EXCITATION-CONTRACTION COUPLING IN SINGLE GUINEA-PIG VENTRICULAR MYOCYTES EXPOSED TO HYDROGEN-PEROXIDE

1. The effects of hydrogen peroxide (H2O2), an in vitro free radical generating system, on excitation-contraction (E-C) coupling were studied in isolated adult guinea-pig ventricular myocytes using Ca2+-sensitive dyes and the patch-clamp technique. 2. In paced myocytes loaded with indo-1 AM, 1 mM H2O2 briefly increased, then decreased the amplitude of intracellular Ca2+ ([Ca2+](i)) transients and cell contractions. Diastolic [Ca2+](i) increased in association with cell shortening. Automaticity also developed, followed shortly by inexcitability. In contrast, paced myocytes exposed to the metabolic inhibitors carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and 2-deoxyglucose (DG), rapidly became inexcitable and exhibited marked diastolic shortening prior to increases in diastolic [Ca2+](i). 3. In patch-clamped myocytes loaded with fura-2, H2O2, reduced the amplitude of the Ca2+ current (I-Ca), the [Ca2+](i) transient, and active cell shortening. H2O2 prolonged the relaxation phase of the [Ca2+](i) transient, and activated an outward membrane current consistent with the ATP-sensitive K+ current (I-K,I-ATP), but did not change the voltage dependence of I-Ca, the peak [Ca2+](i) transient or active cell shortening. These responses were qualitatively similar to patch-clamped myocytes exposed to FCCP and DG. 4. Following exposure to H2O2, I-Ca elicited smaller [Ca2+](i) transients than under control conditions. This was consistent With the observation that H2O2 reduced sarcoplasmic reticulum (SR) stores of Ca2+ by 42 %, when assessed by observing the [Ca2+](i) transients elicited by rapid extracellular application of 5 mM caffeine. In contrast FCCP-DG tended to increase SR Ca2+ stores. 5. Despite the decrease in the caffeine-induced Ca-1(2+) release after H2O2, there was an increase in the Na+-Ca2+ exchange current associated with the caffeine-induced [Ca2+](i) transient. 6. We conclude, therefore, that as with metabolic inhibitors, H2O2 interferes with EC coupling in guinea-pig myocytes by impairing I-Ca and activating I-K,I-ATP. However, unlike metabolic inhibitors, H2O2 stimulates Na+-Ca2+ exchange and depletes SR Ca2+ stores. Furthermore, diastolic [Ca2+](i) becomes elevated while the myocyte is still excitable. These observations suggest that free radicals have primary effects on cardiac CC coupling independent of their depressant effects on metabolism.