Abstract
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 Ca(2+)-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 (ICa), 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 (IK,ATP), but did not change the voltage dependence of ICa, 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, ICa 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 Ca2+i 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 E-C coupling in guinea-pig myocytes by impairing ICa and activating IK,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 E-C coupling independent of their depressant effects on metabolism.