G-protein Signaling in the Heart Scott P. Heximer, Ph.D. Fall 2003

Why study G-protein signaling in cardiac cells? GPCRs are a primary means of communication between The heart and the homeostatic mechanisms for blood Pressure regulation 1) Disease prevention- reduction of hypertrophy 2) Treatment of heart failure - modulating catecholamine effects on heart tissue 3) Antiapoptotic signals - protection of heart tissue

G proteins are molecular switches with intrinsic GTPase activity “OFF” “ON” “OFF”                       GDP   GDP GDP GTP GDP GTP GTP GTP GTP

Cellular Mechanisms for Signal Termination are integrated with physiologic responses to GPCR agonists Receptor phosphorylation and internalization reduces receptor levels facilitates b-arrestin mediated signaling complexes RGS protein function - GTPase activating proteins for Ga subunits - contain multiple modular signaling domains that allow them to act as effectors as well as inhibitors

RGS proteins greatly increase the rate of GTPase hydrolysis in Gasubunits “OFF” “ON” “OFF”                       RGS GDP   RGS GDP GTP GDP GDP RGS RGS GTP RGS GTP GTP GTP

B) Decrease Agonist Sensitivity CLASSIC PARADIGM- RGS proteins are negative regulators of G protein signaling A) Enhance decay kinetics B) Decrease Agonist Sensitivity - RGS + RGS [agonist]

G Proteins: Master Regulators of Cell Function Membrane Excitability Motility Metabolism Differentiation Transcription Translation Proliferation

G-protein signaling pathways are abundant and ubiquitous Biologically relevant signals require receptor, G-protein, and downstream effectors of G-protein signaling Specificity - most receptors couple to a limited # of G-proteins (Ga - most Ga subunits couple to specific effectors - wide number of gene families for each component

Ga -coupled effectors determine final cellular response to agonist

G-proteins are required for normal heart function Sympathetic - Regulation of contractility and heart rate Parasymapathetic - Regulation of heart rate

Conduction of electrical signals to mediate contraction Is conducted by autorhythmic cells

Autorhythmic cells make up the Sinoatrial Node and the electrical conduction system of the heart Different Action Potential firing rates (slower as you get further into the conduction system) allows the SA node to have primary control but provides contingency plan when electrical conduction is blocked

Myocardial Autorhythmic Cells control the Heart Rate myocardial autoarythmic cells have an unstable membrane potential Spontaneously develop action potentials due to the expression of a different set membrane channels that result in drifting pacemaker potential

Molecular Events Leading to Cardiac Cell Contraction in response to Action Potential note the importance of Ca++ handling in the mechanism of contraction and relaxation

Autonomic Nervous System Controls Heart Rate by Controlling the firing rate of Autorythmic Cell APs Norepinephrine- sympathetic neurotransmitter that speeds up pacemaker depolarizarion by altering membrane permeability of ion channels Acetylcholine- sympathetic neurotransmitter that activates K+ efflux while inhibiting Ca++ influx, to result in hyperpolarized pacemaker potential and slowed rate of depolarization NOTE: action potential is always the same duration, only rate of change in pacemaker potential affects rate of AP firing

G-protein-coupled signaling modulates calcium handling during cardiac cell action potential -increased Ca++ leads to greater force of contraction

Cardiac Output is a Measure of Heart Function Cardiac output is the amount of blood pumped/unit time  cardiac output = heart rate x stroke volume Thus, factors that control the rate (SA node AP firing ) or the stroke volume ( myocyte contractility, stretch) Stroke Volume = End Diastolic Volume - End Systolic Volume What happens when stroke volume in one ventricle is reduced compared to the other?

Cardiac Output is controlled by autonomic modulation of heart rate SA node AP firing frequency is regulated by: Sympathetic signals to increase Ion permeability in autoarhythmic Cells (increased heart rate) Parasympathetic inputs that Increase potassium efflux and decrease calcium influx

b1-adrenergic receptor comprises 75 - 80 % of total breceptors Epinephrine and Norepinephrine stimulate b1-adrenergic receptors in cardiomyocytes “OFF” “OFF” “ON”                       GDP   Adenylyl Cyclase GDP GDP GTP GDP GTP GTP GTP GTP b1-adrenergic receptor comprises 75 - 80 % of total breceptors ATP cAMP

couples to Gs and Gi  epinephrine b g AC as b g GDP ai AC GDP PI3K RTK Grb2

aq aq b Angiotensin II couples to PLC b PLCb PKC PIP2 *DAG Ang II PKC PIP2 *DAG aq b g aq + *IP3 PLCb GDP GTP [Ca++]i

G protein-coupled receptors and Heart Disease Myocardial hypertrophy -preventative Heart Failure -treatment

Mice lacking catecholamine-synthesis show marked reduction in hypertrophic response to pressure overload Role for adrenergic receptors in pressure-induced hypertrophic Response Challenges “Wall Stress Hypothesis” that suggests that Hypertrophy is a beneficial Adaptation required to prevent Cardiac damage in response to Higher wall stress i.e. more damage is observed in The non-hypertrophied hearts Adrenergic receptor-mediated signaling pathways involved? Gq Gi Gs

Models for the G protein-mediated Hypertrophic Signaling Pathway -cellular data strongly pointed to a role for Gq-coupled receptors in causing the hypertrophy phenotype

Note similarity to dilated Studying Hypertrophy in Mouse Models Transgenic overexpression of wt Gq is sufficient to cause hypertrophy and decompensatory heart failure in mice GqTg WT Note similarity to dilated cardiomyopathy

Direct Gq inhibition blocks hypertrophic response to TAC MHCa-Gq minigene transgenic MHCa-RGS4 Transgenic

Myocyte Gq family members are required for hypertrophic response to TAC

cardiomyopathy and greatly reduced ejection fraction (heart function) 5- to 15-fold transgenic overexpression of b1 adrenergic receptor leads to dilated cardiomyopathy and greatly reduced ejection fraction (heart function) IN CONTRAST- Up to 200 fold transgenic overexpression of b2 adrenergic receptor has very little effect on cardiac function Does Gi signaling have a protective role during heart failure?

Adenoviral delivery of b1-AR but not b2-AR induces apoptosis in cardiomyocytes

Inhibitors of Gi signaling (PTx) and PI3K (LY) induce apoptosis In isoproterenol-stimulated cardiac myocytes

Gi-coupled crosstalk signaling with RTKs leads to increased Grb2 and Ras activation Transgenic expression of activated Ras expression leads to Cardiac hypertrophy in mice

Deletion of one copy of the Grb2 gene prevents development of cardiac hypertrophy following pressure overload Grb2 +/+ Grb2 +/-

Impact of High Catecholamine Levels During Chronic Heart Failure 1 ARs are selectively downregulated b1 and b2 ARs are uncoupled from G proteins - Increased levels of b-ARK1 - Increased levels of Gi subunits

Transgenic overexpression of bARK-ct prevents development of cardiac dysfunction in mouse models of cardiomyopathy

EMERGING CONCEPTS - GPCRs exist as dimers/oligomers Functional consequences of receptor heterodimerization - new mechanisms for modulating receptor downregulation/trafficking Reduced agonist-dependent b1 AR internalization when 2 AR is coexpressed

Receptor scaffolds that bind bARs PDZ domain-containing EMERGING CONCEPTS IN bAR SIGNALING Receptor scaffolds that bind bARs Non PDZ-containing PDZ domain-containing

Adrenergic Signaling- The Human Connection Adrenergic receptor genes in the human heart are susceptible To variations that can lead to differences in function between Individuals carrying different alleles adrenergic genes show variable sensitivity to SNP usage in the human Genome (e.g. no variants have been identified for the a1B adrenergic Receptor) - nonsynonomous (changes the protein that is coded) synonomous (no apparent changes in the protein) BUT WHAT ABOUT? Slpicing Translation modification RNA stability

Positional Map of Adrenergic Receptor Variants

less coupling to adenylyl cyclase Substitution of Arg389 with Gly in b1 adrenergic receptor results in much less coupling to adenylyl cyclase

b2 adrenergic receptor variant in 4th TM domain reduces Coupling of receptor to Gs In transgenic mice- reduced cardiac contractility (dP/dt) In humans- reduced survival and reduced capacity for exercise

BUT WAIT- Some receptors have more than one SNP Receptor haplotypes may be a more informative method for studying receptor expression and function