Digital-imaging microscopy analysis of calcium release from sarcoplasmic reticulum in single rat cardiac myocytes

Abstract

Digital imaging microscopy of fura-2 fluorescence has allowed us to assess the dynamic patterns of local Ca increase in newly isolated rat myocardial cells. Of the myocytes bathed in a saline solution (1.8 mM Ca2+, 37 degrees C, pH 7.4), 10%-20% exhibited local spontaneous contractions. The resting intracellular free calcium concentration ([Ca2+]i) of these cells was 106 +/- 4 nM versus 77 +/- 3 nM for non-contracting cells. The spontaneous contractile activity appeared to be closely related to internal spontaneous Ca waves that spread across the myoplasm (velocity approximately 50 microns/s, maximal Ca amplitude = 195 +/- 11 nM) along the major axis of the cells. Precise topographical examination of Ca wave propagation indicated a refractory period for internal Ca release. The occurrence of both the generation and propagation of spontaneous Ca increases appeared to be closely dependent on the extent of Ca loading of the cells. Most of our observations were in accordance with the assumption that local Ca overload of the sarcoplasmic reticulum (SR) is the main parameter involved in the spontaneous Ca-release phenomena. Using the same approach, the increase in internal Ca evoked by KCl (50 mM) addition was investigated, and compared with that seen during spontaneous activity. Total [Ca2+]i increase induced by K+ depolarization involved three consecutive local Ca-release patterns: (a) a peripheral Ca enhancement that remained during the total [Ca2+]i increase, (b) subsequent transversal local Ca increases occurring in Z-line regions, (c) longitudinal local Ca increases. In addition, a weak heterogeneous Ca distribution was detected in both peripheral and central parts of resting cardiac cells. Thus, the total Ca increase seemed to result consecutively from a peripheral Ca pool, from junctional SR and from longitudinal structures (possibly longitudinal SR).