The long-term objectives are to investigate the mechanism of excitation contraction (E-C) coupling in heart and to study the contribution of reverse Na-Ca exchange to """"""""triggering"""""""" sarcoplasmic reticular (SR) Ca release in mammalian heart. In particular, defects in the mechanism of E-C coupling may be central to such health related problems as the long QT syndrome, heart failure and various cardiomyopathies.
The specific aims will be approached with the following design and methods. Isolated guinea pig ventricular cells will be voltage clamped with single suction microelectrodes. Ca and in some cases Na currents will be activated by appropriate membrane depolarizations. Ca transients and SR Ca release will be measured with the salt form of the dye Indo-1. The extent to which the inward L-type Ca current is responsible for triggering SR Ca release will be assessed by dialyzing the cell with the newly developed exchanger inhibitory peptide XIP. These experiments will be repeated while the cell is dialyzed with various concentrations of Na. The concentration of intracellular Na will be measured with the Na sensitive indicator SBFI and the relationship between intracellular Na and the extent of triggering of SR Ca release by reverse Na-Ca exchange will be assessed. Experiments will be conducted to ensure that the combination of Ca current and reverse Na-Ca exchange does not directly activate the contractile elements. The voltage dependance of SR Ca release triggered by reverse Na-Ca exchange will be measured and the voltage dependence of reverse exchange current will be compared with this measurement. Methods for rapidly injecting the exchanger inhibitory peptide into the cell will be employed to assess the extent to which reverse Na-Ca exchange triggers SR Ca release during cardiac action potentials, at body temperature, when the concentration of intracellular Na is believed to be physiological and during glycoside treatment. Finally, the extent to which Na current indirectly contributes to SR Ca release by causing Na to accumulate in the vicinity of the exchangers thus stimulating reversal of exchange will be assessed.
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