Time-dependent Effects of Thapsigargin on Cardiac Intracellular Ca~2+ Release and Sarcoplasmic Reticulum Ca~2+ Content

[Objective] Thapsigargin (TG), a sarcoplasmic reticulum (SR) Ca2+-ATPase inhibitor, is commonly employed to deplete SR Ca2+ content. In this study, we investigated the time effects of TG (100 nmol·L-1) on SR Ca2+ content and SR Ca2+ release, and the relationship between them. [Methods] Local field stimulation was delivered to freshly isolated rat single cardiac myocytes to generate action potential-induced Ca2+ transients (ACT) for assessing intracellular Ca2+ release. Caffeine (20 mmol · L-1)-induced Ca2+ transients (CCT) was used to estimate the SR Ca2+ content. Results were recorded by confocal Ca2+ imaging with a Zeiss LSM-410 confocal microscopic system. [Results]Time-dependent effects of TG on ACT peak were: 5. 55±0. 28 (in F/ F0, the normalized fluorescence, where Fo denotes the resting fluorescence), 0 min; 4. 89 ± 0. 36, 10 min; 2. 58±0.38, 17 min; 2. 11 ±0. 19, 25 min; 2.22±0.17, 35 min; and 2.27±0.27,50 min. Time-dependent effects of TG on CCT peak were: 6.73±0. 17, 0 min; 6. 60 ±0.59, 10 min; 6. 24 ±0.44, 17 min; 3.99±0.28, 25 min; 2. 24 ±0.37, 35 min; and 1. 14 ±0. 01, 50 min. Curve of ACT as a function of CCT displayed a steep “J”-like shape. [Conclusion]TG demonstrates a nonlinear time-dependence on cardiac intracellular Ca2+ release and SR Ca2+ content. Partial depletion of SR Ca2+ content severely diminishes Ca2+ release, suggesting that SR Ca2+ release is tightly regulated by Ca2+ from the lumenal side of the SR.