Inhibition of Na⁺/H⁺ exchange preserves viability, restores mechanical function, and prevents the pH paradox in reperfusion injury to rat neonatal myocytes

Rat neonatal myocytes exposed to 2.5 mM CaCN and 20 mM 2-deoxyglucose at pH 6.2 (chemical hypoxia) quickly lose viability when pH is increased to 7.4, with or without washout of inhibitors - a 'pH paradox'. In this study, we evaluated the effect of two Na⁺/H⁺ exchange inhibitors (dimethylamiloride and HOE694) and a Na⁺/Ca²⁺ exchange inhibitor (dichlorobenzamil) on pH-dependent reperfusion injury. Intracellular free Ca²⁺ and electrical potential were monitored by laser scanning confocal microscopy of rat neonatal cardiac myocytes grown on coverslips and co-loaded with Fluo-3 and tetramethylrhodamine methylester. After 30-60 min of chemical hypoxia at pH 6.2, mitochondria depolarized and Ca²⁺ began to increase uniformly throughout the cell. Free Ca²⁺ reached levels estimated to exceed 2 μM by 4h. Washout of inhibitors at pH 7.4 (reperfusion), with or without dichlorobenzamil, killed most cells within 60 min, despite a marked reduction of Ca²⁺ in dichloroben zamil-treated cells. Reperfusion at pH 7.4 in the presence of 75 μM dimethylamiloride or 20 μM HOE694, or at pH 6.2, prevented cell death. HOE694-treated cells placed into culture medium recovered mitochondrial membrane potential. In most cells, this occurred before normal Ca²⁺ was restored. Contracted myocytes re-extended over a 24-h-period. By 48 hours, most cells contracted spontaneously and showed normal Ca²⁺ transients. Our results indicate that Na⁺/H⁺ exchange inhibition protects against pH-dependent reperfusion injury and facilitates full recovery of cell function.