Role of mitochondrial re-energization and Ca2+ influx in reperfusion injury of metabolically inhibited cardiac myocytes

Objective: We used isolated myocytes to investigate the role of mitochondrial re-energization and Ca2+ influx during reperfusion on hypercontracture, loss of Ca2+ homeostasis and contractile function. Methods: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion injury was assessed from hypercontracture, loss of Ca2+ homeostasis ([Ca2+](i) measured with fura-2) and failure of contraction in response to electrical stimulation. Mitochondrial membrane potential was followed using the potentiometric dye tetramethylrhodamine ethyl ester. Results: Metabolic inhibition led to contractile failure and rigor accompanied by a sustained increase in [Ca2+](i). Reperfusion after 10 min metabolic inhibition led to an abrupt repolarization of the mitochondrial membrane potential (after 25.5 +/- 1.2 s), a transient fall in [Ca2+](i), followed by an abrupt hypercontracture (37.1 +/- 1.8 s) in 84% of myocytes. Ca2+ homeostasis (diastolic [Ca2+](i) < 250 nM) recovered in only 23.3 +/- 5.1% of cells and contractions recovered in 15.3 +/- 2.2%. Oligomycin abolished the hypercontracture on reperfusion, but mitochondrial repolarization was unaffected. Preventing Ca2+ influx during reperfusion with Ca2+-free Tyrode or with an inhibitor of Na+/Ca2+ exchange did not prevent the hypercontracture, but increased the percentage of cells recovering Ca2+ homeostasis and contractile function. The presence of 0.5 mu M cyclosporin A did not prevent hypercontracture but increased the percentage of cells recovering Ca2+ homeostasis to 56.2 +/- 3.6% and contractile function to 52 +/- 4.3%. Conclusions: Reperfusion-induced hypercontracture, and loss of Ca2+ homeostasis and contractile function are initiated following mitochondrial re-energization. The hypercontracture requires the production of oxidative ATP but not Ca2+ influx during reperfusion. Loss of Ca2+ homeostasis and contractile function are linked to Ca2+ influx during reperfusion, probably via opening of mitochondrial permeability transition pores. (c) 2005 European Society of Cardiology. Published by Elsevier B.V All rights reserved.