Biophysical properties and functional consequences of reactive oxygen species (ROS)-induced ROS release in intact myocardium

Reactive oxygen species (ROS)-induced ROS release (RIRR) is a fundamental mechanism by which cardiac mitochondria respond to elevated ROS levels by stimulating endogenous ROS production in a regenerative, autocatalytic process that ultimately results in global oxidative stress (OS), cellular dysfunction and death. Despite elegant studies describing the phenomenon of RIRR under artificial conditions such as photo-induced oxidation of discrete regions within cardiomyocytes, the existence, biophysical properties and functional consequences of RIRR in intact myocardium remain unclear. Here, we used a semi-quantitative approach of optical superoxide (O-2(-)) mapping using dihydroethidium (DHE) fluorescence to explore RIRR, its arrhythmic consequences and underlying mechanisms in intact myocardium. Initially, perfusion of rat hearts with 200 mu M H2O2 for 40 min (n = 4) elicited two distinct O-2(-) peaks that were readily distinguished by their timing and amplitude. The first peak (P1), which was generated rapidly (within 5-8 min of H2O2 perfusion) was associated with a relatively limited (10 +/- 2%) rise in normalized O-2(-) levels relative to baseline. In contrast, the second peak (P2) occurred 19-26 min following onset of H2O2 perfusion and was associated with a significantly greater amplitude compared to P1. Spatio-temporal ROS mapping during P2 revealed active O-2(-) propagation across the myocardium at a velocity of similar to 20 mu m s(-1). Exposure of hearts (n = 18) to a short (10 min) episode of H2O2 perfusion revealed consistent generation of P2 by high (>= 200 mu M, 8/8) but not lower (<= 100 mu M, 3/8) H2O2 concentrations (P < 0.03). In these hearts, onset of P2 occurred following, not during, the 10 min OS protocol, consistent with RIRR. Importantly, P2 (+) hearts exhibited a markedly greater (by 3.8-fold, P < 0.001) arrhythmia score compared to P2 (-) hearts. To explore the mechanism underlying RIRR in intact myocardium, hearts were perfused with either cyclosporin A(CsA) or 4'-chlorodiazepam (4'-Cl-DZP) to inhibit the mitochondrial permeability transition pore (mPTP) or the inner membrane anion channel (IMAC), respectively. Surprisingly, perfusion with CsA failed to suppress (P = 0.75, n.s.) or even delay H2O2-induced P2 or the incidence of arrhythmias compared to untreated hearts. In sharp contrast, perfusion with 4'-Cl-DZP markedly blunted O-2(-) levels during P2, and suppressed the incidence of sustained ventricular tachycardia or ventricular fibrillation (VT/VF). Finally, perfusion of hearts with the synthetic superoxide dismutase/catalase mimetic EUK-134 completely abolished the H2O2-mediated RIRR response as well as the incidence of arrhythmias. These findings extend the concept of RIRR to the level of the intact heart, establish regenerative O-2(-) production as the mediator of RIRR-related arrhythmias and reveal their strong dependence on IMAC and not the mPTP in this acute model of OS.