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

Abstract

We used the relation between intracellular Ca²⁺ concentration ([Ca²⁺]_i) and cell shortening during tetanus to evaluate the endogenous characteristics of Ca²⁺ responsiveness of myofibrils in mouse ventricular myocytes. Enzymatically isolated myocytes were loaded with fura-2 AM (4 μM 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 μM) (an inhibitor of the Ca²⁺ 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 Ca²⁺ concentration ([Ca²⁺]_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 μM) (desensitization of the myofibrils to Ca²⁺), and shifted leftward by the Ca²⁺ sensitizing thiadiazinone derivative, EMD-57033 (0.5 μM) (sensitization of the myofibrils to Ca²⁺). β-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 Ca²⁺ in isolated mouse myocytes and can be applied to various mouse models of heart disease.