The overall aim of the proposed research is to understand PKC-e and PKC-S function in the mammalian heart, with an emphasis on how these calcium-independent diacylglycerol-activated serine/threonine kinases regulate calcium handling and contractile properties. The overall hypothesis to be tested is that PKC-e and PKC-S can inhibit or stimulate cardiac contractility and calcium fluxes depending upon the subcellular compartments in which they accumulate.
In Aim 1, native constructs of PKC-e and PKC-S isoforms will be fused with fluorescent proteins and expressed in adult rat ventricular myocytes to establish a link between PKC isoform expression level, sites of translocation, altered systolic calcium and inotropic responses.
In Aim 2, use of dominant negative PKC-e and PKC-S constructs will address the isoform(s) involved in contractile responses to cell-permeable PKC activators and to agonists of G-protein coupled receptors.
In Aim 3, the subcellular localization of diacylglycerol will be controlled independently of agonist receptors with light-activated caged compounds to determine diacylglycerol's functional effects in surface membranes, transverse-tubules and perinuclear regions of adult rat myocytes. The outcome of this research will shed new light on mechanisms of action of PKC-e and PKC-S and their control by agonists such as the endothelin peptides and other agonists operating through G-protein coupled receptors. The endothelin/diacylglycerol/protein kinase C signaling system represents an important regulatory axis in the mammalian heart which is thought to play a central role in control of contractility, intracellular calcium, gene expression, growth, cell death, and the heart's response to chronic stress such as hypoxia/ischemia or high blood pressure. Evidence is also accumulating that this signaling system is altered in failing hearts and may contribute to disease progression. A better understanding of coupling between receptors and PKC isoforms, and the subcellluar compartments in which each isoform acts to regulate basic cardiac function, will ultimately provide a foundation on which to explore signaling defects and other mechanisms of cardiac dysfunction in various forms of heart disease.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL081386-03
Application #
7255456
Study Section
Cardiac Contractility, Hypertrophy, and Failure Study Section (CCHF)
Program Officer
Buxton, Denis B
Project Start
2005-07-01
Project End
2010-06-30
Budget Start
2007-07-01
Budget End
2008-06-30
Support Year
3
Fiscal Year
2007
Total Cost
$308,008
Indirect Cost
Name
University of Wisconsin Madison
Department
Physiology
Type
Schools of Medicine
DUNS #
161202122
City
Madison
State
WI
Country
United States
Zip Code
53715
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