Intracellular calcium level required for calpain activation in a single myocardial cell

We have hypothesized that calpain mediates myocardial injury induced by Ca²⁺ overload. However, in vitro study demonstrated that the calcium requirement for calpain activation is around 10 μM, which is difficult to reach without the cell collapsing. Furthermore, because calpastatin is abundant in the myocardial cell, calpain may not be activated in physiological conditions. To elucidate whether calpain is activated by the calcium concentration reachable in myocardial living cells, we measured the calpain activity and the calcium concentration simultaneously in isolated guinea-pig cardiomyocytes. t-Butoxycarbonyl-Leu-Met-7-amino-4chlorimethylcoumarin (Boc-Leu-Met-CMAC), a fluorescent substrate of calpain, and/or fura red, a calcium indicator, were loaded into isolated cardiomyocytes together, and their fluorescence were measured separately. Intracellular Ca overload was induced by changing the superfusate from normal Tyrode solution to a sodium-free one. After changing the solution, fluorescence intensity of fura red and Boc-Leu-Met-CMAC did not change for a while, then fluorescence intensity of fura red began to rise. This was followed by the fluorescence intensity of Boc-Leu-Met-CMAC starting to rise 160±45 s after [Ca²⁺]_i increase. The relative fluorescence intensity of fura red increased to 1.37±0.32 folds of the control at the point that calpain became active. The calcium concentration at this point was estimated as 451 nM. These results indicate that calpain is activated by the slight rise of Ca concentration in intact cardiomyocytes.