The purpose of this research program is to gain new insights into cellular and molecular mechanisms that regulate expression of ion channels in heart. Although our understanding of mechanisms regulating contractile protein gene expression has advanced rapidly, we know relatively little about regulation of expression of another structure crucial to control of cardiac contractile performance: the L-type calcium channel. A goal of this project is to learn how neural, ionic and paracrine factors determine how many functional copies of the L-type calcium channel are present in the sarcolemma of heart cells. Recent experiments from this laboratory demonstrate that sympathetic innervation in vitro increases calcium channel expression determined by ligand binding and patch clamp studies. In a cultured rat ventricular myocyte model, we will determine by what mechanisms sympathetic innervation and specific neuroeffectors regulate dihydropyridine (DHP) receptor gene expression and the relation between abundance of message, DHP receptor protein, and channel function. A myocyte-sympathetic ganglion co-culture model will be utilized that permits studies on direct myocyte innervation and denervation. Then, in cultures of myocytes alone, three candidate neuroeffectors will be studied: norepinephrine, nerve growth factor and calcitonin gene-related peptide. The role of protein kinases C and A in mediating expression of DHP message and protein will be assessed. There is evidence suggesting that alterations in cytosolic calcium concentration may regulate expression of calcium channels. Accordingly, we will determine whether, and by what means, a decrease or increase in cytosolic calcium concentration alters the abundance of DHP receptors in heart. Furthermore, immunofluorescence and immunocolloidal gold labeling studies will be conducted to determine where in cardiac myocytes DHP receptors are localized. The topology of the DHP receptor in the sarcolemmal membrane will be examined with recently developed site-directed antibodies. Novel probes and powerful contemporary techniques will be used to examine the integrated cellular biological response to altered DHP receptor gene expression. Abnormalities of calcium channel expression have been implicated in. pathophysiological conditions, including hypertrophy, arrhythmias, and cardiomyopathy. Gaining further understanding of mechanisms that regulate calcium channel expression is of fundamental biological importance and will have therapeutic implications.
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