This application requests support for the continued study of the intracellular ionic environment in cardiac tissue. This is important because the intracellular free-Na+ (aiNa) and free-Ca++ (aiCa) activities are not constant, but vary as a function of membrane voltage, stimulation frequency, and time; and can be modified by alterations in the extracellular ionic composition and by certain drugs. Because of the Na/Ca exchange system, changes in aiNa produce changes in aiCa and as a consequence, tension. Thus, modulation of aiNa may be an important mechanism in the regulation of contractility in cardiac muscle. aiNa and aiCa will be measured with ion-selective microelectrodes in sheep Purkinje fiber and ventricular muscle preparations. The membrane voltage (Vm) will be controlled using a voltage clamp, and resting and phasic tension will be monitored. The mechanism of regulation of aiNa will be investigated in stimulated and unstimulated preparations. Of particular interest is the frequency dependent rise in aiNa that occurs with stimulation. The hypothesis that it results from Na+ entry via the Na+ channel will be tested by the use of the Na+ channel blocker TTX, and by studying the relationship between the frequency dependent rise in aiNa and the Na+ channel steady state inactivation curve. The importance of the level of activation of the Na/K pump and extracellular K+ accumulation in the regulation of aiNa will be studied by systematically reducing [K]o. With these protocols, temporal correlations between changes in aiNa, aiCa, and tension may be drawn. Furthermore, where the frequency dependent rise in aiNa is suppressed by Na+ channel inhibition, it should be possible to modify the force-frequency curve. Lastly, the coupling ratio for Na/Ca exchange may be calculated for a wide range of Vm and for steady state and non-steady state conditions. These experiments will provide new information about the regulation of Na+ and Ca++; ions that contribute directly to the complex force-frequency relationship found in cardiac muscle.