The loading of fura-2 into mitochondria in the intact perfused rat heart and its use to estimate matrix Ca²⁺ under various conditions

When rat hearts were perfused with a medium containing 10 µM fura-2/AM for 1 hr at 37°C a significant amount of the derived fura-2 could be detected in subsequently isolated mitochondria. This procedure allowed the measurement of matrix tree Ca²⁺ concentration ([Ca²⁺](m)) of mitochondria rapidly isolated from whole hearts by a method which avoids artefactual redistribution of Ca²⁺ [Ca²⁺](m) in mitochondria prepared from control hearts and incubated with respiratory substrates and EGTA was found to be 172 ± 23 nM (mean ± S.E.M.). When hearts were subjected to either increased mechanical work or treatment with 1 µM L-epinephrine (for 2 mins) [Ca²⁺](m) increased to 916 ± 138 nM and 727 ± 65 nM respectively. The presence of ruthenium red (2.5 µM) in thr perfusion medium prior to and during inotropic intervention diminished these increases in [Ca²⁺](m)(to 316 ± 28 nm and 218 ± 18 nM respectively) but did not affect control values. Addition of Na⁺ ions to incubated mitochondria to enhance mitochondrial Ca²⁺ egress diminished these increases in [Ca²⁺](m) due to pre-treatment with positive inotropes (compared to controls). These changes in [Ca²⁺](m) were broadly parallelled by changes in the active non-phosphorylated form of pyruvate dehydrogenase (PDH) under all circumstances. These results provide further evidence that the activation of PDH by positive inotropes is accomplished by, and at least in part due to, raised mitochondrial matrix free [Ca²⁺] and that such increases can be maintained in isolated and suitably incubated mitochondria.