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

为了建立获得离体心肌细胞的方法，我们采用Langendorff灌注法对豚鼠心脏灌注胶原酶溶液。我们在原理方法上进行了许多改进，获得了相当高比例的完整肌细胞。然而，为了测量细胞悬液的钙荧光，我们试图得到越来越多的完整肌细胞的产量。然而，我们得出的结论是，不可能达到高于70-80%的产量。因此，不能测量细胞悬液的钙荧光和耗氧量。然后，我们将利用心肌切片建立细胞内钙和氧消耗的测量方法。同时测量细胞内钙和氧的消耗是非常困难的。同时，我们决定分别测量它们。首先，建立了耗氧量测量系统。最近，我们得到了合理的耗氧量值，它应该是基础代谢和钙处理能量在兴奋-收缩耦合中的总和。接下来，我们用本台购买的CAF110检测了340nm和380nm处的pca荧光强度关系。在pCa = 8.5 ~ 7.5之间，340nm / 380nm的比值逐渐增大。然后，我们做了以下实验。我们将狗的左心室解剖成大块，放入冰冷的KB溶液中。在切除心外膜部分后，这些块被切成小块（几毫米）。取正常心脏和钙超载心脏的小块，在KB溶液中加入FuraII-AM。3-4小时后，冲洗小块，测量细胞内钙浓度。正常心脏细胞内钙浓度与钙超载衰竭心脏细胞内钙浓度无差异。

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.