Use of Tetanus to Investigate Myofibrillar Responsiveness to Ca2+ in Isolated Mouse Ventricular Myocytes.

We used the relation between intracellular Ca(2+) concentration ([Ca(2+)](i)) and cell shortening during tetanus to evaluate the endogenous characteristics of Ca(2+) responsiveness of myofibrils in mouse ventricular myocytes. Enzymatically isolated myocytes were loaded with fura-2 AM (4 microM for 10 min), and the fura-2 fluorescence ratio at 340 and 380 nm excitation wave length [F(340)/F(380)] and cell length were measured simultaneously. Following treatment with thapsigargin (0.2 microM) (an inhibitor of the Ca(2+) pump of sarcoplasmic reticulum), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction and an instantaneous plot of the fluorescence ratio signal versus cell length (R-L trajectory) was constructed. An increase in the extracellular Ca(2+) concentration ([Ca(2+)](o)) from 0.5 to 2 mM extended the R-L trajectory without a substantial shift of the relation. The R-L trajectory was shifted rightward by the nonselective phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xantine (IBMX, 200 microM) (desensitization of the myofibrils to Ca(2+)), and shifted leftward by the Ca(2+) sensitizing thiadiazinone derivative, EMD-57033 (0.5 microM) (sensitization of the myofibrils to Ca(2+)). Beta-adrenergic stimulant, isoproterenol (5 nM), also shifted the R-L trajectory to the right, suggesting that the membrane receptor could be preserved. These results suggest that the R-L trajectory is a useful method to estimate the myofibrillar responsiveness to Ca(2+) in isolated mouse myocytes and can be applied to various mouse models of heart disease.