Excitation‐contraction coupling in single guinea‐pig ventricular myocytes exposed to hydrogen peroxide.

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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.

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