The L-type Ca2+ channel provides the initial triggering influx of Ca2+ (I/Ca) for excitation-contraction coupling in cardiac muscle. This channel is an extensively regulated protein complex composed of three subunits: alpha1, beta and alpha2-delta. Stimulation of neurohormonal receptors such as endothelin (ET), angiotensin II, and alpha1-adrenergic receptors result in the production of the second messenger diacylglycerol (AG), which can activate protein kinase C (PKC). The effects of activation of PKC on the contractility of the heart and on the L-type Ca2+ channel are controversial. The objectives of this proposal are to clarify the effect of neurohormonal activation of PKC on native cardiac L-type Ca2+ channel are controversial. The objectives of this proposal are to clarify the effect of neurohormonal activation of PKC on native cardiac L-type Ca2+ channels and determine the molecular mechanisms underlying this regulation. Preliminary data using the perforated whole-cell patch clamp technique demonstrate a potent increase in Ica in response to ET and photorelease of a DAG analogue. The hypothesis that PKCepsilon directly stimulates cardiac L-type Ca2+ channel activity by phosphorylation of the alpha1C subunit will be tested. Experiments will utilize isolated rat ventricular myocytes and cultured embryonic stem cell- derived cardiomyocytes, which will be studied using a series of complementary cellular electrophysiology, biochemical, and molecular biology approaches. We propose to address the cardiac myocytes in response to ET and photorelease of caged diC8; 2) Determine the PKC isoform specificity by modulation of cardiac L-type Ca2+ channels; 3) Characterize the phosphorylation of the alpha1C subunit by PKC and determine the functional consequences. Overall, these results will provide a new molecular framework to understand the regulation of L-type Ca2+ channel by the CAG/PKC system. Activation of PKC has been implicated in myocardial hypertrophy and heart failure as well as in ischemic heart disease. Understanding the detailed regulation of the L-type Ca2+ channel by PKC may provide insights and potential novel therapeutic strategies for these disease processes.
Showing the most recent 10 out of 103 publications
