salicylates and dihydroethidium

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 ventricu

Topics: Animals; Antioxidants; Arrhythmias, Cardiac; Cyclosporine; Diazepam; Ethidium; Fluorescence; Fluorescent Dyes; Hydrogen Peroxide; In Vitro Techniques; Intracellular Membranes; Mitochondrial Membrane Transport Proteins; Mitochondrial Permeability Transition Pore; Myocardium; Organometallic Compounds; Oxidants; Oxidative Stress; Rats; Salicylates; Superoxides; Voltage-Dependent Anion Channels

Mice that lack the mitochondrial form of superoxide dismutase (SOD2) incur severe pathologies and mitochondrial deficiencies, including major depletion of complex II, as a consequence of buildup of endogenous reactive oxygen species (Melov, S., Coskun, P., Patel, M., Tuinstra, R., Cottrell, B., Jun, A. S., Zastawny, T. H., Dizdaroglu, M., Goodman, S. I., Huang, T. T., Miziorko, H., Epstein, C. J., and Wallace, D. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 846-851 and Li, Y., Huang, T. T., Carlson, E. J., Melov, S., Ursell, P. C., Olson, J. L., Noble, L. J., Yoshimura, M. P., Berger, C., Chan, P. H., Wallace, D. C., and Epstein, C. J. (1995) Nat. Genet. 11, 376-381). These problems can be greatly attenuated or rescued by synthetic antioxidant treatment, such as with the catalytic antioxidant EUK189 (Hinerfeld, D., Traini, M. D., Weinberger, R. P., Cochran, B., Doctrow, S. R., Harry, J., and Melov, S. (2004) J. Neurochem. 88, 657-667). We have used heart mitochondria from sod2 null mice to better understand mitochondrial reactive oxygen species production both in the absence of SOD2 and following in vivo antioxidant treatment. Isolated heart mitochondria from 5-day-old sod2 null animals respiring on the complex II substrate succinate exhibited statistically significant higher levels of mitochondrial O2* (157%, p < 0.01) but significantly less H2O2 (33%, p < 0.001) than wild type littermates. Treatment of sod2 nullizygous mice with EUK189 proportionately increased the levels of complex II and H2O2. Increased production of O2* resulting from complex II normalization had no effect on steady state levels due to the rapid conversion to H2O2, a process presumably aided by the presence of the EUK189, an SOD mimetic.

Topics: Animals; Antioxidants; Catalysis; Ethidium; Genotype; Hydrogen Peroxide; Immunoblotting; Mice; Mice, Transgenic; Mitochondria; Mitochondria, Heart; Organometallic Compounds; Oxazines; Oxidative Stress; Oxygen; Reactive Oxygen Species; Salicylates; Submitochondrial Particles; Superoxide Dismutase; Superoxides