Hydrogen Peroxide Activates Mitogen-Activated Protein Kinases and Na⁺-H⁺ Exchange in Neonatal Rat Cardiac Myocytes

From the Departments of Physiology (A.S., K.L.B., A.M.S., J.B., P.A.L.), Medicine (K.L.B., A.M.S.), and the Cardiovascular Institute (A.S., K.L.B., A.M.S., J.B., P.A.L.), Loyola University Chicago, Stritch School of Medicine, Maywood, Ill.

Correspondence to Pamela A. Lucchesi, PhD, Department of Physiology, Loyola University Medical School 110-5225, 2160 South First Ave, Maywood, IL 60153. E-mail plucche{at}luc.edu

Abstract—Reperfusion of cardiac tissue after an ischemic episode is associated with metabolic and contractile dysfunction, including reduced tension development and activation of the Na⁺-H⁺ exchanger (NHE). Oxygen-derived free radicals are key mediators of reperfusion abnormalities, although the cellular mechanisms involved have not been fully defined. In the present study, the effects of free radicals on mitogen-activated protein (MAP) kinase function were investigated using cultured neonatal rat ventricular myocytes. Acute exposure of spontaneously beating myocytes to 50 µmol/L hydrogen peroxide (H₂O₂) caused a sustained decrease in contraction amplitude (80% of control). MAP kinase activity was measured by in-gel kinase assays and Western blot analysis. Acute exposure to H₂O₂ (100 µmol/L, 5 minutes) resulted in sustained MAP kinase activation that persisted for 60 minutes. Catalase, but not superoxide dismutase, completely inhibited MAP kinase activation by H₂O₂. Pretreatment with chelerythrine (10 µmol/L, 45 minutes), a protein kinase C inhibitor, or genistein (75 µmol/L, 45 minutes) or herbimycin A (3 µmol/L, 45 minutes), tyrosine kinase inhibitors, caused significant inhibition of H₂O₂-stimulated MAP kinase activity (51%, 78%, and 45%, respectively, at 20 minutes). Brief exposure to H₂O₂ also stimulated NHE activity. This effect was completely abolished by pretreatment with the MAP kinase kinase inhibitor PD 98059 (30 µmol/L, 60 minutes). These results suggest that low doses of H₂O₂ induce MAP kinase–dependent pathways that regulate NHE activity during reperfusion injury.

Key Words: signal transduction • mitogen-activated protein kinase • hydrogen peroxide • Na⁺-H⁺ exchange

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