title: Biophysical properties and functional consequences of reactive oxygen species (ROS)-induced ROS release in intact myocardium.
abstract: 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 μM H(2)O(2) 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 H(2)O(2) 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 H(2)O(2) 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 ~20 μm s(-1). Exposure of hearts (n = 18) to a short (10 min) episode of H(2)O(2) perfusion revealed consistent generation of P2 by high (≥200 μM, 8/8) but not lower (≤100 μM, 3/8) H(2)O(2) 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 H(2)O(2)-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 H(2)O(2)-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.
authors.affiliation: Cardiovascular Institute, Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA.
authors.forename: Nora
authors.initials: N
authors.lastname: Biary
authors.forename: Chaoqin
authors.initials: C
authors.lastname: Xie
authors.forename: Justin
authors.initials: J
authors.lastname: Kauffman
authors.forename: Fadi G
authors.initials: FG
authors.lastname: Akar
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology / Q000627:therapeutic use; D001145:Arrhythmias, Cardiac / Q000188:drug therapy / Q000378:metabolism / Q000503:physiopathology*; D016572:Cyclosporine / Q000494:pharmacology; D003975:Diazepam / Q000031:analogs & derivatives / Q000494:pharmacology; D004996:Ethidium / Q000031:analogs & derivatives; D005453:Fluorescence; D005456:Fluorescent Dyes; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000378:metabolism / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000502:physiology; D000083162:Mitochondrial Permeability Transition Pore; D009206:Myocardium / Q000378:metabolism*; D009942:Organometallic Compounds / Q000494:pharmacology / Q000627:therapeutic use; D016877:Oxidants / Q000494:pharmacology; D018384:Oxidative Stress; D051381:Rats; D012459:Salicylates / Q000494:pharmacology / Q000627:therapeutic use; D013481:Superoxides / Q000378:metabolism*; D050994:Voltage-Dependent Anion Channels / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: C488690:4'-chlordiazepam; D000975:Antioxidants; C433928:EUK-134; D005456:Fluorescent Dyes; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D009942:Organometallic Compounds; D016877:Oxidants; D012459:Salicylates; D050994:Voltage-Dependent Anion Channels; C067883:dihydroethidium; D013481:Superoxides; D016572:Cyclosporine; D006861:Hydrogen Peroxide; D004996:Ethidium; D003975:Diazepam