MITOCHONDRIAL CALCIUM TRANSPORTING PATHWAYS DURING HYPOXIA AND REOXYGENATION IN SINGLE-RAT CARDIOMYOCYTES

Objective: Mitochondrial [Ca2+]([Ca2+](m)) rises in parallel with cyto solic [Ca2+]([Ca2+](c)) following ATP-depletion rigor contracture indu ced by hypoxia in isolated cardiomyocytes. We investigated the pathway s involved in the hypoxia induced changes in [Ca2+](m) by using known inhibitors of mitochondrial Ca2+ transport, namely ruthenium red, an i nhibitor of the Ca2+ uniporter (the normal influx route) and clonazepa m, an inhibitor of Na+/Ca2+ exchange, (the normal efflux route). Metho ds: [Ca2+](m) was determined from indo-1/am loaded rat myocytes where the cytosolic fluorescence signal had been quenched by superfusion wit h Mn2+. [Ca2+](c) was measured by loading myocytes with indo-1 pentapo tassium salt during the isolation procedure. Cells were placed in a sp ecially developed chamber for induction of hypoxia and reoxygenated 40 min after rigor development. Results: 50% of control cells hypercontr acted upon reoxygenation; this correlated with a [Ca2+](m) or [Ca2+](c ) higher than approximately 350 nM at the end of rigor. Clonazepam com pletely abolished the rigor-induced rise in [Ca2+](m) but not [Ca2+](c ). On reoxygenation [Ca2+](m) increased over the first 5 min and remai ned elevated whereas [Ca2+](c) fell. In the presence of ruthenium red a dramatic increase in [Ca2+](m) occurred 5-10 min after rigor develop ment (the indo-1 fluorescence signal was saturated); [Ca2+](c) also in creased but to a lesser extent. On reoxygenation, [Ca2+](m) fell rapid ly even though cells hypercontracted and [Ca2+](c) remained elevated. Conclusions: During hypoxia following rigor development Ca2+ uptake in to mitochondria occurs largely via the Na+ /Ca2+ exchanger rather than the Ca2+ uniporter whereas on reoxygenation the transporters resume t heir normal directionality. (C) 1998 Elsevier Science B.V. All rights reserved.