Research InterestsMechanisms of heterotrimeric G protein signaling, defining the roles of G proteins and their regulators in the cardiovascular system.
Vice Chair, Research, Department of Physiology
Keywords: G-protein signaling/RGS protein function/vascular smooth muscle cell funtion/sinoatrial myocyte function/atrial myocyte function
Detailed Description: Many physiologic processes are mediated by a group of switch-like heterotrimeric G proteins. G proteins are normally coupled to receptors on the cell surface to act as intracellular relays between environmental stimuli and the rest of the cell. Our work defines the biologic importance for precise kinetic regulation of G-protein-mediated signaling events.
Regulation of G-protein signaling pathways:
The G-protein heterotrimer is composed of a GDP-bound G alpha subunit and a G beta gamma heterodimer. In the absence of an extracellular stimulus, the G-protein is coupled to a plasma membrane-spanning receptor (G protein-coupled receptor; GPCR). Receptor activation results in the exchange of GTP for GDP on the G alpha subunit and the dissociation of GTP-bound G alpha from the G beta gamma heterodimer. This condition marks the activated ("ON") state during which time the G alpha and G beta gamma subunits are free to engage appropriate downstream effector pathways. Effector signaling is terminated following G alpha catalysed hydrolysis of GTP and reformation of the quiescent receptor-coupled heterotrimer.
RGS proteins are a family of GTPase activating proteins (GAPs) for G alpha subunits. By increasing the intrinsic rate of GTP hydrolysis for G alpha subunits, RGS proteins impact GPCR-mediated signaling pathways by:
i) promoting faster signal termination kinetics following removal of a physiologic GPCR agonist; and ii) decreasing GPCR agonist sensitivity (i.e. higher agonist concentrations are needed to achieve the same degree of signaling). Our work is aimed at defining the molecular mechanisms that regulate the function of RGS proteins in vivo . Using a combination of physiology, biochemistry, cell biology, pharmacology, and genetics we examine how subcellular localization, G-protein selectivity and interaction with other cellular signaling components regulates the function of RGS proteins in living organisms.
Cardiac Myocyte Function in Disease Models:
G-protein signaling is involved in the regulation of imprtant cardiac cell functions including inotropy and pacemaking within the sinoatrial node. Our work has shown that proper regulation of parasympathetic signals in the sinoatrial node requires the function of RGS proteins, specifically RGS4. RGS4 knockout mice show profound sensitivity to parasympathetic activity at the level of heart rate control. Our recent work has been aimed at understanding the effect of altered RGS4 function on pacemaking and arrhytmogenesis in cardiac tissues.
VSMC Function in Disease Models:
Hypertension is a leading risk factor for cardiovascular disease in humans. My laboratory is interested in using genetic mouse models to understand the molecular pathways that regulate blood pressure at the level of the peripheral vasculature. Recently we discovered that mice lacking RGS2, a potent inhibitor of Gq signaling, are profoundly hypertensive and show prolonged vasoconstrictor signaling in the peripheral vasculature. We are currently determining the role of the vasculature, kidney and central nervous system in mediating this phenotype. We are using calcium imaging to study vasoconstrictor responses primary cultured VSMCs in vitro , vessel bath technologies to study vascular contractile function in situ and blood pressure measurements in surgically implanted mice to study whole animal physiology. Future studies are aimed at defining new therapeutic targets for the treatment of hypertension and heart disease.
Modulation of RGS protein location and function by DHHC isoforms (palmitoyl-CoA transferases):
The RGS family of proteins contain cysteine residues in their amino termini that may be selectively palmitoylated. The palmitoylation status of these residues has important consequences on the ability of these proteins to associate with the plasma membrane or intracellular endosomal pools. We are currently using a multitude of microscopic and cell signaling tools to characterize the biological function of differentially palmitoylated RGS at both the plasma membrane and within intracellular pools.
Cell and tissue culture: Artery cultures, cardiomyocytes, smooth muscle cells, endothelial cells, atrial tissue, perfused heart (Langendorf), sinoatrial node cells
Procedures: Adenovirus, behavioural tests, cAMP RIA, confocal microscopy, EEG, electrophysiology, Fura2 calcium imaging, gene expression analysis, immunohistochemistry, inositol phosphate measurement, in-vitro electrophysiology, in-vivo electrophysiology, isolated atria preparation, isolated vessel preparation, mouse femoral artery injury, patch clamp, radiotherapy, rat carotid balloon injury, RIA, RT-PCR, siRNA, vagotomy, vessel cannulation, voltage clamp, western blot.
Amplifier (ADI BioLab), analytical balances, benchtop centrifuge, blood pressure telemetry units for mice (DSI), blotting apparatus, calcium imaging system (PTI), culture hood, culture incubators, departmental beta and gamma counters, dissecting microscope, ECG telemetry units for mice (DSI), fluorescence microscope, gel apparatus, infusion apparatus, low and high-speed centrifuge (2x Sorvall RC5B), low and ultralow freezers, microwave oven, Millar 1.4F solid state blood pressure catheters, mini vortexer, monochromator, setups for electropherosis, stimulator, stirrer/hot plate, telemetry workstation (DSI), water baths.
Within the Department of Physiology:
Outside the Department of Physiology:
Committee member/officer of national/international scientific organizations:
Councillor, Canadian Society for Atherosclerosis Thrombosis and Vascular Biology (CSATVB)
Publications and Awards
1. Atrial Fibrillation Promotion by Endurance Exercise: Demonstration and Mechanistic Exploration in an Animal Model. Guasch E, Benito B, Qi X, Cifelli C, Naud P, Shi Y, Mighiu A, Tardif JC, Tadevosyan A, Chen Y, Gillis MA, Iwasaki YK, Dobrev D, Mont L, Heximer S, Nattel S. J Am Coll Cardiol. 2013 Apr 10.S0735-1097(13)01408-3. S0002-9440(13)00188-0.