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 [Ca(2+]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+]i-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 microM 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.
Rodrigo
Glenn C
GC
Department of Cell Physiology and Pharmacology, University of Leicester, PO Box 138, Leicester LE1 9HN, UK. gcr4@le.ac.uk
Standen
Nicholas B
NB
D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006352:Heart Ventricles; D007783:Lactones / Q000494:pharmacology; D008297:Male; D008564:Membrane Potentials; D008929:Mitochondria, Heart / Q000378:metabolism*; D009200:Myocardial Contraction; D015428:Myocardial Reperfusion Injury / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000648:ultrastructure; D010085:Oxidative Phosphorylation; D051381:Rats; D017208:Rats, Wistar; D013141:Spiro Compounds / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007783:Lactones; D013141:Spiro Compounds; C121329:sanglifehrin A; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D015632:1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; D002118:Calcium