CHANGES IN MITOCHONDRIAL-FUNCTION INDUCED IN ISOLATED GUINEA-PIG VENTRICULAR MYOCYTES BY CALCIUM OVERLOAD

1. Changes in [Ca2+](i) and pH(1), mitochondrial membrane potential (P si(m)) and mitochondrial [NADH] have been measured independently using fluorescent techniques in single isolated guinea-pig ventricular myoc ytes subjected to Ca2+ overload. 2. The changes in NADH autofluorescen ce on the inhibition or uncoupling of respiration are consistent with the signal emanating from the mitochondrial NADH.3. Removal of Ca2+ an d Mg2+ from the bathing Tyrode solution induced a modest fall in both [Ca2+](i) and pH(i), a small slowly developing depolarization of Psi(m ), and an initial fall followed by a rise in mitochondrial [NADH]. 4. In myocytes that maintained an intact sarcolemma, return to Ca2+-conta ining fluid elicited a strong but brief intracellular acidification, a rise in [Ca2+](i) which generally recovered more slowly to stabilize above the initial level in Tyrode solution, a steep fall in mitochondr ial [NADH] and a brief transient recovery followed by a large sustaine d depolarization of Psi(m). NADH autofluorescence and mitochondrial de polarization often reached values that were not further increased by u ncoupling respiration although recovery of NADH was elicited by inhibi tors of respiration. 5. These changes were reduced when the Ca2+ overl oad was less severe as evidenced by a reduced hypercontracture upon Ca 2+ repletion. A similar reduction could be routinely achieved by eleva tion of [Mg2+](i) during the period of Ca2+ depletion. 6. These result s suggest that the well-established depletion of energy-rich phosphate s that occurs on Ca2+ overload is due to the combined effects of the f ailure of the citric acid cycle to provide sufficient mitochondrial NA DH for the respiratory chain and an uncoupling of respiration from ATP production due to depolarization of Psi(m). The former effect could r esult from the depletion of sarcoplasmic amino acids and the latter fr om increased Ca2+ cycling across the mitochondrial wall provoked by th e elevated [Na+](i) and [Ca2+](i).