IONIC MECHANISM OF THE EFFECTS OF HYDROGEN-PEROXIDE IN RAT VENTRICULAR MYOCYTES

1. Whole-cell and amphotericin-perforated patch-clamp techniques have been used to study the effects of hydrogen peroxide (H2O2) on action p otentials and underlying ionic currents in single myocytes from the ve ntricles of adult rat hearts. 2. The results obtained differed markedl y depending on the recording method utilized. Conventional whole-cell recordings, in which the myoplasm is dialysed with the contents of the pipette, failed to show any significant effects of H2O2 on the action potential or cell shortening. In contrast, when action potentials wer e recorded with the amphotericin-perforated patch method, H2O2 (50-200 mu M) produced a marked prolongation of the action potential and an i ncrease in cell shortening. 3. Voltage-clamp recordings with the ampho tericin-perforated patch method showed that H2O2 caused no significant changes is either the Ca2+-independent transient outward K+ current ( I-to) or the inwardly rectifying K+ current (I-K1). 4. Application of tetrodotoxin (TTX; 8 x 10(-6) M), a Na+ channel blocker, largely inhib ited the effects of H2O2 on the action potential. Moreover, anthopleur in A (4 x 10(-7) M), which augments Na+ current (I-Na) by slowing its inactivation, mimicked the effects of H2O2 on the action potential of ventricular myocytes. These effects on I-Na were also blocked almost c ompletely by TTX. 5. The hypothesis that H2O2 can augment I-Na by slow ing its kinetics of inactivation was tested directly using ensemble re cordings from cell-attached macropatches. These results demonstrated a significant enhancement of late opening events when H2O2 (200 mu M) w as included in the recording pipette. A corresponding slowing of inact ivation of the ensemble I-Na was observed. 6. The possibility that pro tein kinase C (PKC) is an intracellular second messenger for the obser ved effects of H2O2 was examined using the blocker bisindolylmaelimide (BIS; 10(-7) M). Bath application of BIS prior to H2O2 exposure signi ficantly delayed and also attenuated the development of the action pot ential prolongation. 7. These results demonstrate marked electrophysio logical effects of H2O2 in rat ventricle. The dependence of these effe cts on recording methods suggests involvement of an intracellular seco nd messenger, and the results with the PKC inhibitor, BIS, support thi s possibility. The most prominent effect of H2O2 on the ionic current which underlie the action potential is a slowing of inactivation of th e TTX-sensitive I-Na. Recent molecular studies have demonstrated a PKC phosphorylation site on the rat cardiac Na+ channel isoform and have also shown that PKC activation can slow inactivation of I-Na.