The relation between Ca^<2+>-transient and Ca^<2+>-handling energy in excitation-contraction coupling

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To establish the method to obtain isolated cardiac myocytes, we perfused guinea pig heart with collagenase solution by Langendorff perfusion method. We improved many points in principle method, and got intact myocytes at considerably high percentage. However, to measure calcium fluorescence of cell suspension, we tried to get more and more yields of intact myocytes. However, we concluded that higher than 70-80% of yield could not be attained. Therefore, neither calcium fluorescence nor oxygen consumption of cell suspension could be measure. Then, we are going to establish the measurement of intracellular calcium and oxygen consumption by using cardiac muscle slices. Simultaneous measurement of intracellular calcium and oxygen consumption was very difficult. Also, we determined to measure them separately. At first, the measuring system for oxygen consumption could be established. Recently, we got reasonable values of oxygen consumption, which is supposed to be the sum of basal metabolism and calcium handling energy in excitation-contraction coupling. Next, by using CAF110 which was bought by this grand, we examined pCa-intensity of fluorescence relation at 340nm and 380nm. The ratio of 340nm to 380nm gradually increased between pCa = 8.5 and 7.5. Then, we did the following experiments. We dissected canine left ventricles into big blocks and put them into ice-cold KB solution. After removal of epicardial parts, these blocks were cut into small pieces (several mm). Small pieces from normal hearts and Ca overload hearts were loaded with FuraII-AM in KB solution. After 3-4 hrs, small pieces were washed out and measured their intracellular calcium concentration. No difference was observed between intracellular calcium concentration of normal hearts and Ca overload failing hearts.