Effect of Vacuolar Proton ATPase on pH_i, Ca²⁺, and Apoptosis in Neonatal Cardiomyocytes During Metabolic Inhibition/Recovery

From the Research Service (E.K.-P., J.A.N., H.L.L., R.L.E., R.A.G.), Veterans Affairs San Diego Healthcare System, San Diego, Calif; the Division of Biochemistry (R.A.G.), Department of Molecular & Experimental Medicine, The Scripps Research Institute, La Jolla, Calif; and the Department of Medicine (E.K.-P., H.L.L., R.L.E.), University of California, San Diego, School of Medicine, San Diego, Calif.

Correspondence to Robert L. Engler, MD, ACOS/Research, VA San Diego Health Care System (151), 3350 La Jolla Village Dr, San Diego, CA 92161. E-mail rengler{at}ucsd.edu

Abstract—Recently, we found that vacuolar proton ATPase (VPATPase) operates in cardiomyocytes as a complementary proton-extruding mechanism. Its activity was increased by preconditioning with resultant attenuation of intracellular acidification during ischemia. In this study, we examined whether VPATPase-mediated proton efflux during metabolic inhibition/recovery may spare Na⁺ overload via Na⁺-H⁺ exchange, attenuate Na⁺-Ca²⁺ exchange, and decrease apoptosis. Neonatal rat cardiomyocytes were subjected to 2- to 3-hour metabolic inhibition with cyanide and 2-deoxyglucose and 24-hour recovery. The effect of VPATPase inhibition by 50 nmol/L bafilomycin A₁ on apoptosis, pH_i, and [Ca²⁺]_i was studied by flow cytometry with propidium iodide, seminaphthorhodafluor (SNARF)-1-AM, and indo-1-AM staining, respectively. VPATPase inhibition increased the amount of apoptosis measured after 24 hours of recovery and abrogated the protective effect of inhibition of Na⁺-H⁺ exchange by (5-N-ethyl-N-isopropyl)amiloride (EIPA). Dual blockade of VPATPase and Na⁺-H⁺ exchange was additive in effect with EIPA on pH_i during metabolic inhibition/recovery and recovery from the acid challenge with sodium propionate. VPATPase blockade increased the rate of accumulation of intracellular Ca²⁺ at the beginning of metabolic inhibition and abrogated the delaying effect of EIPA on intracellular Ca²⁺ accumulation. These results indicate that VPATPase plays an important accessory role in cardiomyocyte protection by reducing acidosis and Na⁺-H⁺ exchange–induced Ca²⁺ overload.

Key Words: apoptosis • vacuolar proton ATPase • bafilomycin A₁ • metabolic inhibition • cardiomyocyte

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