Previously we have shown, that inhibition of Ca²⁺-uptake by mitochondria at the beginning of reperfusion protects cardiomyocytes against reperfusion injury. The aim of present study was to investigate, whether an uncoupling of the mitochondrial respiratory chain at the beginning of reperfusion leads to reduced repolarisation of the mitochondrial membrane potential (DY_m) and to analyze, whether this reduces the mitochondrial Ca²⁺-uptake, leading to reduction in reperfusion injury. Isolated adult rat cardiomyocytes were superfused anoxically (60 min; 100% N₂; no glucose; pH 6.4) and then reperfused with a normoxic buffer (20 min; 21% O₂; 2.5 mM glucose; pH 7.4).The DY_m was detected with the fluorescence indicator JC-1. Mitochondrial Ca²⁺ was measured by the fluorescence indicator Fura-2, while the cytosolic Ca²⁺ was quenched with Cobalt. The percentage of necrotic cells at the end of reperfusion was detected with propidium iodide dye (37.5 mM). During reperfusion, an uncoupling of the mitochondrial respiratory chain was induced by application of Carbonylcyanid-m-chlorophenylhydrazon (CCCP; 100 nM).

The application of CCCP during reperfusion significantly reduced the repolarisation of DY_m (JC-1 fluorescence at the end of reperfusion in presence of 100 nM CCCP: 266.40 ± 12.96 % of end-anoxic JC-1 fluorescence vs. 371.45 ± 22.91 % in control: n >= 16, p < 0.05 for all further measurements). Mitochondrial Ca²⁺-uptake was also significantly reduced by CCCP (Mitochondrial Ca²⁺-concentration in the 10^th min of reperfusion in presence of 100 nM CCCP: 91.28 ± 4.55 % of end-anoxic mitochondrial Ca²⁺-concentration vs. 115.97 ± 6.29 in control). The percentage of necrotic cells at the end of reperfusion was significantly reduced from 23.26 ± 3.91 % in control to 13.50 ± 1.80 % in presence of 100 mM CCCP during reperfusion. The data of present study show, that the uncoupling of the mitochondrial respiratory chain by CCCP during reperfusion reduced the repolarisation of DY_m and the mitochondrial Ca²⁺-uptake at the beginning of reperfusion. This results into a reduction in reperfusion-induced necrosis of cardiomyocytes.