Effects of Simulated Ischemia and Reperfusion on the Sarcoplasmic Reticulum of Digitonin-Lysed Cardiomyocytes

ATP-dependent, inorganic phosphate-supported Ca uptake by digitonin-lysed adult rat ventricular cardiomyocytes was used to evaluate the effects of simulated ischemia and reperfusion on the physically intact sarcoplasmic reticulum. Mitochondrial reactions were inhibited with rotenone and oligomycin. Ca accumulation in the presence of the calcium efflux inhibitors, procaine (10 mM) and ruthenium red (30 μM), was used to characterize unidirectional uptake kinetics. A decrease in pH from 7.2 to 6.6 increased the [Ca] K0.5 from 0.5 to 2.0 μM and reduced the apparent Vmax by 28%. In the absence of procaine and ruthenium red, at a free [Mg] of 0.5 mM, maximum net uptake occurred at pCa 6.2 when pH was 7.2 and at pCa 6.0 when pH was 6.6. At lower pCa, net Ca accumulation declined. Increasing free [Mg] from 0.5 to 1 mM at pH 6.6 or to 2.5 mM at pH 7.2 increased net Ca accumulation in the absence of procaine and ruthenium and shifted maximum uptake to pCa 5.6 and 6.0, respectively. Increases in cytosolic free [Mg] thought to occur during myocardial ischemia are therefore capable of inhibiting calcium efflux from the sarcoplasmic reticulum. Reducing [ATP] from 10 to 1 mM reduced maximum net Ca uptake by 30% both in the presence and absence of efflux inhibitors. Preincubation of intact myocytes under conditions designed to simulate ischemia and reperfusion decreased Ca uptake ≥.50%. The data indicate that myocardial ischemia and reperfusion can alter both Ca accumulation and calcium release by the sarcoplasmic reticulum.