The overall goal of this project is to gain fundamental new insights into mechanisms which determine expression of two of the proteins that are pivotal in regulation of calcium concentration in cardiac myocytes: the Na/Ca exchanger and the alpha-1 subunit of the L-type calcium channel. The channel provides the predominant calcium influx pathway; the exchanger provides the predominant calcium efflux pathway. Therefore, alterations in expression of these proteins have profound effects on cell calcium homeostasis and excitation-contraction coupling. There is increasing evidence that abnormal expression of the exchanger and calcium channel play a role in the contraction/relaxation abnormalities present in human heart failure.
Specific aims i nclude testing the hypothesis that tonically increased calcium concentration in cultured adult rat myocytes produces a coordinate increase in expression of the Na/Ca exchanger and of the L-type calcium channel. It will be determined whether activation of protein kinase A and protein kinase C signaling pathways produces coordinate alterations in expression and function of the Na/Ca exchanger and calcium channel in myocytes. The in vitro experiments on exchanger and calcium channel expression will be extended to in vivo studies in the rat. Rats will receive chronic infusions of norepinephrine and expression and function of the Na/Ca exchanger and calcium channels will be examined. The hypothesis will be tested that denervation and reinnervation in vivo produces coordinate regulation of expression of the exchanger and calcium channel. Rats will undergo chemical sympathetic denervation, myocytes will be isolated, and message levels, protein abundance, and function of the DHP receptor and exchanger will be determined biophysically. Other rats will be studied following reinnervation and subsequent expression of the exchanger and DHP receptor will be examined. Experimental approaches include a combination of molecular, ligand binding, and biophysical techniques that will relate cell calcium and second messages to function of the exchanger and calcium channel and will determine the net effect on calcium homeostasis and contractile properties of single cells. Taken together, these studies will determine (1) whether expression of the exchanger and calcium channel is coordinately regulated, (2) whether calcium-dependent, protein kinase C, and protein kinase A dependent second messenger systems play an important role, and (3) the in vitro and in vivo consequences of channel and exchanger alteration on calcium homeostasis and cell function.
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