ESTIMATION OF MYOFIBRILLAR RESPONSIVENESS TO CA2+ IN ISOLATED RAT VENTRICULAR MYOCYTES

To estimate myofibrillar responsiveness to Ca2+, we used the relation between cell length and intracellular [Ca2+] ([Ca2+](i)) during tetani c contractions of isolated ventricular myocytes. Enzymatically isolate d rat ventricular myocytes were loaded with fura-2 AM (4 mu M for 10 m in) and excited alternately at 340 nm and 380 nm. The ratio (R) of fur a-2 fluorescence at these wavelengths [F(340)/F(380), an index of [Ca2 +](i)] and cell length (L) were measured simultaneously. Following tre atment with thapsigargin (0.2 mu M), myocytes were stimulated at 10 Hz for 10 s to produce a tetanic contraction every min and an instantane ous plot of R vs L (R-L trajectory) was constructed. The R-L trajector y followed the same path during cell shortening and re-lengthening, su ggesting that dynamic equilibrium between R and L was achieved during tetanus. Increasing the extracellular [Ca2+] from 1 to 8 mM extended t he R-L trajectory without a substantial shift of the relation. The Ca2 +-sensitizing thiadiazinone derivative, EMD57033 (1 mu M), shifted the R-L trajectory to the left (sensitization of the myofibrils to Ca2+), whereas the non-selective phosphodiesterase inhibitor, 3-isobutyl-1-m ethylxantine (200 mu M), shifted the R-L trajectory to the right (dese nsitization of the myofibrils to Ca2+), in agreement with previous res ults obtained using skinned preparations. We conclude that the R-L tra jectory is useful for estimating the myofibrillar responsiveness to Ca 2+ in isolated myocytes and may be beneficial for the evaluation of in otropic agents.