The role of the cytosolic Ca (Cai) transient in determining the configuration of the cardiac action potential was further investigated. For this purpose, rat ventricular myocytes loaded with the Ca(2+) sensitive fluorescent dye indo-1 were used. Myocytes were loaded with either 1) the acetoxy-methyl ester of indo-1 (indo-1 AM), which provides qualitative information about changes in the Cai transient or 2) the Ca(2+) sensitive, free acid form of indo-1, which allows intracellular Cai to be quantified. The magnitude of the Cai transient was graded by various physiological and pharmacological interventions and membrane voltage or current was recorded using patch-type microelectrodes. Earlier work in indo-1 AM loaded cells had shown that phase-plane loops of membrane potential (Vm) vs indo-1 ratio from a stimulus train conformed to a common trajectory during the slow tail of repolarization, despite a beat-dependent decrease in the magnitude of the Cai transient. It was found that phase plane loops of the Vm vs. indo-1 ratio from spontaneous diastolic Cai oscillations also conformed to this trajectory. Experiments with indo-1 free acid, which was incorporated into the cell by inclusion in the microelectrode filling solution, showed that in rested beats peak Cai can exceed 2 microM and then decrease to 0.4 - 0.6 microM in the steady state. Evidence was found that both electrogenic Na/Ca exchange and a Ca(2+)-dependent ion channel current may participate in the modulation of the cardiac action potential by the Cai transient.
