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

2. 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

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

4. 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

6. 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

7. 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]

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

9. 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

10. Ga -coupled effectors determine final cellular
response to agonist

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

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

13. 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

14. 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

15. 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

16. 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

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

18. 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?

19. 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

20. 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
% of total breceptors
ATP
cAMP

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

22. 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

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

24. 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

25. 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

26. 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

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

28. Myocyte Gq family members are required for
hypertrophic response to TAC

29. 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?

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

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

32. 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

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

34. 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

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

36. 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

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

38. 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

39. Positional Map of Adrenergic Receptor Variants

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

41. 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

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