© 1998 by European Society of Cardiology
Copyright © 1998, European Society of Cardiology
Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes
aDivision of Cardiology, Johns Hopkins University Hospital, Baltimore MD 21205, USA
bBristol Heart Institute, Bristol Royal Infirmary, Bristol BS2 8HW, UK
cDepartment of Biochemistry, University of Bristol, Bristol BS8 1TD, UK
dLaboratory of Cardiovascular Science, National Institutes of Health, Baltimore MD 21224, USA
* Corresponding author. Address for correspondence: Bristol Heart Institute Department of Cardiac Surgery, Level 7, Bristol Royal Infirmary, Bristol BS2 8HW, UK. Tel: +44 (117) 928 3586; Fax: +44 (117) 928 3581; E-mail: elinor.griffiths@bristol.ac.uk
Objective: Mitochondrial [Ca2+] ([Ca2+]m) rises in parallel with cytosolic [Ca2+] ([Ca2+]c) following ATP-depletion rigor contracture induced by hypoxia in isolated cardiomyocytes. We investigated the pathways involved in the hypoxia induced changes in [Ca2+]m by using known inhibitors of mitochondrial Ca2+ transport, namely ruthenium red, an inhibitor of the Ca2+ uniporter (the normal influx route) and clonazepam, an inhibitor of Na+/Ca2+ exchange, (the normal efflux route). Methods: [Ca2+]m was determined from indo-1/am loaded rat myocytes where the cytosolic fluorescence signal had been quenched by superfusion with Mn2+. [Ca2+]c was measured by loading myocytes with indo-1 pentapotassium salt during the isolation procedure. Cells were placed in a specially developed chamber for induction of hypoxia and reoxygenated 40 min after rigor development. Results: 50% of control cells hypercontracted upon reoxygenation; this correlated with a [Ca2+]m or [Ca2+]c higher than approximately 350 nM at the end of rigor. Clonazepam completely abolished the rigor-induced rise in [Ca2+]m but not [Ca2+]c. On reoxygenation [Ca2+]m increased over the first 5 min and remained elevated whereas [Ca2+]c fell. In the presence of ruthenium red a dramatic increase in [Ca2+]m occurred 5–10 min after rigor development (the indo-1 fluorescence signal was saturated); [Ca2+]c also increased but to a lesser extent. On reoxygenation, [Ca2+]m fell rapidly even though cells hypercontracted and [Ca2+]c remained elevated. Conclusions: During hypoxia following rigor development Ca2+ uptake into mitochondria occurs largely via the Na+/Ca2+ exchanger rather than the Ca2+ uniporter whereas on reoxygenation the transporters resume their normal directionality.
KEYWORDS RuR, ruthenium red; BDM, 2,3-butanedione monoxime; FCCP, carbonylcyanide p-trifluoromethoxyphenylhydrazone; 
m, mitochondrial membrane potential; pH, pH gradient across the inner mitochondrial membrane
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