Effects of phosphocreatine on SR Ca2+ regulation in isolated saponin-permeabilized rat cardiac myocytes

Abstract

The effects of phosphocreatine (PCr) on sarcoplasmic reticulum (SR) Ca2+ regulation were investigated in saponin-permeabilized rat ventricular myocytes. Cells were perfused continuously with weakly Ca2+-buffered solutions approximating to the intracellular milieu. Ca2+ release from the SR was detected using Fura-2 or Fluo-3. Withdrawal of PCr reduced the frequency of spontaneous Ca2+ release by 12.8 ± 3.4 % (n = 9) and the amplitude of the spontaneous Ca2+ transient by 17.4 ± 3.1 % (n = 9). Stepwise reductions in [PCr] progressively increased the time for the spontaneous Ca2+ transient to rise from 25 to 100 % of the maximum value (TP75) and to fall by 75 % of the peak level (DT75). Following complete PCr withdrawal, the TP75 and the DT75 were 147.1 ± 13.2 and 174.8 ± 23.2 % of the control values, respectively. Experiments involving confocal microscopy showed that PCr withdrawal decreased the propagation velocity of spontaneous Ca2+ waves. PCr withdrawal also reduced the frequency and amplitude, but increased the duration of spontaneous Ca2+ sparks. Rapid application of 20 mM caffeine was used to assess the SR Ca2+ content at the point of spontaneous Ca2+ release. In the absence of PCr, the amplitude of the caffeine-induced Ca2+ transient was 18.4 ± 2.7 % (n = 9) lower than in the presence of 10 mM PCr. This suggests that PCr withdrawal reduces the maximum SR Ca2+ content that can be sustained before spontaneous Ca2+ release occurs. These results suggest that local ADP buffering by PCr is essential for normal Ca2+ regulation by the SR. Prolongation of the descending phase of the spontaneous Ca2+ transient is consistent with a reduction in the efficiency of the SR Ca2+ pump due to ADP accumulation. The fact that spontaneous Ca2+ release occurs at a lower SR Ca2+ content in the absence of PCr suggests that the Ca2+ release mechanism may also be affected. These effects may be of relevance in circumstances where PCr depletion and Ca2+ overload occur, such as myocardial ischaemia or anoxia.