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

I. S. Harper, J. M. Bond, E. Chacon, J. M. Reece, B. Herman, J. J. Lemasters

Research output: Contribution to journalArticlepeer-review

60 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)430-442
Number of pages13
JournalBasic research in cardiology
Volume88
Issue number5
DOIs
StatePublished - Sep 1993

Keywords

  • HOE694
  • Na/Ca exchange
  • Na/H exchange
  • chemical hypoxia
  • confocal microscopy
  • dichlorobenzamil
  • dimethylamiloride
  • intracellular calcium
  • ischemia/reperfusion injury
  • neonatal myocytes

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