90% of its maximal value and allowed to equilibrate
for 30 minutes. The standard Krebs salt solution (112.5mM NaCl, 4.7mM KCl, 1.2mM MgCl2,
51
1.2mM KH2PO4, 25mM NaHCO3, 12mM Glucose, (1.5mM CaCl2 (papillary) 1mM CaCl2
(trabeculae)) was used and gassed with 95% O2 and 5% CO2 to a pH of 7.4.
Perfusionchamber
27C
A
B
Forcetransducer
MotorArm
Increasinglength
Area ofsarcomeremeasurement
Figure 2.1 Papilary muscle force-length apparatus.
(A) Isolated papillary muscles are mounted in a perfusion chamber and stimulated with platinum
electrodes. (B) The papillary muscle undergoes incremental length changes by the motor server
arm while the changes in force generation are aquired through a force transducer. Sarcomere
length is recorded as outlined in Section 2.2.2.1.
52
When muscle contracts, the contractile proteins undergo structural changes resulting in
shortening of the myocyte, leading to pressure generation. Shortening can be monitored by
measuring the sarcomere length. After the equilibration period of 30 min, the force length
relation was determined. Muscle preparations were put to slack length whereby no force was
detected and the baseline for the force transducer was set. Sarcomere length was measured
throughout the protocol using a high speed video length (HSVL) detection system from Aurora
Scientific (papillary muscles) and laser diffraction for trabeculae. Micromanipulators were used
to increase the length of the muscle over the protocol. Small changes in length were made and
muscle was allowed to equilibrate for 1 min between length changes. Length was decreased from
2.25um to slack and then increased from slack to Lmax (2.3um).
Force and sarcomere length raw data were analyzed and force was normalized to the
cross sectional area (mN/mm2 ) and plotted against the diastolic sarcomere length. Passive
tension data was fitted to the exponential equation (y=e(x-xo/t)+a), whereby y is the passive
tension, xo is the average resting passive tension, x is the measured sarcomere length, a is the
measurement offset and t is the compliance constant of the myocardium. Statistical significance
was determined by Adrian Pasculucae at the Samuel Lunenfeld Research Institute using F
statistics of the experimental and control data normalized linear model. Developed tension
comparisons were compared by generalized additive model to generate splines.
2.2.2.2 Isolated Cardiomyocyte Methods
Isolated cardiomyocyte experiments enable one to investigate function independent of systemic
compensatory mechanisms and local paracine signaling from surrounding tissue. To investigate
excitation contraction coupling (Chapter 4), single myocyte experiments were preformed, while
myofilament mechanics were investigated by force-calcium experiments.
2.2.2.2.1 Dissociation of Murine Cardiomyocytes
Adult mice ages 8-12 wks were used for the studies. Mice were euthanized by cervical
dislocation and the heart was rapidly removed and placed in chilled calcium free Tyrodes
53
solution (137mM NaCl, 5.4mM KCl, 10mm HEPES, 0.5mM NaH2PO47H2O, 1.0mM
MgCl26H2O and 10mM Glucose, pH 7.4(NaOH)). The aorta was then cannulated with a blunted
18 gauge needle and perfused for 3-4 minutes with calcium free Tyrodes solution at 37οC.
Collagenase containing Tyrodes solution was then perfused (1 mg/ml, CLS2 Worthington’s) for
10-12 minutes at 37οC. The ventricular free wall was dissected free and myocytes were
mechanically dispersed in high K + containing KB solution (100mM K-glutamate, 10mM K-
asparate, 2.5mM KCl, 20mM Glucose, 10mM KH2PO4, 2mM MgSO47H2O, 20mM Taurine,
5mM Creatine, 0.5mM EGTA, 5mM HEPES and 0.1% albumin, pH 7.2 (KOH)). Myocytes were
used within 6 hours of being isolated.
2.2.2.2.1.1 Single Myocyte Morphometry
Using the 5x objective microscope, fields of plated cardiomyocytes were photographed, and
length and width measurements were recorded, after calibration with micrometer, using GNU
image manipulation program.
2.2.2.2.1.2 Single Myocyte Contractility
Freshly isolated myocytes were plated on glass perfusion plate and allowed to settle for 2
minutes. Calcium containing Tyrodes (1 mM CaCl) heated to 32οC was perfused for 20 minutes
to ensure proper equilibration of calcium and temperature. Cells were then whole field stimulated
by platinum electrodes (5-7volts, 1 Hz, 5 ms duration). Cells were allowed to equilibrate (~5
minutes) to ensure stable contraction and then myocyte sarcomere length measurements were
captured using the HSVL program (Aurora Scientific, Canada) at 0, 3 and 5 minutes for 10
second intervals. Only stable cells with no spontaneous contractions were selected for analysis
(Figure 2.2).
2.2.2.2.1.3 Single Myocyte Calcium Transients
54
Freshly isolated myocytes were incubated with Indo AM calcium indicator dye (Molecular
Probes, final concentration 1 uM with 0.45% Pluronic Acid) for 5 minutes at room temperature.
Cells were then plated on a glass perfusion dish and perfused with calcium containing Tyrodes
solution (1mM CaCl, 32οC) for at least 20 minutes. Cells were whole field stimulated with
platinum electrodes (5-7 volts, 1 Hz, 5ms duration). The selected cell was allowed to equilibrate,
and calcium transients were recorded at 0, 3 and 5 min of the protocol for 10 second intervals
and captured with the Felix software program. At 5 minutes, single myocyte contractility was
also recorded using the HSVL program. Only cells with stable contraction with no spontaneous
contraction were selected for analysis. Background subtraction to control for autofluroescence
and varied background was evaluated by 1) recording of fluorescence in the absence of a cell 2)
55
Figure 2.2 Configuration of single cardiomyocyte apparatus
(A) Single myocyte contractility apparatus. Single myocytes are plated on the perfusion chamber
and stimulated with platinum electrodes. Sarcomere Shortening is captured by the high speed
video sarcomere length program by Aurora Scientific. Specific detail is outlined in Section
2.2.2.2 (B) Calcium Transient apparatus. Single myocytes are plated on a perfusion chamber and
stimulated with platinum electrode. Fluroscence signals were captured and analzed as outlined in
Section 2.2.2.
56
unloaded cells and 3) manganese perfusion. Amplitude of the calcium transient was expressed as
a ratio of the emissions (405/485nm) corrected for background.
2.2.2.2.2 Force-Calcium Relationship in Skinned Myocytes
I did these experiments in the laboratory of Dr. Pieter deTombe at the University of
Illinois at Chicago as previously described (Belin et al. 2007). Briefly, mice were euthanized by
cervical dislocation and the heart was rapidly excised and placed in ice cold PBS solution. After
excess blood was removed, ~10 mg pieces of left ventricle free wall were dissected and quickly
placed in liquid nitrogen. The frozen biopsies were then kept at –80οC until they were used for
mechanical isolation of single myocytes. To isolate the single myocytes, the sample was placed
in relaxing solution (5.55 mM Na2ATP, 7.11mM MgCl2, 2.0mM EGTA, 108.01mM KCl, 8.91
mM KOH and 10.0mM Imidazol, pH 7.0 and Ionic strength 149.17) with the addition of protease
inhibitor cocktail (Sigma) , 10mM DTT and 0.3% Triton. On ice, the sample was homogenized
for 35seconds at 1500rpm with a polytron and then incubated on ice for 10-15 minutes. After
centrifugation at 560g for 1 min at 4οC, the supernatant was decanted and the pellet resuspended
in 2ml of relaxing solution. Centrifugation and resuspension of the pellet was carried out 3 times
with a final resuspension in 500ul of relaxing solution on ice.
Once the myocytes were isolated, a small aliquot was placed on a microscope mounting
cover slip, and myocytes were selected based on size (100-150um long and ~30um thick) and
striation uniformity. Selected myocytes were attached to a 5mN force transducer arm (Aurora
Scientific, Canada) and a motor server arm by silicon adhesive (Dow corning, ML USA). Once
the adhesive had cured, the myocyte was moved to an adjacent well containing relaxing solution
(5.95mM Na2ATP, 6.41mM MgCl2, 10.0mM EGTA, 100.0 mM BES, 10.0mM creatine
phosphate, 50.25mM potassium prop, protease inhibitor cocktail (Sigma) and 10mM DTT, pH
7.0), and the sarcomere length of the myocyte preparation was set to 2.25um using a spatial
Fourier transform, whereby the peak power spectrum corresponded to a mean sarcomere length.
57
The myocyte then alternated between relaxing solution wells and activation solution
(5.95mM Na2ATP, 6.20mM MgCl2, 10.0mM Ca+2EGTA, 100.0mM BES, 10.0mM creatine
phosphate and 29.98mM potassium prop, protease inhibitor cocktail (Sigma) and 10mM DTT,
pH 7.0) wells with varing calcium concentrations at 15οC . For each calcium concentration, the
preparation was released to slack after peak isometric tension development to obtain a baseline
before switching to the relaxing solution. The difference in peak tension and the baseline tension
is the total force developed for the given calcium concentration. As force generation in the heart
is highly cooperative, whereby force generation is highly dependant on the amount of
intracellular calcium available, the data was fitted using the equation:
P=[Ca+2]nH/(KnH+[Ca+2]KnH)
Whereby P= tension , K= calcium concentration required for half-maximal activation, and nH is
the Hill coefficient for cooperatively of the myofilaments. Data was analyzed as mean ± SEM.
2.3 Cellular Biology and Signal Transduction Methods
To understand the molecular changes that underpin the physiological responses seen at the whole
heart level, the tissue level and at the cellular level, a variety of molecular and biochemical
techniques were employed.
2.3.1 Histological Analysis and Microscopy
Excised hearts were washed with PBS and fixed in 10% buffered formalin and paraffin
embedded. 8μm sections were stained with hemotoxylin and eosin, masson trichrome stain or
picrosirius red by the histology core at the Centre for Phenogenomics (Toronto, Canada) and
slides were scanned at 20x lens using the Mirax microdigital slide scanner (Carl Ziess
MicroImaging). Picrosirius red with 10% PMA was used for visualization of perimysial fiber
58
content using the Optigrid structured illumination microscopy (Qioptiq LINOS) as previously
described (Fedak et al. 2003). Using the 20x objective lens multiple areas of the myocardium
were captured and analyzed for percent area of collagen (Image J software) to infer collagen
content. Only sections absent of interstitial fibrosis and perivascular fibrosis were analyzed.
2.3.2 Electron Microscopy
Electron Microscopy was done by Doug Holmyard at the electron microscopy facility at Mount
Sinai Hospital, Toronto, Canada. Briefly, left ventricle free wall specimens were rapidly
dissected and cut into 2mm3 pieces and placed in fixative solution containing 2%
paraformaldehyde and 2.5% glutaraldehyde in 0.15M sodium phosphate buffer, pH7.4. The
specimens were post fixed for 1 hr in sodium phosphate buffer containing 1% osmium tetroxide
and 1.25% potassium feroocyanide then dehydrated in a series of alcohol steps and embedded in
Epon Araldite. Thin sections were obtained and stained with uranyl acetate and lead citrate.
Random fields were scanned at various magnifications and photographed for subsequent
analysis.
2.3.3 Western Blotting and Immunoprecipitation
Hearts used for biochemical analysis were excised, rinsed in cold PBS and immediately frozen in
liquid nitrogen for subsequent analysis. Whole hearts or isolated cardiomyocytes were
homogenized in chilled radioimmunoprecipitation buffer containing 50mM Tris pH7.4, 150
NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton, 1% NP –40, 100mM sodium fluoride,
1mM sodium orthovandadate, 1x protease cocktail containing leupeptin, aprotinin and PMSF.
Lysates were clarified by centrifugation at 14 000x g for 15 minutes at 4C. Protein
Quantification was carried out following standard protocol for the BCA kit (Pierce) and proteins
(40ug) were boiled in 2x sample buffer, resolved by SDS PAGE and transferred to nitrocellulose.
For Immunoprecipitations, Flag M2 antibody immobilized on agarose beads (Sigma) was used to
enrich for ShcA containing fraction, and beads were washed 3 times and boiled in sample buffer,
resolved by SDS PAGE and transferred to nitrocellulose. Membranes were blocked in 5% BSA
59
and incubated with the appropriate primary antibody overnight, washed and incubated with the
appropriate HRP conjugated secondary antibody for 1 hr. Blots were visualized after incubation
with Chemiluminesence reagents. Additional antibodies used were: p44/42 and phospho-p44/42
Erk (Cell Signaling Technologies), GAPDH (Biosciences).
2.3.4 Zymography
Left Ventricle free wall was rapidly dissected from hearts, washed in PBS and placed in liquid
nitrogen. Heart specimens were then homogenized in RIPA buffer and clarified 2x by
centrifugation at 14 000g for 20 minutes at 4C. 50 ug of protein was loaded onto an 8% Tris
glycine gel containing 0.5% gelatin A substrate (Sigma). Zymography sample buffer contained:
0.625M Tris pH 6.8, 10% glycerol, 2% SDS and 2% bromophenol blue. The gel was then
washed 3times for 20 minutes each at room temperature with 2.5% Triton wash. The gel was
then incubated at 37C for the desired time in incubation buffer containing 50mm Tris pH 7.4,
150mM NaCl and 5mM CaCl2. The gel was then stained with Coomassie and destained to
resolve the digested bands.
2.3.5 RNA Isolation and Real Time RT PCR
Hearts were excised and rinsed in DEPC-treated PBS and quickly frozen in liquid nitrogen.
Ventricle tissue was homogenized in Trizol reagent (Invitrogen) to isolate RNA and cDNA
synthesis was carried out following the Superscript II cDNA synthesis kit (Invitrogen). Real time
RT PCR template detection was carried out by SYBRgreen (Applied Biosystems) and read using
the Applied Biosystems 7900HT machine. Data was normalized to GAPDH internal control and
expressed as fold increase or decrease from control samples (Schmittgen et al. 2008). Primers for
real time RT PCR were described previously (Schoenfeld et al. 1998).
2.3.6 Preparation of Tamoxifen
60
Injected tamoxifen was prepared as previously described (Sohal et al. 2001). Briefly, Tamoxifen
citrate (Sigma) was sonicated in peanut oil (Sigma) at a concentration of 5mg/ml. 20mg/kg/day
was injected in the peritoneal cavity for 5 days. Mice were studied 7 days post injection.
61
Chapter 3
ShcA is required to maintain cardiac structure and function
A version of this appeared in:
The ShcA phosphotyrosine docking protein uses distinct mechanisms to regulate myocyte and
global heart function (manuscript submitted)
Attributions:
Cardiac Catheterization studies were done in collaboration with the Surgical Technician at the
Heart and Stroke/Richard Lewar Centre of Excellence, Dr.M.Golum Kabir
Histology and Electron Microscopy were done in collaboration with the facilities at the Samuel
Lunenfeld Research Institute
62
3 ShcA is required for Cardiac Structure and Function
3.1 Introduction
Despite heart failure (HF) being a leading cause of death and morbidy in the western world,
current treatment strategies have not significantly impacted on long-term survivial (Ho et al.
1993; O'Connell et al. 1994; Russell et al. 2008). Recent studies have established tyrosine kinase
(TK) signaling to be integral to cardiac function, as disruption of various TKs in animal models
leads to a dilated cardiomyopathy phenotype. In particular, loss of myocyte-specific signals
downstream of the receptor TK ErbB2 results in a dilated cardiomyopathy(Crone et al. 2002;
Ozcelik et al. 2002), while myocyte-specific deletion of FAK, a non-receptor TK, results in
eccentric remodeling in response to hemodynamic overload (DiMichele et al. 2006) and aging
(Peng et al. 2006). The clinical relevance of TK signaling is supported by clinical trials in which
tyrosine kinase inhibitors such as Herceptin, which targets ErbB2, and Gleevec, an inhibitor of
kinases such as Abl and the platelet-derived growth factor receptor, have shown cardiac side
effects in a subset of oncology patients (De Keulenaer et al.; Slamon et al. 2001; Sparano 2001;
Chu et al. 2007; Force et al. 2007). Therefore, through dissection of signaling networks
downstream of TKs, we expand our understanding of cardiac function and potentially contribute
to novel therapeutic strategies (Chien 1999; MacLellan et al. 2000; Katz 2002).
An important molecular mechanism for adding complexity and specificity to TK
signaling utilizes the docking protein ShcA. This broadly expressed protein is one of 4 Shc
family members and is expressed in the forms of 66, 52 and 46 kDa polypeptides capable of
binding to phosphotyrosine (pTyr)–containing motifs on activated TKs both through an N-
terminal pTyr binding (PTB) domain and a C-terminal Src Homology 2 (SH2) domain (Pelicci et
al. 1996; Migliaccio et al. 1997; Ravichandran 2001; Jones et al. 2007). Once recruited to
activated TKs through such pTyr recognition domains, ShcA can itself undergo tyrosine
phosphorylation in the central CH1 (collagen homology 1) region, thereby stimulating the
activation of specific cytoplasmic signaling pathways. Notably, phosphorylation of the tyrosine
residues 239/240 and 313 in the CH1 region creates two consensus pY-X-N motifs that bind the
63
SH2 domain of the Grb2 adaptor, leading to stimulation of the Erk-MAP Kinase (MAPK) and
phosphatidylinositol 3’kinase (PI3K) pathways (McGlade et al. 1992; van der Geer et al. 1996;
Walk et al. 1998; Ravichandran 2001). Protein-protein interactions mediated by phosphorylation
of the CH1 tyrosines of ShcA has been shown to be important in the development of a functional
monosynaptic stretch reflex circuit (Hardy et al. 2007) and ErbB2-induced breast cancer in the
mouse (Ursini-Siegel et al. 2008).
The first biological role for ShcA was demonstrated by Lai and Pawson in 2000.
Surprisingly, mice homozygous for the ShcA null allele are embryonic lethal at E12.5, and
displayed decreased trabeculation, hypoplastic endocardial cushions and poorly developed
vasculature. Subsequent studies demonstrated that ShcA is required specifically in the
developing myocardium and that embryonic cardiovascular development requires ShcA PTB
domain recruitment to activated TKs; however, downstream signaling through the CH1 pTyr
sites were not essential (Hardy et al. 2007). While p66 ShcA has been shown to be important in
mediating protective cardiovascular effects (Cosentino et al. 2008), it is unknown how p52 and
p46 ShcA impact on cardiac structure and function in the postnatal myocardium.
The observation that ShcA is pivotal in the developing heart led us to investigate the role
of ShcA in the postnatal myocardium. To this end, we conditionally excised ShcA in ventricular
cardiomyocytes, whereby the loss of ShcA in the myocardium results in a dilated
cardiomyopathy phenotype that evolves over the course of one year. The remodeling process that
results in eccentric remodeling, suggests ShcA signaling is uniquely required to maintain cardiac
structure and function in the post-mitotic myocardium, in the absence of utrastructural changes
or exaggerated fibrosis.
3.2 Results
3.2.1 Generation of ventricular cardiomyocyte - specific ShcA null mice
To generate ventricular cardiomyocyte-specific ShcA null mice we employed a Cre/loxP strategy
by intercrossing mice with a ShcA floxed (ShcAflx) allele (Hardy et al. 2007) with mice
possessing the myosin light chain 2v (Mlc2v) Cre KI (Mlc2vKI/wt ) allele (Chen et al. 1998). The
64
ShcA floxed allele has a triple flag-tagged ShcA cDNA (ShcA minigene) fused with exon 3 of
the endogenous ShcA gene. LoxP sites 5’ of the first coding exon and 3’ of the poly-
adenylation site (pA) of the ShcA minigene were used for conditional excision by the Cre
recombinase (Figure 3.1A). Upon Cre mediated
Figure 3.1 Ventricular cardiomyocyte-specific deletion of ShcA.
(A) Schematics of ShcA floxed and deleted (CKO) alleles. (B) PCR of genomic DNA showing
recombination of the floxed allele in the heart of ShcA CKO mice only (C) Timeline of ShcA
excision in the heart as seen by western blot analysis of protein levels at the timepoints indicated.
Heart lysates collected at 12 weeks of age showed significant reduction in the levels of ShcA
protein suggesting excision of the ShcA gene by Cre recombinases (D) Lysates from partially
purified cardiomyocytes demonstrated effective recombination as ShcA protein levels were
65
significantly reduced. (E) Mlc2v Cre is specific to cardiomyocytes as lysates prepared from
spleen and lung showed no reduction in ShcA protein levels in the presence or absence of Mlc2v
Cre. PND, postnatal day; WK, week
66
recombination, the first coding exon, the intervening ShcA cDNA and exon 3 are excised leaving
a Neor cassette in-frame, thus ensuring a functionally inactive allele (Figure 3.1A and B).
Mating of ShcAflx/flx Mlc2vwt/wt and ShcAflx/wt Mlc2vKI/wt mice yielded the expected 1:1:1:1
Mendelian ratio. As expected, ShcAflx/flx Mlc2vKI/wt mice (designated ShcA CKO) showed
selective deletion of ShcA in ventricular cardiomyocytes (Chen et al. 1998). ShcAflx/flx
Mlc2vwt/wt mice were employed as littermate controls (Table 3.1) and were indistinquishable
from ShcAflx/wtMlc2vKI/wt mice (LVEDD: 4.00± 0.11 mm and % FS: 44.89± 2.38; n=4 ) at 6
months of age, as previously shown (Chen et al. 1998; Peng et al. 2006).
Mlc2v Cre recombinase-mediated excision of ShcA, as inferred by the level of ShcA
protein, was detected at a minimal level at 2 weeks of age as previously reported (Chen et al.
1998; Peng et al. 2006), while excision markedly increased by 12 weeks of age (Figure 3.1C),
consistent with previous studies (Peng et al. 2006). ShcA CKO ventricular lysates at 12 weeks
of age contained some residual ShcA protein, likely from non- myocyte sources (fibroblasts,
smooth muscle cells and endothelial cells), since robust loss of ShcA protein levels was observed
in partially purified left ventricle cardiomyocytes at similar timepoints (Figure 3.1D). Lysates
from spleen and lung confirmed the specificity of excision, as ShcA levels were comparable to
control lysates in these tissues (Figure 3.1E).
3.2.2 ShcA is required for the maintenance of cardiac structure and function
Homozygous ShcA-/- mice die at embryonic day (E) 11.5 (Lai et al. 2000), while mice
with germline ablation of the p66 ShcA isoform are long lived (Migliaccio et al. 1999). In
contrast, ShcA CKO mice survive embryonic development, but demonstrate significant cardiac
dysfunction with age. At 6 weeks of age, echocardiography data showed no significant
difference in cardiac dimensions (LVEDD (n>6): 3.79± 0.05 mm for ShcA Con vs. 3.92 ± 0.05
mm for ShcA CKO), or cardiac contractility as measured by fractional shortening of the left
ventricle (%FS (n=6): 48.30 ± 1.17 for ShcA Con vs. 45.49 ± 1.02 for ShcA CKO). In addition,
the heart weight-to-body weight (HW/BW) ratio for ShcA Con mice (5.30±0.25) was not
different from ShcA CKO animals (5.00±0.15) (n>7). However, at 12 weeks, ShcA CKO mice
developed decreased fractional shortening and distended chamber morphology without evidence
67
of concentric hypertrophy (Figure 3.2A and B, Table 3.1). This cardiac dysfunction combined
with the absence of antecedent cardiac
Figure 3.2 ShcA is required for the maintenance of cardiac function.
(A) Echocardiography time course of cardiac contractility as measured by % fractional
shortening showing a decline in function of ShcA CKO hearts over the course of 1 year
compared to controls. (B) Echocardiography time course of chamber morphology as measured
by left ventricular end diastolic dimensions (mm) showing a progressive increase in chamber
dimension over the course of 1 year in ShcA CKO hearts compared to controls. See
Supplemental Table I for complete data. (C) Representative histology of transverse heart sections
stained with hemotoxylin and eosin at 1 year of age; ShcA CKO hearts show enlarged chamber
dimensions compared to littermate controls. 6.25 x magnification (D) Isolated cardiomyocyte
dimensions of ShcA CKO and control mice. The mean ± SEM of 3 independent experiments are
shown. (E) Quantitative real time RT PCR of heart failure markers showing an increase in
mRNA expression of atrial naturetic factor (ANF) and Skeletal Actin (Sk Actin) in ShcA CKO
mice compared to controls at 3 months of age (n=4). Data was normalized to GAPDH internal
control and expressed as a fold change (arbitrary units, AU) relative to control mRNA
expression. No difference was detected in mRNA expression levels of Sarcoplasmic reticulum
ATPase pump 2A (SERCA), brain naturetic peptide (BNP) and beta –myosin heavy chain (B-
MHC). (F) Hemodynamic measurements of ShcA CKO (n=5) and control mice (n=5) at
20minutes after 3.0 mg/kg body weight IP injection of dobutamine at 3 months of age. Values
expressed as mean ±SEM of the percent change from baseline measurements. dP/dT max,
maximal first derivative of LV pressure development; dP/dT min, minial first derivative of LV
pressure development. HR, heart rate; bpm, beats per minute. *P<0.05 compared to littermate
control values for the given timepoint.
68
Figure 3.2. ShcA is required for the maintenance of cardiac function
69
6 weeks 12 weeks 6 months 1 Year
Con ShcA CKO Con ShcA CKO Con ShcA CKO Con ShcA CKO
HR (bpm) 473.2±4.02 484.2±11.8 502.7±8.70 509.4±8.4 571.9±19.9 526.2±9.7 530.5±10.04 506.6±13.8
LVEDD (mm) 3.78±0.05 3.93±0.06 3.91±0.06 4.25±0.05* 3.97±0.07 4.41±0.09 * 4.02±0.1 5.08±0.13 *
LVESD (mm) 1.86±0.07 2.16±0.06* 2.17±0.06 2.58±0.05 * 2.10±0.12 2.78±0.09 * 2.17±0.14 3.69±0.16 *
% FS 49.16±1.44 45.52±1.4 44.65±0.87 39.23±0.89 * 47.12±2.6 36.4±1.33 * 46.7±2.3 27.87±1.7 *
VCF 0.98±0.022 0.913±0.056 0.96±0.03 0.84±0.0* 1.105±0.07 0.814±0.04 * 1.028±0.855 0.619±0.047 *
Ant wall (mm) 0.69±0.02 0.68±0.02 0.66±0.01 0.66±0.02 0.64±0.02 0.64±0.03 0.66±0.02 0.66±0.03
Post wall (mm) 0.61±0.2 0.69±0.02 0.66±0.02 0.61±0.02 0.71±2.5 0.73±0.03 0.66±0.03 0.61±0.02
n 6 10 14 9 5 7 8 11
LVEDD, left ventricle end diastolic dimension; LVESD, left ventricle end systolic dimension; %FS, %fractional shortening;
VCF,velocity of circumferential shortening. * p<0.05 from littermate control for given timepoint.
Table 3.1. Echocardiography timecourse data.
70
hypertrophy at 6-8 weeks of age, suggests that ShcA CKO mice enter a dilated cardiomyopathy
phenotype in parallel with the kinetics of maximal ShcA excision in adult cardiomyocytes.
Indeed, isolated cardiomyocytes from ShcA CKO mice at 12 weeks of age had increased cell
lengths indicating eccentric remodeling (Figure 3.2D), while ShcA CKO hearts had elevations in
atrial natiuretic factor (ANF) and skeletal actin transcripts (Figure 3.2E), two genetic markers of
cardiac pathology, Nevertheless, responsiveness to the adrenergic agonist dobutamine was
unaltered in ShcA CKO mice compared to controls (Figure 3.2F). While ShcA has been reported
to be critical in MAPK activation, no significant changes in phospho-Erk, indicative of Erk
activation, were noted at 6 months (Figure 3.3A). This could be due to the heterogeneous cell
population within the myocardium and the multiple converging signals leading to MAPK
activation (Bueno et al. 2002).
By one year of age, ShcA CKO mice had a further decline in cardiac function with
coincident enlargement of the left ventricle chamber dimensions (Figure 3.2A, B and C, Table
3.1) in conjunction with an increase in HW/BW ratios (Figure 3.3B). Despite severe ventricle
dilation accompanied by a trend of increasing lung weight-to-body weight ratios (Figure 3.3B),
electron microscopy revealed no evidence of myofibrillar disarray or aberrant intercalated disc
structure or mitochondrial abnormalities (swollen or disrupted cisternae) (Figure 3.3C). Masson
trichrome staining revealed only minimal myocardial interstitial fibrosis in ShcA CKO mice at 1
year of age (Figure 3.3D). As interstitial fibrosis and myocardial ultrastructure changes are
typically present in advanced remodeling states in association with myocyte loss (Sabbah et al.
2000), these studies suggest that the loss of ShcA impacts on factors leading to cardiac dilation
without promoting myocyte loss or interstitial fibrosis.
71
Figure 3.3 ShcA CKO mice undergo eccentric remodeling with preserved cyto-
ultrastructure and minimal fibrosis.
(A) Representative western blot detection of pErk in heart lysates at 6 months of age. pErk
levels were similar between ShcA CKO and control hearts. (B) Gravimetric data showing
increased heart weight (HW) to body weight (BW) in ShcA CKO mice (n=5) compared to
controls (n=5). Lung weight (LungW) to body weight ratios showed an increasing trend in ShcA
CKO mice compared to controls. (C) Transmission electron microscope showing preserved
ultrastructure and normal mitochondrial morphology at 1 year of age in ShcA CKO and
littermate controls. Scale=2 μm. (D) Histological analysis of masson trichome stained transverse
sections showing minimal interstitial fibrosis with peri-venous fibrosis in Shc CKO hearts
compared to littermate controls at 1 year of age. Magnification for upper panel 6.25x, lower
panel 20x. *P< 0.05 from littermate controls.
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Figure 3.3. ShcA CKO mice undergo eccentric remodeling with preserved cyto-
ultrastructure and minimal fibrosis.
73
3.3 Discussion
Our study demonstrates that the loss of ShcA in the ventricular cardiomyocyts leads to a
dilated cardiomyopathy that evolves over the course of one year. The dilated cardiomyopathy is
characterized by progressive chamber dilation mediated by eccentric remodeling without
excessive deposition of interstitial fibrosis or ultrastructural changes. This study extends our
understanding of the biological requirement for ShcA, which was previously established for
embryonic heart development, neuromuscular development and oncogenic signaling (Lai et al.
2000; Hardy et al. 2007; Ursini-Siegel et al. 2008), and demonstrates that ShcA is essential in the
postnatal myocardium.
This study also highlights the important and predominant role of the p52 ShcA and p46
ShcA isoforms in maintaining cardiac structure and function. Germline ablation of the p66 ShcA
isoform results on a small cardiac chamber size at baseline and results in protection against
angiotensin II- induced ventricular remodeling (Graiani et al. 2005); however, these studies
demonstrate that the loss of all 3 isoforms results in a dilated heart. Therefore, from these data,
one can infer that p52 and p46 ShcA are critical in the maintenance of cardiac structure and
function, independent of the protective properties resulting from a loss of p66 ShcA.
While in vitro data has shown ShcA to be central to Erk-MAPK activation in response to
mitogenic stimuli, we did not see a loss of Erk activation. Other signal transduction pathways
important in cardiac maintenance and hypertrophy were also interrogated, but did not show a
significant effects attributed to the loss of ShcA. This could be due to the heterogenous cell type
composition within the myocardium as well as multiple converging inputs regulating the
dynamic nature of cardiac function. Critical to advancing the role of ShcA in the myocardium
requires identification of downstream signaling targets important in modulating cardiac structure
and function.
These studies highlight the essential role of ShcA signaling within the postnatal
myocardium, but also set the foundation to investigate the mechanism by which the loss of ShcA
leads to a dilated cardiomyopathy, and how the loss of ShcA in cardiomyocytes affects their
ability to contract. Lastly, as ShcA is a modular protein, it will be of great interest to understand
74
how the individual domains and signaling motifs contribute to the dilated cardiomyopathy
phenotype seen in the ShcA CKO mouse.
75
Chapter 4
The loss of ShcA in the myocardium uncouples single myocyte function and global heart
function
A version of this appeared in:
The ShcA phosphotyrosine docking protein uses distinct mechanisms to regulate myocyte and
global heart function. (Submitted manuscript)
Attributions:
Cardiac Surgical Operations were done in collaboration with Dr.M. Golum Kabir at the Heart
and Stroke/Richard Lewar Centre of Excellence.
76
4 The loss of ShcA in the myocardium uncouples single myocyte function and global
heart function
4.1 Introduction
The loss of ShcA in the myocardium leads to a dilated cardiomyopathy phenotype; however, the
mechanism by which the loss of ShcA precipitates the phenotype is unclear. Dilated
cardiomyopathies are associated with early functional changes that encompass systolic
dysfunction, diastolic dysfunction, cytoskeletal deficiencies and aberrent electromechanical
coupling (Backx et al. 1995; Giordano et al. 2001; Houser et al. 2003; Ji et al. 2004). The loss of
ShcA in the cardiomyocyte population obligates investigations of cellular and tissue function to
gain insight into the complex nature of the dilated cardiomyopathy phenotype.
Given the chamber dilation and depressed systolic function seen at the whole heart level,
we investigated the effect of ShcA deficiency on baseline cardiomyocyte contractility with
respects to calcium handling and myofilament interactions. Cardiomyoycte contractility is
regulated by excitation contraction coupling (ECC) (Figure 4.1), whereby depolarization of the
cardiomyocyte triggers a series of ion movements that culminate in a rise in intracellular
calcium, allowing for cross bridge formation and subsequent force generation. When an action
potential causes depolarization of the myocyte membrane, voltage gated calcium channels
(dihydropyridine receptor, Ca+2 L-type) have an increased probability of opening, allowing for a
local rise in calcium ( ~ 10-20μM). This rise of calcium triggers a large efflux of calcium from
the juxtaposed sarcoplasmic reticulum (SR) via the ryanodine receptor (RyR), in a process
termed calcium induced calcium release (CICR) (Bers 2002). The rapid release of SR calcium
(~300mM) removes the inhibition imposed by the troponin-tropomyosin complex to initiate
myofilament crossbridge formation and the subsequent generation of force (Figure 4.1A and B).
Relaxation of force requires the calcium to be pumped back into the SR by the SR ATPase
pump, Serca2a, whose function is gated by phosphorylation of phospholamban (PLN) (Bers
2008).
77
(A) Calcium entry into the cardiomyocyte initiates CICR (Section 4.1). (B) With increased calcium in systole, calcium is available to bind
to TnC and induce a conformation change within the Tm-Tn complex and allows for myosin binding to actin and subsequent force
generation. (C) Temporal relation of calcium and force development and the relationship of force and calcium in the cardiomyocyte.
Ca+2
L-type Calcium Channel
RyanidineReceptor
↑ [Ca+2]
Serca2a
CICR
Sarcoplasmic reticulum
Myofilaments
Diastole Systole
Actin
Tm-Tncomplex
myosin
Force generation
Ca+2 Force
Forc
e
Ca+2Time
A
B
C
Figure 4.1 Excitation Contraction Coupling in Cardiomyocytes.
78
Myofilaments are the end effectors of excitation contraction coupling. The large rise in
intracellular calcium initiated by the AP, allows for calcium to bind to various components of the
myofilament and initiate contraction. The myofilaments within the cardiac sarcomere are
composed of 2 main filaments, myosin (thick filaments) and actin and its associated proteins,
tropomysin (Tm) and hetero-trimeric troponin (Tn) (thin filaments).
The thin filaments are the main site for Ca+2 regulation of contraction. Actin is the
backbone thin filament and is comprised of monomeric G (globular)-actin that polymerizes to
form F( filamentous)-actin, two of which associate to form a double standed structure. Actin
contains sites for myosin head binding that initiate contractile force (Gordon et al. 2000).
Actomyosin binding is regulated in part by Tm and the Tn complex. Tm is coiled coil molecule
that spans 7 monomeric actin molecules in F-actin and overlaps with its neigbouring Tm in a
head to tail configuration (Gordon et al. 2000). Tm is expressed from 2 genes, α and β, and
cardiac Tm is predominantly α,α (tobacman, ls, 1996). The hetero-trimeric Tn complex
triangulates with Tm, actin and Tn subunits to regulate contraction. Tn subunits consist of Tn-C,
which binds Ca+2, Tn-I, which binds actin to inhibit crossbridge formation, and Tn-T, which
links Tn to Tm (Solaro 1995; Gordon et al. 2000). Cardiac Tn-C contains 4 calcium binding
sites, 2 high affinity ( for which Ca+2 is always bound) and 1 low affinity sites (Gordon et al.
2000)
Myosin is composed of two heavy chains and 4 light chains. The two heavy chains
associate as a coiled coil structure for most of their length (rod section), and after the hinge
region ( S2 fragment), there is a projecting N-terminal globular head termed subfragment 1 (S1
fragment) (Gordon et al. 2000). The S1 fragment contains the actin binding domain and the ATP
hydrolysis site and also associates with a pair of light chain molecules. The rod section of
myosin associates with C protein, whereby altered phosphorylation of C protein can modify thick
filament structure and ultimately contraction (Gordon et al. 2000). Titin, the giant elastic protein,
interacts with myosin , C protein and actin and is thought to generate passive tension in the
sarcomere and dictate interfilament lattice spacing (Granzier et al. 2005).
In diastole, intracellular calcium is low and Tn-I exerts an inhibitory effect such that Tn-
T is associated with Tm and prevents actomyosin crossbridges. In systole, when Ca+2 binds the
low affinity site on Tn-C, it induces a conformational change, such that Tn-C and Tn-I associate
79
more closely, thus weakening Tn-I-Tn-T interactions. This removes the constraint on Tm that
causes it to shift, no longer blocking the actomyosin crossbridge sites. This increases the
probability of the actin sites to be occupied by myosin heads in a three state model of thin
filament activation (Solaro 1995; Gordon et al. 2000) (Figure 4.1B). The degree by which
myofilaments generate force at a given calcium concentration is the calcium sensitivity, and this
is represented by the concentration of calcium required to generate half maximal force
generation when interrogating the force-calcium relationship (Figure 4.1C). Alteration in calcium
sensitivity is associated with myofilament isoform shifts and altered phosphylation of key
residues of various myofilaments and is also implicated in cardiomyopathies(Willott et al.;
Solaro 2008).
While factors intrinsic to the cardiomyocyte, such as ECC, can affect global cardiac
function. There are also properties of the myocardium, extrinsic to the myocyte that also
influence cardiac performance. The main properties are electrical synchronisity of
AP.propogation and the mechanical properties of the tissue, such as the properties contributed by
the extracellular matrix. Action potential depolarization initiates in the sino-atrial node and
propogates through the atria to the atrio-ventricular node. From here, AP depolarization spreads
through the Bundle of His, the purkinje fibres and then through the ventricles in a stereotypic
pattern to ensure the heart functions as a syncytium. Abberent propagation of depolarization or
repolarization result in cardiac dysfunction, as effective contraction is compromised (Lehnart et
al. 2007).
Another mechanism by which the heart is coupled is through the extracellular matrix. The
extracellular matrix is a heterogenous microenvironment that contains structural molecules, such
as collagen, matricellular molecules, such as osteoponin and proteoglycans, and also serves as a
reservoir for signaling molecules, such as HB-EGF (Spinale 2007). Deficiencies in many aspect
of the ECM can potentially contribute to cardiac demise (see Table 4.1). For example, collagen,
a major ECM structural molecule, when disrupted can rapidly induce cardiac dilation and
dysfunction (Spinale 2007). Collagen fibrils encase myocytes (endomysial fibres) and perimysial
fibres run along myocytes, typically spanning 4-5 myocytes (Streeter et al. 1969). It is thought
that the uncoiling of the perimysial collagen fibres contributes to the stiffness or mechanical
diastolic properties of the myocardium (MacKenna et al. 1996; MacKenna et al. 1997;
Matsubara et al. 2000), and it is the loss of these fibres that results in cardiac dilation and
80
increased distensibility seen in heart failure (Caulfield et al. 1992). Alterations is collagen
mechanical properties can also be affected by changes in collagen crosslinking (Avendano et al.
1999; Herrmann et al. 2003). Indeed, turnover of collagen by matrix metalloproteinases (MMPs),
tissue zinc-dependant proteases, have been implicated in various disease models (Spinale
2007).While the mechanical contributions of other components of the ECM are still emerging
(Fomovsky et al.), the literature has demonstrated an important and dynamic role for the ECM in
regulating cardiac structure and function.
This chapter addresses both the cell autonomous and non cell autonomous effects of
ShcA deficiency on cardiomyocytes and myocardial mechanical tissue properties, respectively,
to provide insight into the molecular defects contributing to the dilated cardiomyopathy
phenotype seen in the ShcA CKO mouse. Surprisingly, cardiomyocyte function was enhanced in
ShcA CKO mice compared to controls, suggesting global systolic dysfunction is not attributed to
poor cardiomyocyte function. Rather, ShcA CKO mice have deficiencies in myocyte/matrix
interactions that lead to mechanical uncoupling, that precipitate the dilated cardiomyopathy
phenotype.
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Table 4.1. ECM components that contribute to mechancal integrity of the myocardium
82
Gene Deletion Enzymatic substrate or endproduct (Spinale 2007)
Phenotype in animal model
MMP 2 denatured fibrillar collagen, basement membrane proteins, proteoglycans
Reduced dilation post MI and pressure overload(Matsumura et al. 2005) Over expression:DCM with age(Bergman et al. 2007)
MMP 3 All basement membrane proteins, elastin and proteoglycans
Impaired MI scar maturation (Morita et al. 2006)
MMP 7 Fibrillar collagen (I and III) Increased post MI survival (Lindsey et al. 2006)
MMP 9 denatured fibrillar collagen, basement membrane proteins, proteoglycans
Attenuates contractile dysfunction in HF (Moshal et al. 2008) Reduced LV enlargement post MI (Ducharme et al. 2000; Romanic et al. 2002)
MMP13 Fibrillar collagen and ECM proteins
No ablation study to date
MMP 14 (MT1) Diverse array of collagens and proteins
Non cardiac phenotype
Tissue inhibitor of metalloproteinase 1
Increased LV dilation and dysfunction post MI (Ikonomidis et al. 2005)
Tissue inhibitor of metalloproteinase 3
LV dilation with age (Fedak et al. 2004)
Osteopontin LV dilation and dysfunction post MI (Trueblood et al. 2001)
Thrombospondin Cardiac rupture after MI and increased load (Schroen et al. 2004)
Hyaluronic acid synthase 2
Synthesis of hyaluronic acid Failure to form endocardial cushions in embryonic heart development (Camenisch et al. 2000; Camenisch et al. 2002)
83
4.2 Results
4.2.1 The loss of ShcA signaling results in enhanced single myocyte contractility
To address whether the dilated cardiomyopathy phenotype resulting from ShcA deficiency is
associated with changes in baseline cardiomyocyte function, we measured isolated single
myocyte sarcomere length shortening in 12 week old mice. Surprisingly, despite a global
reduction of systolic function, ShcA CKO cardiomyocytes had enhanced contractility compared
to controls (8.85±0.51% vs. 6.78±1.16 % respectively; p<0.05; Table 4.2). Calcium transient
experiments demonstrated no significant change in amplitude between ShcA CKO and littermate
controls (Table 4.2), suggesting altered calcium sensitivity of the myofilament (Kobayashi et al.
2005; Solaro 2008). Indeed, the calcium sensitivity of ShcA CKO myocytes was enhanced, but
was not obviously associated with a decrease in serine 23/24 phosphorylation of Troponin I
(Table 4.2, data not shown). These results demonstrate that the enhanced contractility seen at
baseline in ShcA CKO mice is due to changes in myofilament properties that are regulated by
ShcA signaling.
As the elevation in contractility could be a compensatory mechanism resulting from
developmental defects mediated by the variable excision of ShcA in the postnatal period by the
Mlc2v Cre recombinase KI mouse (Chen et al. 1998; Peng et al. 2006), we utilized the tamoxifen
inducible Cre transgenic mouse (MerCreMer) driven by the alpha MHC promoter to acutely
excise ShcA (Sohal et al. 2001; Andersson et al. 2009). ShcA MCKO (ShcAflx/flx MerCreMer +/wt) and their littermate controls, ShcA MCON (ShcAflx/flx MerCreMerwt/wt and ShcAflx/wt
MerCreMer+/wt), were injected with tamoxifen for 5 days. ShcA MCKO mice showed no
evidence of chamber dilation or depressed systolic dysfunction 7 days post injection (Table 4.4);
however, 7 days after the tamoxifen protocol, ShcA MCKO cardiomyocytes showed elevated
baseline contractility compared to controls (6.42 ±0.34% vs. 7.76±0.36%, respectively; n=5
hearts with > 25 cells, p=0.016). The single myocyte data suggest that the defect in isolated
84
myocyte function is a cell autonomous effect due to the loss of ShcA that can be uncoupled from
whole heart function.
85
Table 4.2. Single myocyte assays
Assay Parameter Control ShcA CKO
Contractility % Sarcomere shortening 6.90±0.58 8.85±0.30*
dSL/dT max (μm/sec) 3.57±0.16 4.93±0.34*
dSL/dT min (μm/sec) 2.65±0.22 3.73±0.31*
TR50 (sec) 0.032±0.005 0.033±0.002
TR80 (sec) 0.052±0.006 0.064±0.005
Ca+2 Transients Amplitude (F405/485nm) 0.323±0.018 0.335±0.019
Force Ca+2 Curve Fmax (mN/mm2) 18.04±0.55 19.81±1.015
EC50 (μmoles/L) 2.46±0.08 2.18±0.13*
Hill Coefficient 3.57±0.34 3.74±0.64
Contractility and transient assays have a minimum of 3 hearts per group with a minimum of 15
cells. Force-Ca+2 experiments have a minimum of 6 hearts per group. dSL/dT max, maximum
first derivative of sarcomere shortening; dSL/dTmin, minimum first derivative of sarcomere
shortening; TR50, time to 50% relaxation ; TR80 , time to 80% relaxation. F, Fluorescence of
calcium transient; Fmax , maximal force; EC50, Ca+2 concentration at 50% maximal force. *
p<0.05 from littermate controls at given time point.
86
4.2.2 The loss of ShcA leads to deregulation of the ECM components in the heart
The presence of elevated single myocyte contractility despite decreased global systolic function
suggests a mechanical uncoupling within the myocardium; therefore, we investigated whether
the chamber dilation in ShcA CKO mice results from impaired ECM-myocyte interactions. To
this end, we examined extracellular matrix (ECM) properties by measuring the force-sarcomere
length relationship in isolated ventricular papillary preparations and by examining perimysial
connective tissue surrounding individual myocytes at early time points of disease. In response to
sarcomere length stretch, ShcA CKO papillary muscles had reductions in passive tension due to
higher compliance (p<0.001) compared to controls (compliance parameter (t) = 0.42 vs. 0.20,
respectively; Figure 4.2A), consistent with a disrupted ECM (Granzier et al. 1995; MacKenna et
al. 1996; Fedak et al. 2003; Spinale 2007). As papillary muscles can be heterogenous in shape
leading to a lack of uniformity of sarcomere length, a small cohort of ultrathin trabeculae muscle
preparations were also examined and gave similar results (data not shown). Consistent with the
mechanical results, perimysial collagen fibres stained with picrosirius red showed decreased
complexity and structural collagen content, at 12 weeks of age in ShcA CKO mice (Figure 4.2B
and C). In further support of mechanical uncoupling, ShcA CKO papillary muscles also showed
reduced developed tension (p<0.001), suggesting that poor force transmission could potentially
contribute to the systolic dysfunction seen in the whole heart. While the cause of the altered
ECM structure and function in ShcA CKO myocardium is unclear, MMP activity in six month
old ShcA CKO hearts was elevated compared to controls (Figure 4.2D), suggesting activation of
remodeling signaling pathways . Overall, our data supports the conclusion that, despite causing
enhanced cardiomyocyte contractility, ShcA excision leads to a progressive dilated
cardiomyopathy phenotype as a result of impaired maintenance of the ECM
87
Figure 4.2 The loss of ShcA leads to deregulation of the ECM components in the heart.
(A) Papillary passive tension, expressed as mN/mm2 (miliNewtons normalized to cross sectional
area), across increasing sarcomere lengths (μm) as described in Methods. All data for individual
papillary muscles (≥5 independent experiments per group) were plotted and fitted using a
exponential equation (y=e(x-xo)/t +a), where x=sarcomere length (μm), xo = average resting
tension, a= measurement offset, t= compliance constant. A significant reduction in ShcA CKO
compliance (t=0.42) was demonstrated compared to control (t=0.20); p<0.001 (B) Visualization
of perimysial collagen fibres stained with Picrosirius Red using Optigrid structured illumination
microscopy. ShcA CKO hearts demonstrated a decreased complexity of collagen staining
compared to controls (63x Magnification). (C) Collagen area was quantified from multiple
images from each heart and demonstrated a decrease in % collagen area in the ShcA CKO hearts
compared to controls (n=3). (D) Zymography of hearts from ShcA CKO (n=3) and their
littermate controls (n=3) at 6 months demonstrate ShcA CKO mice have increased MMP activity
as the ventricle chambers dilate. Increased activity was noted for proMMP9 and an unidentified
MMP species at ~200kDa. The postive control sample was supernatant from mouse embryonic
fibroblasts stimulated with concanavalin A for 24hrs. Values are means ± SEM with *p<0.05
compared to littermate controls for given time point.
88
Figure 4.2. The loss of ShcA leads to deregulation of the ECM components in the heart.
89
4.2.3 ShcA CKO mice undergo an eccentric remodeling response with transverse aortic constriction
To test the hypothesis that ShcA is critical for the maintenance of mechanical integrity of the
heart through ECM-myocyte interactions, 8 week old ShcA CKO mice and their littermate
controls were subjected to biomechanical stress by transverse aortic constriction (TAC). The
introduction of acute mechanical stress to the hearts of ShcA CKO mice, prior to overt dilation,
would be predicted to induce a rapid transition into HF. After 4 weeks of TAC, control mice
mounted the expected concentric hypertrophy response (Figure 4.3A, Table 4.3), accompanied
by multifocal interstitial fibrosis (Figure 4.3B) and preserved overall heart function (Figure
4.3A), Table 4.3). ShcA CKO mice, in contrast, showed a rapid decline in cardiac function that
quickly transitioned into congestive HF after the 4 weeks of TAC, characterized by severe
chamber enlargement, depressed fractional shortening, minimal ventricular wall thickening, and
elevated lung weights in ShcA CKO mice (Figure 4.3 A and B, Table 4.3 and 4.5). Despite
marked chamber enlargement and impaired cardiac function, ShcA CKO mice had a similar level
of interstitial fibrosis compared to controls after TAC (Figure 4.3B). These changes in ShcA
CKO hearts were accompanied by elevation of matrix-metalloproteinase 2 (MMP2) activity at 1
week TAC and subtle depressions in pErk signaling (Figure 4.3C and D).
As ShcA excision with the Mlc2v Cre recombinase KI mouse occurs slowly in postnatal
development (Chen et al. 1998; Peng et al. 2006), we acutely excised ShcA using the tamoxifen
inducible Cre transgenic mouse driven by the alpha MHC promoter (Sohal et al. 2001). ShcA
MCKO and their littermate controls, ShcA MCON, were injected with tamoxifen for 5 days, and
7 days post injection both groups underwent the TAC procedure (n≥5). Although acute ligand
mediated excision of ShcA can show some inherent variability in excision (Figure 4.3E)(Sohal et
al. 2001), ShcA MCKO showed dilated chamber dimensions upon echocardiography with
decreased systolic function compared to ShcA MCON mice after 4 weeks of TAC (Figure 4.3F,
Table 4.4), while hypertrophy, indicated by anterior wall thickness (Table 4.4), and HW/BW
ratios (5.86±0.35 vs. 4.55±0.14; n=5) were not different. These results suggest that ShcA is not
initially required for mounting a hypertrophic response, but is necessary for preventing
ventricular dilation.
90
Figure 4.3 ShcA CKO mice undergo an eccentric remodeling response with TAC.
(A) Echocardiography data shows a decline in ShcA CKO cardiac function with TAC, as
demonstrated by % fractional shortening of ShcA CKO and control mice after a 4 week TAC
protocol; see Table 4.3 for complete echocardiography analysis. (B) Histological analysis of
transverse heart sections stained with masson trichrome showing dilation of ShcA CKO hearts
compared to controls after TAC. (C) Zymography of ShcA CKO and control hearts after a 4
week TAC protocol showing enhanced MMP2 activation in ShcA CKO mice after TAC. The
positive control (+) sample was supernatant from mouse embryonic fibroblasts stimulated by
concanavalin A for 24hrs. (D) Western blot analysis of ShcA CKO and control hearts for pERK
activation after TAC. ShcA CKO hearts after 4 weeks of TAC show subtle depressions in pERK
activation compared to controls. Data shows 3 independent hearts per group. (E) Western blot
analysis showing loss of ShcA protein in heart and isolated cardiomyocytes in the presence of
MerCreMer transgene driven by the αMHC promoter compared to littermate controls, 7 days
after tamoxifen injection protocol as explained in Methods. (F) Histological analysis of
transverse sections stained with masson trichrome showing chamber dilation in ShcA MCKO
hearts compared to controls after 4 weeks of TAC. See Table 4.4 for complete echocardiography
study. Values are means ±SEM with ^p<0.05 compared to same genotype sham, * p<0.05
compared to littermate control values for the given time point. WK, week
91
Figure 4.3. ShcA CKO mice undergo an eccentric remodeling response with TAC
92
Table 4.3. Echocardiography and cardiac catheterization data for TAC studies
Parameter Sham 1 wk TAC 4 wk TAC
Con ShcA CKO Con ShcA CKO Con ShcA CKO
HR (bpm) 500.1±1.24 517.2±1.46 501.62±1.05 501.54±1.9 491.3±10.8 525.5±6.25
LVEDD(mm)
3.94±0.01 4.34±0.014* 3.85±0.01 5.05±0.01*^ 4.56±0.13 5.80±0.19*^
LVESD(mm)
2.16±0.01 2.64±0.016* 2.07±0.016 3.93±0.02*^ 2299±0.18 5.11±0.20*^
% FS 45.6±0.180 39.5±0.24* 47.04±0.306 22.69±0.32*^ 35.2±2.59 12.08±1.5*^
VCF (circ/sec)
0.94±0.004 0.817±0.0057* 0.97±0.0067 0.49±0.0066*^
0.77±0.06a 0.27±0.031*^
Ant wall (mm)
0.70±0.03 0.67±0.04 0.99±0.05^ 0.69±0.04 * 0.92±0.042^ 0.64±0.05*
Post wall (mm)
0.67±0.03 0.65±0.03 1.13±0.04^ 0.59±0.04* 0.83±0.039^ 0.64±0.03*
N 13 9 12 10 11 9
HR (bpm) 433.88±6.27 444.3±4.56 415.1±10.4 398.6±9.5 460.5±10.2 386.2±24.8
LVESP 101.4±1.04 96.5±1.16 148.9±1.42^ 147.1±1.7^* 136.7±5.8^ 91.5±3.0*
LVEDP 10.88±0.73 8.26±0.57 13.6±2.16^ 17.5±2.3^* 21.2±1.1^ 21.1±0.89^
Dp/dt max 7479.3±153 6200.2±88.6* 6763.6±139 6943.6±388 6664.9±354 3371.8±250^*
Dp/dt min -6582.9±16 -5978.1±118* -5695.1±228 -5792.8±397 -5977.7±35 -3041±191^*
N 10 10 5 5 8 7
^ p<0.05 compared to same genotype sham; * p<0.05 compared to littermate control value for
the given timepoint.
93
Table 4.4. Echocardiography data of TAC study for acute model of ShcA excision
Parameter 7 days P.I. # Sham 4 wk TAC
ShcA MCON
ShcA MCKO
ShcA MCON
ShcA MCKO
ShcA MCON
ShcA MCKO
HR (bpm) 446.7±14.56 473.8±11.09 467.8±7.07 473.0±10.73 431.5±13.48 455.6±11.38
LVEDD (mm)
4.06±0.09 0.383±0.005 4.16±0.011 4.36±0.007 4.25±0.008 4.75±0.019^*
LVESD (mm)
2.29±0.09 0.210±0.008 2.67±0.012 2.66±0.015 2.80±0.012 3.43±0.03^*
% FS 43.75±1.53 45.33±1.67 36.07±1.67 39.17±2.92 34.51±1.44^ 28.32±2.80^*
VCF (circ/sec)
0.809±0.103 0.819±0.039 0.731±0.028 0.774±0.051 0.663±0.03^ 0.581±0.03^*
Ant wall (mm)
0.69±0.02 0.73±0.02 0.69±0.02 0.73±0.02 0.83±0.02^ 0.83±0.05 ^
Post wall (mm)
0.63±0.03 0.61±0.02 0.69±0.03 0.58±0.03 0.68±0.03 0.65±0.01
P.I., post injection; # No significant difference was noted at 7 days post injection between ShcA wt/flx MerCre/Mer +/wt and ShcA flx/flx MerCreMer wt/wt. ^ p<0.05 compared to same genotype sham; * p<0.05 compared to littermate control value for the given timepoint.
94
Table 4.5. Gravimetric data for ShcA CKO and control mice after TAC
Parameter Sham 4 wk TAC
Con ShcA CKO Con ShcA CKO
BW (g) 30.69±0.06 29.80±0.79 29.96±0.90 27.36±0.97
Left Ventricle (mg) 100.0±4.1 103.2±10.1 170.0±6.3^ 170.7±8.4^
LVW/TL (mg/mm) 0.611±0.025 0.633±0.056 1.028±0.035^ 1.027±0.051^
LungW/TL (mg/mm) 1.14±0.032 1.09±0.077 1.56±0.115^ 2.53±0.220^*
BW, body weight; LVW/TL, left ventricle weight/tibial length; LungW/TL, lung weight/tibial
length. ^ p<0.5 compared to same genotype sham, * p<0.05 compared to littermate control for
given timepoint.
required for mounting a hypertrophic response, but is necessary for preventing ventricular
dilation, possibly by ensuring the integrity of the extracellular matrix.
4.3 Discussion
The enhanced cardiomyocyte contractility found in ShcA deficient cardiomyocytes was
surprising in the context of global cardiac dysfunction. The absence of changes to calcium
handling but alterations in the calcium sensitivity suggests ShcA impacts on homeostatic
myocyte contractility at the level of the myofilaments. Regulation of the force-calcium
relationship occurs through phosphyorlation and isoform shifts of the major myofilaments. In
particular, calcium sensitivity is regulated by kinase and phosphatases, such as beta adrenergic
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modulation of serine 23/24 of troponin I and other residues of troponin C and tropomyosin (de
Tombe et al. 2000; Solaro et al. 2002; Solaro 2008) . While little data is available regarding the
role of TK signaling and contractility, genistein, a broad spectrum TK inhibitor has been shown
to increase calcium sensitivity at the level of the myofilaments(Min et al. 2002). The ability for
ShcA to interact with multiple phosphatases (Table 1.1 and 1.2) and influence various signal
transduction pathways, suggest ShcA signaling, potentially through upstream TKs, is important
in single myocyte contractile function. The acute excision of ShcA using the α MHC-
MerCerMer mouse also resulted in hypercontractile cardiomyocytes suggesting that this is a cell
autonomous effect of the loss of ShcA in cardiomyocytes.
The ShcA CKO mice have enhanced cardiomyocyte contractility despite whole heart
cardiac dysfunction, suggesting an uncoupling of global regulators of cardiac function from
myocyte function. When cardiomyocyte function is compromised global cardiac function is
expected to be reduced (Backx et al. 1995; Houser et al. 2003); however, just as importantly,
blood flow change as a result of altered vascular function (Giordano et al. 2001), disruption of
normal propagation of electrical signals (Ji et al. 2004) and uncoupling of force transmission
from the myocyte along collagen struts (Spinale 2007) can all contribute to impaired cardiac
function, thereby precipitating heart failure. he absence of necrosis with fibrosis, arrhythmias or
sudden death in the ShcA CKO mice suggests that cardiomyopathy in these mice did not result
from myocyte loss and stimulation of fibrosis. While the activation of the MMP2 and MMP9 in
ShcA-deficient mice may result from generic stimulation of a final common pathway associated
with adverse cardiac remodeling (Heymans et al. 1999; Ducharme et al. 2000; Spinale 2007), the
early detection of mechanical changes in the passive tension in parallel with the excision of
ShcA suggests disruption of pathways involved in myocyte/myocyte or myocyte/matrix
coupling. Indeed, several matrix-associated TKs have docking sites for the PTB and SH2
domains of ShcA (Mainiero et al. 1995; Vogel et al. 1997; Ravichandran 2001), so that ShcA,
through reciprocal signaling between cellular compartments (Hardy et al. 2007; Ursini-Siegel et
al. 2008) could potentially affect mechanical coupling through adhesion interactions, secretion of
matricelluar proteins or direct remodeling of the matrix (Bowers et al.; Spinale 2007). It will be
of great interest to explore more deeply the underlying mechanism through which ShcA impacts
on global heart structure and function. The presence of enhanced cardiomyocyte function in the
context of a dilated cardiomyopathy reiterates that HF is a mosaic syndrome and requires
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stratification that is based on the precipitating etiology to effectively impact on prognosis (Liew
et al. 2004).
Overall, these data suggest single myocyte contractilily and global cardiac function can
be uncoupled, and highlight the signaling complexity underlying cardiac physiology. These data
also highlight a central role for ShcA in mediating TK signaling in the myocardium. As ShcA
has a modular domain structure, it will be of interest to understand the role of ShcA PTB and
SH2 domains coupling to upstream TKs and of the CH1 pTyr sites in activation downstream
pathways in regulating cardiac structure and function.
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Chapter 5
ShcA uses distinct signaling mechanisms to regulate myocyte contractility and global heart
function
A version of this appeared in:
The ShcA phosphotyrosine docking protein uses distinct mechanisms to regulate myocyte and
global heart function. (Submitted manuscript)
Attributions:
Cardiac Catheterization and Transverse Aortic Constriction studies were done in collaboration
with the Surgical Technician at the Heart and Stroke/Richard Lewar Centre of Excellence,
Dr.M.Golum Kabir
Histology was done in collaboration with the facilities at the Samuel Lunenfeld Research
Institute
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5 ShcA uses distinct signaling mechanisms to regulate myocyte contractility and global
heart function
5.1 Introduction
Adaptor or docking proteins are molecules containing multiple signaling domains and motifs that
generally lack kinase activity (Pawson et al. 1997). The presence of multiple potential signaling
mechanisms allows for the introduction of complexity to protein-protein interactions. ShcA has 2
pTyr containing recognigtion domains, the N terminal PTB domain and the C terminal SH2
domain (Introduction Section 3.2). Other SH2 domain containing adaptors are Grb2, Crk and
Nck, while other PTB containing adaptors are the Dok and IRS family of proteins. In the
myocardium, ShcA then has the potential to interact with a multitude of TKs to regulate cardiac
structure and function (Table 1.1-3)
ShcA also have various signaling motifs that contribute to downstream signal
propogation. In the CH2 domian, Serine 36 has been identified as critical in mediating oxidative
stress signaling (Trinei et al. 2009) , while the Y239/240 and Y313 of the CH1 region, when
phosphorylated, generate two consensus pY-X-N motifs that bind the SH2 domain of the Grb2
adaptor, leading to stimulation of the Erk-MAP Kinase (MAPK) and phosphatidylinositol
3’kinase (PI3K) pathways (Introduction Section 4.2). Alternative ShcA signaling mechanisms
include adaptin binding in the CH1 region, SH3 domain containing proteins recruited to the
proline rich CH1 region, or serine/threonine phosphorylation motifs within the ShcA protein
(Pelicci et al. 1992; Weng et al. 1994; Bonfini et al. 1996; Okabayashi et al. 1996). In addition,
recent data suggests that IQGAP links ShcA to the actin cytoskeleton through a non-canonical
interaction with the PTB domain (Smith et al.). Therefore, ShcA has several alternative
downstream signaling mechanisms that could be utilized to impact on cardiac function.
To understand how ShcA mediates it effects in the myocardium, we used the ShcA
mutant allele series (Hardy et al. 2007), which is comprised of ShcA knock-in (KI) alleles each
containing discrete point mutations that allowed us to evaluate the functional roles of ShcA PTB
and SH2 domains in coupling to upstream TKs in the postnatal heart and of the CH1 pTyr sites
in activating downstream pathways. Analysis of specific KI mutants indicate that PTB domain
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inactivation produced similar cardiac dysfunction found in ShcA CKO mice, indicating that
PTB-pTyr interactions are essential for ShcA function in the myocardium, while the SH2 domain
has a more modest role in hypertrophy signaling in response to biomechanical stress and aging.
In contrast, the CH1 pTyr sites are dispensable in maintaining normal cardiac structure and
function, but are required for preventing cardiac dysfunction induced by biomechanical stress.
Our results therefore indicate that ShcA is a critical hub protein that transmits pTyr-dependant
signals to control adult myocardial function and response to stress through a variety of distinct
molecular mechanisms.
5.2 Results
5.2.1 Generation of mice with ventricular specific ShcA point mutations
Given its modular domain architecture, ShcA can potentially signal downstream of multiple
tyrosine kinases important in both cardiac maintenance and the disease model of pressure
overload (Ravichandran 2001). Therefore, to evaluate the individual contributions of the various
domains and signaling motifs of ShcA, we utilized a ShcA mutant allelic series (Hardy et al.
2007) to generate mice in which the KI mutations (δKI) are restricted to cardiomyocytes. To test
the importance of the PTB or SH2 domains of ShcA in recognizing pTyr motifs of activated TKs
in the postnatal myocardium, mutant PTB and SH2 alleles were evaluated with respect to cardiac
structure and function. To understand signaling mechanism downstream of ShcA in the post
mitotic myocardium, we evaluated CH1 pTyr signals in cardiac structure and function using the
phospho-null 3F allele (see below).
Mice with ShcA KI alleles containing individual point mutations were intercrossed with
ShcAflx/flx Mlc2vKI/wt mice to give ShcAδKI/flx Mlc2vKI/wt. In non-cardiac tissue, the KI mutant
allele yields no phenotype owing to expression of the fully functional floxed allele. However,
Cre recombinase excision of the floxed allele in ventricular cardiomyocytes unmasks the
properties of the mutated allele (cardiac specific KI (CKI))(Figure 13A)(Hardy et al. 2007). The
ShcA KI alleles have a single flag tagged ShcA cDNA fused with exon 3, each with discrete
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point mutations (δKI). The individual point mutations preclude 1) pTyr binding of the PTB
domain to
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Figure 5.1 Schematic of ShcA allele strategy to generate ventricle cardiomyocyte specific
ShcA KI mutants.
(A) Breeding strategy to generate mice containing one mutant ShcA δKI allele and one ShcA
floxed allele. In the presence of Mlc2v Cre, the floxed allele becomes functionally null,
unmasking the individual mutant domain ShcA δKI allele. (B) Individual ShcA δKI alleles are
outlined denoting the point mutations. (C) Representative western blot analysis showing
reduction in protein levels of the ShcA floxed allele (3x flag) and expression of the individual
mutant ShcA δKI allele (1x flag) only in the presence of Mlc2v Cre in SH2 CKI hearts
compared to littermate controls.
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Figure 5.1. Schematic of ShcA allele strategy to generate ventricle cardiomyocyte specific
ShcA KI mutants.
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upstream TKs by mutating arginine 175 to glutamate (δPTB CKI; ShcAδPTB/flx Mlc2v CreKI/wt ),
2) pTyr binding of the SH2 domain by mutation of arginine 397 to lysine (δSH2 CKI;
ShcAδSH2/flx Mlc2v CreKI/wt) and 3) downstream signaling from the tyrosines in the CH1 region
by mutating tyrosines 239/240/313 to phenylalanine (3F CKI; ShcA3F/flx Mlc2v CreKI/wt) (Figure
13B and C). Mice were viable and recovered at the expected 1:4 Mendelian frequency. Controls
were littermates with the genotypes ShcAδKI/flx Mlc2v Crewt/wt , ShcAwt/flx Mlc2v Crewt/wt or
ShcAwt/flx Mlc2v CreKI/wt.
5.2.2 The PTB domain of ShcA couples to upstream TKs to maintain cardiac structure
and function
Like the ShcA CKO mice, δPTB CKI mice demonstrated a dilated cardiomyopathy phenotype at
an early age. Echocardiography and histological analysis revealed enlarged left ventricle
chamber dimensions and reduced fractional shortening (Figure 5.2A and B, Table 5.1). Over the
course of 1 year, ventricle dilation progressed, while cardiac function declined. A trend in the
elevation of HW/BW ratios in δPTB CKI mice at 1 year of age, suggests a remodeling heart
(Figure 5.3A). Masson trichrome staining of hearts at 1 year revealed minimal areas of interstitial
fibrosis, as in ShcA CKO hearts (data not shown). As the δPTB CKI mice displayed phenotypic
defects at 8 weeks of age (LVEDD: 4.01± 0.06 mm for δPTB Con vs. 4.51± 0.10 mm for δPTB
CKI and %FS: 50.7 ± 1.31 for δPTB Con vs. 39.6 ± 1.68 for δPTB CKI; n>7), TAC studies were
not carried out. The early presentation of the dilated cardiomyopathy in the δPTB CKI mice
could result from subtle gene dosage effects originating from the presence of one functionally
null allele that precludes PTB coupling to upstream TKs during development. Thus, cardiac
chamber dimensions and cardiac function require ShcA coupling to upstream TKs through the
pTyr binding activity of its PTB domain.
δSH2 CKI mice were followed for 1 year to see if ShcA SH2 domain signaling is
required to maintain cardiac structure and function. By 6 months of age, δSH2 CKI mice
demonstrated subtle cardiac dysfunction, and by 1 year of age, systolic function was reduced and
chamber dimensions were slightly enlarged (Figure 5.2A and B, Table 5.1). At one year of age,
no significant difference in HW/BW ratio was noted (Figure 5.3A). As the cardiac function of
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Figure 5.2 TK-ShcA signaling is required to maintain cardiac structure and function, while
downstream signaling from the CH1 pTyr sites is dispensable.
(A) Echocardiography data showing % fractional shortening of the mutant ShcA allele series.
δPTB CKI mice have reduced cardiac function as early as 3 months, while 3F CKI mutants have
normal cardiac function. Over the course of 1 year, δSH2 CKI mice have a progressive decline in
cardiac function. (B) δPTB CKI mice have enlarged cardiac chambers (left ventricle end
diastolic dimensions) that progress over the course of 1 year. In contrast, 3F CKI mice maintain
chamber dimensions similar to controls. δSH2 CKI have a small increase in chamber dimensions
over the course of 1 year compared to controls. (C) Histological cross sections of hearts from the
various mutant ShcA allele mice. 6.25 magnification. (D) Heart Weight to Body weight ratios of
the various mutant ShcA allele mice. ^ p < 0.05 compared to same genotype control shams, *
p<0.05 compared to littermate controls at given time points.
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Figure 5.2 TK-ShcA signaling is required to maintain cardiac structure and function, while
downstream signaling from the CH1 pTyr sites is dispensable
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δSH2 CKI mice was unchanged at 2-3 months of age, (Figure 5.2A, Table 5.1), the mice were
subjected to the TAC protocol. δSH2 CKI mice had similar remodeling of the left ventricle
chamber dimensions compared to controls, but showed reduced cardiac function and blunted
hypertrophy response compared to controls after 4 weeks of TAC (Figure 5.2 C and D, Table
5.3). These results demonstrate that SH2 domain-mediated interactions play a role in
hypertrophy signaling in response to biomechanical stress and aging.
5.2.3 ShcA phosphotyrosine derived signaling is required in hemodynamic overload
While in vitro and in vivo studies have shown the CH1 pTyr sites are important in downstream
signaling, genetic analysis has indicated that phosphorylation of tyrosines 239/240 and 313 is
dispensable for embryonic heart development (Hardy et al. 2007). Consistent with this, phospho-
null 3F CKI mice did not develop significant cardiac dilation and cardiac dysfunction (Figure
5.2A-D,. Table 5.1) over a 1 year period. However, 3F CKI mice subjected toTAC at 2-3 months
rapidly advanced into HF, as demonstrating by dilated ventricle chambers coupled with reduced
cardiac function (Figure 5.3 A and B, Table 5.2). These data suggest that the tyrosine
phosphorylation sites in the CH1 region of ShcA are dispensable in maintaining normal heart
function. However, in response to biomechanical stress, ShcA signaling requires phosphorylation
of CH1 tyrosine sites associated with recruitment of SH2 domain proteins such as the Grb2
adaptor (Walk et al. 1998; Velazquez et al. 2000).
These data suggest that the tyrosine phosphorylation sites in the CH1 region of ShcA are
dispensable in maintaining normal heart function, but become important in the context of a
mechanical stress, such as hemodynamic overload. Therefore, ShcA maintains cardiac function
through coupling to upstream TKs and, in the context of hemodynamic overload, requires
recruitment of downstream SH2 domain proteins such as Grb2 to CH1 pTyr sites. Of interest, the
lack of requirement for CH1 pTyr signaling in the maintenance of normal cardiac function
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Figure 5.3 TK-ShcA signaling is required to maintain cardiac structure and function, while
downstream signaling from the CH1 pTyr sites is necessary after hemodynamic overload.
(A) % Fractional shortening demonstrates that 3F CKI mice have reduced cardiac function after
4 weeks of TAC compared to controls, while δSH2 CKI have a slight reduction in cardiac
function. (B) Left ventricle end diastolic dimensions are increased in 3F CKI mice compared to
controls after 4 weeks ofTAC, while δSH2 CKI have preserved chamber morphology. Individual
KI controls were not significantly different and pooled for graphing purposes, individual
echocardiograpy data is found in Table 5.3. (D) Single myocyte contractility demonstrates
hypercontractility in δPTB CKI and 3F CKI isolated single myoyctes, while δSH2 CKI isolated
single myocytes have contractility similar to controls. Single myocyte contractility consists of
>3 independent experiments preformed with >15 cells. Individual KI controls were pooled
(n=9, >45 cells) (See Table 5.4). ^ p < 0.05 compared to same genotype control shams, * p<0.05
compared to littermate controls at given timepoints.
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Figure 5.3 TK-ShcA signaling is required to maintain cardiac structure and function,
while downstream signaling from the CH1 pTyr sites is necessary after hemodynamic
overload
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Table 5.1. Echocardiography data for 1 year timecourse
n Heart Rate (bpm) LVEDD (mm) LVESD (mm) % FS VCF (circ/sec) Ant Wall (mm) Post Wall (mm)
12 week
δPTB CON 6 500.2±14.8 3.85±0.07 2.07±0.06 46.4±0.98 0.98±0.03 0.72±0.02 0.61±0.04
δPTB CKI 10 513.5±6.21 4.50±0.10* 2.90±0.11* 34.6±1.22* 0.76±0.03* 0.64±0.02* 0.65±0.03
3F CON 12 517.0±11.0 4.00±0.06 2.12±0.05 46.8±0.99 1.04±0.038 0.79±0.02 0.69±0.02
3F CKI 7 501.1±10.9 3.87±0.06 2.19±0.05 44.0±1.0 0.93±0.022 0.79±0.02 0.65±0.02
δSH2 CON 8 512.1±9.59 3.94±0.07 2.12±0.07 46.3±1.24 1.03±0.03 0.74±0.02 0.67±0.02
δSH2 CKI 6 502.2±11.7 3.78±0.04 2.08±0.09 45.0±2.14 0.97±0.05 0.74±0.04 0.69±0.04
6 Months
δPTB CON 6 521.3±10.77 4.01±0.05 2.02±0.06 49.7±1.34 1.06±0.042 0.71±0.03 0.71±0.02
δPTB CKI 14 522.8±10.44 4.64±0.10* 3.13±0.13* 33.4±1.32* 0.75±0.036* 0.69±0.02 0.71±0.02
3F CON 5 477.0±1.72 4.06±0.07 2.23±0.11 46.0±2.21 0.96±0.04 0.69±0.03 0.59±0.02
3F CKI 7 458.6±12.6 3.95±0.08 2.13±0.08 46.4±1.09 0.94±0.03 0.70±0.03 0.60±0.02
δSH2 CON 7 529.5±18.0 3.94±0.08 2.11±0.10 46.9±1.96 1.08±0.07 0.73±0.02 0.74±0.04
δSH2 CKI 9 527.2±15.6 3.99±0.09 2.31±0.10 42.17±1.44 0.93±0.03* 0.77±0.01 0.70±0.02
Continued
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1 Year n Heart Rate (bpm) LVEDD (mm) LVESD (mm) % FS VCF (circ/sec) Ant Wall (mm) Post Wall (mm)
δPTB CON 6 545.2±14.78 4.05±0.09 2.08±0.12 49.1±2.10 1.14±0.057 0.77±0.04 0.76±0.07
δPTB CKI 9 518.4±12.8 5.00±0.17* 3.26±0.22* 31.9±1.79* 0.78±0.035* 0.69±0.03 0.69±0.02
3F CON 8 525.2±9.34 4.06±0.09 2.04±0.09 50.1±1.33 1.07±0.055 0.81±0.04 0.68±0.02
3F CKI 7 506.5±5.86 4.03±0.04 2.14±0.06 46.8±1.53 1.02±0.034 0.76±0.03 0.65±0.02
δSH2 CON 7 555.5±10.8 3.96±0.09 2.07±0.14 48.6±2.52 1.14±0.07 0.87±0.03 0.70±0.02
δSH2 CKI 7 515.7±14.7 4.38±0.09* 2.81±0.15* 36.43±2.47* 0.83±0.07* 0.77±0.02* 0.63±0.03*
^ p<0.05 from same genotype sham; * p<0.05 from littermate control for the given time point
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Heart Rate (bpm)
LVEDD (mm)
% FS Ant Wall (mm)
Heart Rate (bpm)
LVESP (mmHg)
LVEDP (mmHg)
dP/dT max (mmHg/s)
dP/dt min (mmHg/s)
12 wk Sham
3F CON 517.1±11.0 4.00±0.06 46.8±0.99 0.79±0.02 443.8±29.3 113.6±4.28 5.32±0.75 9144.5±121 -8243.4±161
3F CKI 501.1±10.9 3.87±0.06 44.0±1.0 0.79±0.02 399.7±22.2 108.7±4.79 8.59±1.52 6795.7±425* -6690.3±387*
δSH2 CON 512.1±9.59 3.94±0.07 46.3±1.24 0.74±0.02 500.1±18.5 109.3±6.39 16.4±2.83 8387.1±464 -7609.0±618
δSH2 CKI 502.2±11.7 3.78±0.04 45.01±2.14 0.74±0.04 453.8±26.7 106.9±4.58 11.74±2.44 8594.2±989 -7827.3±753
4 wk TAC
3F CON 502.9±10.3 4.37±0.1^ 35.9±1.62^ 0.92±0.02^ 448.5±18.9 150.2±11.9^ 16.7±4.65 6561.9±791^ -6875.9±908^
3F CKI 498.7±11.2 5.05±0.11^* 18.1±1.71^*
0.73±0.02* 466.1±29.7 129.8±11.5^ 23.3±3.66^ 5545.8±806^ -5124.3±562^
δSH2 CON 530.4±11.7 4.35±0.11^ 38.3±1.61^ 0.94±0.03^ 499.8±11.3 178.2±11.16^ 20.20±3.44 8807.1±1091 -8614.1±896
δSH2 CKI 487.3±9.22 4.41±0.11^ 29.5±1.49^*
0.85±0.02^*
545.6±9.48 135.9±11.77^*
16.79±2.41 7294.7±533^* -7194.7±768*
^ p<0.05 from same genotype sham; * p<0.05 from littermate control for the given time point
Table 5.2. Echocardiography and cardiac catheterization for TAC study
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suggests that an alternative signaling mechanism is the predominant mode by which ShcA
directs TK signaling in the absence of stress.
5.2.4 Myocyte contractility requires ShcA phosphotyrosine-based signaling
As the loss of ShcA in cardiomyocytes enhanced myocyte contractility, the three ShcA mutant
allele CKI mouse lines were subjected to single myocyte contractility assays and analysis of
global calcium transients. Both the δPTB CKI and 3F CKI myocytes were hypercontractile
compared to their littermate controls independent of changes in calcium handling, while δSH2
CKI myocytes were not different from control myocytes (Figure 5.3C, Table 5.5). This suggests
that ShcA requires PTB domain coupling to upstream TKs and subsequent signaling through the
CH1 pTyr sites to maintain homeostatic contractility at the myofilament level. This cell
autonomous function of ShcA therefore utilizes the well established signaling through pTyr
residues in the CH1 region, in contrast to the spontaneous dilated cardiomyopathy phenotype, in
which pTyr signaling from the CH1 region is dispensable. Together, these data suggest that in
the myocardium, ShcA signals downstream of TKs to differentially impact on cardiomyocyte
contractility and global heart morphology.
5.3 Discussion
The ability of ShcA to impact on different aspects of cardiovascular function highlights the
modular nature of the ShcA protein and its ability to direct signaling by recruitment to specific
TKs through the PTB and SH2 domains. Indeed, loss of signals derived from PTB domain
docking to upstream tyrosine kinases phenocopies the loss of ShcA in the myocardium, while
loss of signals involving the SH2 domain are required to augment the response to TAC and
hypertrophy associated with age.
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Table 5.3. Contractility and calcium transient assays for ShcA mutant domain KI mice
Parameter Pooled Controls
PTB CKI 3F CKI SH2 CKI
% Sarcomere shortening 7.17±0.24 9.04±0.41* 9.44±0.55* 6.72±0.26
dSl/dT max 2.85±0.18 4.26±0.25* 3.65±0.27* 2.93±0.25
dSl/dT min 3.89±0.20 5.53±0.34* 4.96±0.40* 3.92±0.22
TR50 0.039±0.002 0.035±0.002 0.038±0.002 0.034±0.003
TR80 0.063±0.003 0.056±0.003 0.061±0.003 0.055±0.004
Amplitude (F405/485nm) 0.316±0.011 0.329±0.02 0.281±0.014 0.307±0.02
For each group n=3, minimum 15 cells. Pooled KI controls (n=9, minimum 45 cells). dSL/dT
max, maximum first derivative of sarcomere shortening; dSL/dTmin, minimum first derivative of
sarcomere shortening; TR50, time to 50% relaxation ; TR80 , time to 80% relaxation. F,
Fluorescence of calcium transient. * p<0.05 to littermate control for given time point.
The PTB domain of ShcA has been shown to interact with multiple receptor TKs critical
in cardiac function, such as VEGFR3, ErbB2 and ErbB4 (Segatto et al. 1993; Wolf et al. 1995;
Fournier et al. 1996); in particular, loss of ErbB2 in cardiomyocytes produces a similar
phenotype to ShcA in cardiac ablation studies (Crone et al. 2002; Ozcelik et al. 2002). The loss
of ShcA in other tissues also phenocopies conditional ErbB2 loss (Dankort et al. 2001;
Andrechek et al. 2002; Hardy et al. 2007) and supports the idea that ShcA is a preferential
scaffold for ErbB2 signaling, especially as it contains multiple ShcA consensus binding motifs
(Segatto et al. 1993; Schulze et al. 2005). Of clinical relevance, Herceptin, an effective tyrosine
kinase inhibitor of ErbB2-ErbB3 heterodimers, causes HF in a subset of patients (De Keulenaer
et al.); it is suggestive then that ShcA could function in a pathway that is regulated by Herceptin,
perhaps influencing matrix/myocyte interactions. The clinical utility of Neuregulin (Jiang et al.),
the ligand of heterodimers containing ErbB2 (Shi et al. 2009), in improving cardiac function in
diseased myocardium suggests the ErbB2-ShcA signaling network could be of great interest
therapeutically.
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The slow evolution of a HF phenotype in the SH2 CKI mice, that does not follow the
timecourse of the loss of ShcA in the myocardium, suggests that SH2 coupling to various
upstream TKs has a supportive role in cardiac structure and function, distinct from PTB domain-
mediated signaling. Indeed, the SH2 domain has been shown to bind to the platelet derived
growth factor receptor β (Yokote et al. 1994), FAK (Hecker et al. 2002) and the epidermal
growth factor receptor (Pelicci et al. 1992), unique role for ShcA SH2-mediated signaling in
response to biomechanical stress and aging.
Downstream signaling induced by phosphorylation of tyrosine residues in the CH1 region
of ShcA appears to have evolved as a mechanism to increase signal complexity in multicellular
organisms (Luzi et al. 2000). These tyrosines are covalently modified by activated tyrosine
kinases when ShcA is recruited by either its PTB or SH2 domain and have been shown to be
critical in Ras mediated MAPK activation and PI3K activation through ShcA-Grb2 interactions
(Ravichandran 2001). To understand the requirement for downstream signaling from the pTyr
residues in the CH1 region of ShcA in the adult myocardium, we evaluated the phospho-null
mutant 3F CKI mouse for effects on cardiac structure and function. Of interest, the pTyr sites
required for Grb2 recruitment were not required for normal cardiac function, but become
functionally important in situations of mechanical stress, such as hemodynamic overload. A
small percentage of 3F CKI mice (<20%) did spontaneously dilate with age, but this seems to be
an isolated phenomenon and could be attributed to some unknown stress. Alternative ShcA
signaling mechanisms include adaptin binding in the CH1 region, SH3 domain containing
proteins recruited to the proline rich CH1 region, or serine/threonine phosphorylation motifs
within the ShcA protein (Pelicci et al. 1992; Weng et al. 1994; Bonfini et al. 1996; Okabayashi et
al. 1996). In addition, recent data suggests that IQGAP links ShcA to the actin cytoskeleton
through a non-canonical interaction with the PTB domain (Smith et al.). Therefore, ShcA has
several alternative downstream signaling mechanisms that could be utilized to impact on cardiac
function.
Thus, in the postnatal myocardium, ShcA is able to use distinct signaling pathways to
impact on isolated cardiomyocyte and global heart function. For example, the PTB domain is
required to maintain cardiac function essential for suppressing the dilated cardiomyopathy
phenotype; however, this is independent of downstream signaling from the CH1 pTyr sites.
Nonetheless, the CHI pTyr sites become important in regulating cardiomyocyte contractility and
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coordinating signals in response to hemodynamic stress. These data show that ShcA is a key
docking protein for TK signaling in the postnatal myocardium that coordinates signaling
networks underlying cardiac physiology.
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Chapter 6
Summary and Future Directions
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6 Summary and Future Directions
6.1 Summary
ShcA is a key protein that acts immediately downstream of receptor tyrosine kinases, especially
members of the ErbB family, to organize cytoplasmic signaling pathways. It has a modular
architecture, containing an N-terminal phosphotyrosine-binding (PTB) domain, a C-terminal
SH2 domain, and a central region with phosphotyrosine sites that bind to the adaptor protein
Grb2 and thus regulate the Ras-MAP kinase pathway. Using Cre/LoxP recombination, removal
of murine ShcA from the myocardium leads to a dilated cardiomyopathy, accompanied by
eccentric remodeling, indicating that it is a critical molecule in controlling cytoplasmic signaling
pathways in the heart. Of interest, individual ShcA-deficient cardiomyocytes show an enhanced
contractility, in contrast to the defect at the level of the whole heart. Loss of ShcA in the
myocardium apparently precipitates a dilated cardiomyopathy phenotype through defects in
signaling pathways governing interactions of cardiomyocytes with the extracellular matrix. To
understand more fully the molecular mode of action of ShcA, we examined the effects of an
allelic series of ShcA point mutants on cardiac function, which either eliminate the
phosphotyrosine-recognition domains through which ShcA interacts with activated receptors, or
inhibit its ability to couple to selective downstream pathways. The resulting data indicate that the
ability of ShcA to bind activated receptors through its PTB domain is essential for physiological
cardiac function, whereas the integrity of the SH2 domain is required to augment cardiac
function. The phosphotyrosine sites that couple to Grb2 are more important for stress-induced
responses and cardiomyocyte contractility. Our results identify ShcA as an important
cytoplasmic protein for phosphotyrosine signaling in the heart, demonstrate that it has distinct
cell autonomous and non-cell autonomous roles, and show that different domains and motifs in
this modular protein are of distinct significance in regulating normal and stress responses. These
findings significantly extend our understanding of phosphotyrosine-based signaling in the control
of heart function, and are of both basic and clinical interest.
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Figure 6.1 Model of ShcA signaling in the myocardium
(A) ShcA uses the PTB domain to dock to activated TKs critical in maintaining cardiac structure
and function, while the SH2 domain is important in hypertrophy signaling in response to
hemodynamic overload and aging. ShcA uses an alternative mode of signaling to maintain
cardiac function, as mutation of the CH1 pTyr sites did not elicit a dilated cardiomyopathy
phenotype. (B) ShcA pTyr signaling downstream of the CH1 region is required for appropriating
signals after henodynamic overload and signaling that regulates myocyte contractility.
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6.2 Future Directions
These studies demonstrated a role for ShcA in the postnatal myocardium and highlighted the
complex regulation and signaling underlying cardiac physiology. However, many unanswered
questions remain. Some areas for potential study are outlined below.
6.2.1 What are the specific TK intereactions of the PTB and SH2 domains of ShcA within
the myocardium?
Ventricular specific ShcA KI mice with mutations in the various domains and motifs
demonstrated a requirement for the binding of the PTB domain of ShcA to upstream TKs to
maintain cardiac structure and function, while the integrity of the SH2 domain is required to
augment cardiac function. However, the specific TKs that ShcA interacts with are unknown. One
obvious candidate is the ErbB2 receptor, as cardiac ablation studies phenocopy the loss of ShcA
or PTB binding to upstream TKS in the myocardium, and the presence of multiple ShcA
consensus binding motifs (Introduction section 3.2). To investigate the TK – ShcA pTyr
interactions, various approaches could be undertaken. An unbiased and systematic approach
would utilize mass spectrometry to identify TK-ShcA interactions. Whole heart lysates with Flag
IPs allows for identification of TKs binding to either domain of ShcA. Contamination of other
cell types is a drawback, especially given the abundance of fibroblasts in the myocardium. To
circumvent this, pooling isolated single myocytes could also be done, however introduction of
contamination during the isolation procedure, low yields and the lack of cellular adherence could
obscure physiological interactions. After various attempts, I feel the best approach would be
through isolation of ShcAflx/flx neonatal myocytes. This would ensure an endogenous Flag
epitope to IP and also allows for removal of the ShcA floxed allele through Cre recombinase (α-
MHC MerCreMer) as a control. As an adjunct or alternative approach, isolated neonatal
myocytes could be stimulated with various growth factors, such as Neuregulin, or plated on
various ECM (collagen or fibronectin) and harvested for mass spectrometry. These studies would
enable a myocyte specific ShcA-pTyr network to be established.
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6.2.2 What is the effect of ErbB2-ShcA signaling in the myocardium?
Given the potential for ShcA to be signaling downstream of the ErbB2 receptor in the
myocardium, one must use a genetic approach to precisely isolate the ErbB2-ShcA pathway. I
propose using a genetic strategy employed in these studies and generation of a KI ErbB2 mutant
allele. Mice heterozygous for the ErbB2 floxed allele (Crone et al. 2002) and a KI ErbB2 allele
with point mutations of the ShcA consensus binding sites, when coupled with Cre recombinases
expressed from the MLC2v promoter, will allow for direct interrogation of this signaling axis in
the myocardium. Previously, a hypomorphic ErbB2 KI mouse with one mutated ShcA consensus
binding site was generated (Chan et al. 2004), however no cardiovascular phenotype was
uncovered. Schulze and collegues (Schulze et al. 2005) identified additional ShcA binding
motifs on the ErbB2 receptor, suggesting that the introduction of multiple point mutations to the
ErbB2 receptor may be required to generate a phenotype. Readouts could include apoptosis,
matrix interactions and contractility, in addition to full cardiovascular phenotypic analysis. This
study will have the highest potential to translate directly to address clinical concerns of TKIs.
6.2.3 What are the clinical correlates to the loss of ShcA or TK signaling in the
myocardium?
Dilated cardiomyopathy is a major reason for pediatric cardiac transplantation, along side
complex congential heart disease. While mutations in myofilament, cytoskeletal, and renin-
angiotensin-aldosterone system (RAAS) proteins have been documented, it would be of interest
to screen for mutations in TKs and ShcA (Fatkin et al.). These studies could be done in
collaboration with the Pediatric Cardiology Transplant Team and the Hospital for Sick
Children’s Molecular Genetics Program. Single nucleotide polymorphisms within ShcA have
been evaluated in early onset cardiovascular disease, based on the p66 ShcA literature, but no
significant polymorphisms were detected (Sentinelli et al. 2006). Screening for novel TK
mutations or ShcA mutations could demonstrate novel causes of pediatric dilated
cardiomyopathies.
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6.2.4 How does the loss of ShcA regulate myofilament homeostasis?
The hypercontractile cardiomyocytes found in the ShcA CKO mice was unexpected and
revealed a cell autonomous effect of ShcA in regulating myofilament homeostasis. The actual
molecular mechanism by which ShcA does this is not apparent. Initial probing experiments did
not show alterations in Serine 23/24 phosphorylation of troponin I, consistent with enhanced
calcium sensitivity. To further screen and identify changes, proQ diamond staining (Pierce) of
myofilament preparations was carried out, but no obvious changes were noted. Many different
phosphatases and kinases regulate myofilament interactions (de Tombe et al. 2000; Solaro 2008),
and therefore studies addressing the underlying interactions could be carried out in collaboration
with Dr. P. deTombe. To address this initially, I infused various inhibitors and activators (PKC,
MAPK) in an ex vivo isolated heart perfusion system to assay changes in phosphorylation of
troponin I. No consistent changes were seen. An alternative approach would be incubating the
isolated skinned myocytes of the tamoxifen-inducible ShcA MCKO with inhibitors or activators
and looking for shifts in force-calcium parameters; this could potentially be a more robust
measure. Initial candidates could include inhibitors/activators of Erk-MAPK pathway, PKC, and
various phosphatases (Solaro 2008).
6.2.5 What are the downstream signaling consequences attributed the loss of ShcA in
cardiomyocytes?
In vitro experiments have established ShcA in mitogenic Erk activation; however, no changes in
the phosphorylation of Erk was noted at 6 months of age. As well, other pathways were
interrogated and showed no consistent changes. To try to define the effects of ShcA on MAPK
signaling in general, I used the ShcAflx/flx mouse embryonic fibroblasts and excised ShcA using
tamoxafen inducible Cre recombinase mouse (Badea et al. 2003). Initial experiments show nice
excision with little adverse effects to cells. This system allows for detection of signal
transduction changes in response to a variety of stimuli. A more robust measure could be
changes in c-fos or c-myc either through lucificerase assays or real time RT PCR. This system
could be translated then to the neonatal cardiomyocyte culture system mentioned above to shed
insight into cardiomyocyte specific changes. In particular, neuregulin, EGF, PDGF, and collagen
stimulation would be of great interest.
122
6.2.6 What is the molecular mechanism by which the loss of ShcA affects the mechanical
integrity of the myocardium?
The experiments mentioned above will provide insight into ShcA interactions potentially
impacting on mechanical integrity. For example, mass spectrometry could identify interaction of
the discoidin domain receptor, while matrix adhesion experiments could identify abnormalities in
ShcA-deficient cardiomyocytes. Alternatively, GF stimulation could reveal differences in
secretion of molecules important in regulating the integrity of the ECM. Initial studies in MEFs
did not reveal an increase in MMP activity (by zymography) with generic stimulation using
Concanavalin A or neuregulin, however further optimization and the use of real time RT PCR
readouts could be more robust . One particular candidate of interest would be the regulation of
hyaluronic acid/proteoglycans in the myocardium by ShcA. Microarray analysis of ShcA-
deficient hearts identified the downregulation of Hyaluronidase synthase 2 (Has2), that was
verified with real time RT PCR. Loss of Has2 in the embryonic period, results in hypoplastic
endocardial cushions and a dilated heart, similar to the ShcA null mouse (Camenisch et al. 2000).
Subsequent studies showed that Has2 induction is part of an ErbB2-ErbB3 signaling pathway
(Camenisch et al. 2002). I initially stained for proteoglycans, but little staining was seen in the
myocardium. To enhance retention of proteoglycans post fixation, cetyl pyridinium chloride is
required during histological processing thereby improving the ability to quantify differences
using Alcian blue stain. While it is unknown how proteoglycans affect the mechanical properties
of the myocardium, much can be derived from cartilage literature. Proteoglycans are required to
give tensile strength and are the major water carrying molecule, thereby contributing to the
protective properties against mechanical force (Fomovsky et al.). Given the high amount of
proteoglycans in papillary muscles, it would be of interest to see if ShcA is involved in a Has2-
ErbB2/B3 pathway, especially given the embryonic similarities.
6.2.7 What is the role of Grb2 signaling in the myocardium?
To expand the adaptor protein literature in the myocardium, generation of a ventricular specific
Grb2 KO should be carried out. While Grb2 null mice are embryonic lethal at early time points
123
(Cheng et al. 1998), heterozygotes are viable and have been evaluated in myocardial hypertrophy
signaling(Zhang et al. 2003). In response to hemodynamic overload, Grb2 heterozygotes
demonstrate an absence of hypertrophy, without dilation, that correlates with deficiencies in p38
MAPK activation. Therefore, it would be of great interest to more fully appreciate the role of
Grb2 in the myocardium. The Grb2 floxed allele was generated in the Pawson laboratory and
crossing it to various cardiac specific Cre mice will demonstrate the role for Grb2 in maintaining
cardiac function.
6.3 Concluding Remarks
These studies demonstrate the requirement of ShcA in pTyr signaling in the myocardium. In the
postnatal myocardium, ShcA is able to use distinct signaling pathways to impact on isolated
cardiomyocyte and global heart function. ShcA can signal through the pTyr sites in the CH1
region to regulate cardiomyocyte contractility and coordinate signals in response to
hemodynamic stress. In addition, ShcA is also able to use alternative modes to propagate signals,
once docked to activated TKs, as seen with the dilated cardiomyopathy phenotype. These data
show that ShcA is an integral docking protein for TKs in the postnatal myocardium and enhance
our understanding of the complex signaling networks underlying cardiac physiology.
124
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Appendices (if any)
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Copyright Acknowledgements (if any)
