The heterotrimeric G-protein Gq regulates cardiomyocyte growth in vitro and in vivo. Our recent work suggests that phosphoinositides (PI) play a key role in this response. We hypothesis that PI and the small G-protein Rho initiate signals for cardiomyocyte survival.
The first aim of this proposal is to determine what stimuli activate Rho, examine the compartmentation and targets of Rho signaling, and explore the functional consequences of Rho activation. Agonists for GPCRs, stretch, and extracellular matrix (ECM) proteins will be examined for their ability to activate Rho and modulate its localization in caveolae. FAK and Akt phosphorylation and myofibrillogensis will be examined as targets of Rho activation. In vivo studies will determine whether pressure overload affects Rho localization and activity.
The second aim i s to examine the regulation of phosphatidyinositol 4,5 bisphosphate (PIP2) formation and its compartmentation, define functional effects of PIP2 and examine the requirement for PIP2 in cardiomyocyte survival pathways. Rho has been shown to activate PI4P 5 kinase, thus interventions that activate Rho will be tested for their ability to increase PIP2. PIP2 localization in caveolae will be demonstrated and its regulation examined. PIP2 will be upregulated by adenoviral expression of PI4P 5 kinase, and decreased by 5' phosphatase expression. The requirement for PIP2 in cytoskeletal assembly, and activation of FAK and P13 kinase will be tested using the PLCdelta-PH domain to bind PIP2.
The third aim i s to examine the regulation of PIP3 formation by Gq-and Rho-dependent pathways, determine whether the phosphoinositide-dependent kinase (PDK-I) substrates Akt, S6 kinase, PKC and PKN are activated and assess involvement of PIP3 and PDK- 1 in protection against apoptosis. Proposed experiments will modulate PIP3 levels and express wt and mutant PDK-1 in cardiomyocytes and in vivo.
The fourth aim i s to examine pathways by which GLUT-1 and GLUT-4 are activated, demonstrate their effect on glucose uptake and define the requirement of glucose uptake for cardiac survival. Proposed studies examine the control of GLUT-4 translocation, GLUT-1 expression, glucose uptake and the effect of altering these on the ability of cardiomyocytes to survive Gq-induced apoptosis.
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