The relationship between mitochondrial state, ATP hydrolysis, [Mg2+], and [Ca2+](i) studied in isolated rat cardiomyocytes

1. As ATP has a higher affinity for Mg2+ than ADP, the cytosolic magnesium concentration rises upon ATP hydrolysis. We have therefore used the Mg2+-sensitive fluorescent indicator Magnesium Green (MgG) to provide an index of changing ATP concentration in single rat cardiomyocytes in response to altered mitochondrial state. 2. In response to FCCP, [Mg2+](i) rose towards a plateau coincident with the progression to rigor, which signals ATP depletion. Contamination of the MgG signal by changes in intracellular free Ca2+ concentration (the K-D of MgG for Ca2+ is 4.7 mu M) was excluded by simultaneous measurement of [Ca2+](i) and [Mg2+](i) cells dual loaded with fura-2 and MgG. The response to FCCP was independent of external Mg2+, confirming an intracellular source for the rise in [Mg2+](i). 3. Simultaneous measurements of mitochondrial NAD(P)H autofluorescence and mitochondrial potential (Delta psi(m); JC-1 fluorescence) and of autofluorescence and MgG: allowed closer study of the relationship between [Mg2+](i) and mitochondrial state. Oligomycin abolished the FCCP-induced rise in [Mg2+]i without altering the change in autofluorescence. Thus, the rise in [Mg2+](i) in response to FCCP is consistent with the release of intracellular Mg2+ following ATP hydrolysis by the mitochondrial F1F0-ATPase, 4. The rise in [Mg2+](i) was correlated with cell-attached recordings of ATP-sensitive K+ channel (K-ATP) activity. In response to FCCP, an increase in K-ATP channel activity was seen only as [Mg2+](i) reached a plateau. In response to blockade of mitochrondrial respiration and glycolysis with cyanide (CN-) and 2-deoxyglucose (DOG), [Mg2+](i) rose more slowly but again K-ATP channel opening increased only when [Mg2+](i) readied a plateau and the cells shortened. 5. Oligomycin decreased the rate of rise of [Mg2+](i), delayed the onset of rigor and increased the rate of mitochondrial depolarization in response to CN--DOG. Thus, with blockade of mitochondrial respiration, Delta psi(m) is maintained by the mitochondrial F1F0-ATPase at the expense of ATP reserves. 6. In response to CN--DOG, the initial rise in [Mg2+](i) was accompanied by a small rise in [Ca2+](i). After [Mg2+](i) reached a plateau and rigor developed, [Ca2+](i) rose progressively. On reperfusion, in hypercontracted cells, [Ca2+](i) recovered before [Mg2+](i) and [Ca2+](i) oscillations were sustained while [Mg2+](i) decreased. Thus on reperfusion, full recovery of [ATP](i) is slow, but the activation of contractile elements and the restoration of [Ca2+](i) does not require the re-establishment of millimolar concentrations of ATP.