Mitochondrial calcium transporting pathways during hypoxia and reoxygenation in single rat cardiomyocytes.
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.
Griffiths
E J
EJ
Division of Cardiology, Johns Hopkins University Hospital, Baltimore, MD 21205, USA. elinor.griffiths@bristol.ac.uk
Ocampo
C J
CJ
Savage
J S
JS
Rutter
G A
GA
Hansford
R G
RG
Stern
M D
MD
Silverman
H S
HS
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000008:administration & dosage / Q000494:pharmacology; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D002998:Clonazepam / Q000008:administration & dosage / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D005456:Fluorescent Dyes; D000860:Hypoxia / Q000378:metabolism*; D007211:Indoles; D008856:Microscopy, Fluorescence; D008929:Mitochondria, Heart / Q000187:drug effects / Q000378:metabolism*; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D012430:Ruthenium Red / Q000008:administration & dosage / Q000494:pharmacology; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors
D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D007211:Indoles; D019831:Sodium-Calcium Exchanger; D012430:Ruthenium Red; D002998:Clonazepam; C048960:indo-1; D010100:Oxygen; D002118:Calcium