Basic Research in Cardiology

, Volume 88, Issue 5, pp 430–442 | Cite as

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
Original Contributions

Summary

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.

Key words

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

Abbreviations

DCB

dichlorobenzamil

DMA

dimethylamiloride

2-DOG

2-deoxy-D-glucose

HOE694

3-methylsulfonyl-4-piperidinobenzoyl guanidine hydrochloride

KRH

Krebs-Ringer-HEPES buffer containing 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 1,2 mM MgSO4, 2 mM CaCl2, and 25 mM Na-HEPES buffer

PI

propidium iodide

TMRM

tetramethylrhodamine methylester

ΔΨ

electrical potential difference

Ψ

electric potential

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Copyright information

© Steinkopff-Verlag 1993

Authors and Affiliations

  • I. S. Harper
    • 1
    • 2
  • J. M. Bond
    • 1
  • E. Chacon
    • 1
  • J. M. Reece
    • 1
  • B. Herman
    • 1
  • J. J. Lemasters
    • 1
  1. 1.Laboratories for Cell Biology, Department of Cell Biology & Anatomy, School of MedicineUniversity of North Carolina at Chapel HillChapel HillUSA
  2. 2.Experimental Biology ProgrammeMedical Research CouncilTygerbergSouth Africa

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