Mitochondrial Ca²⁺ transients in cardiac myocytes during the excitation-contraction cycle: Effects of pacing and hormonal stimulation

Using laser scanning confocal microscopy, our objective was to measure mitochondrial, nuclear, and cytosolic free ionized Ca²⁺ in adult rabbit cardiac myocytes loaded with Ca²⁺ -indicating fluorophores. When myocytes were loaded with Flou 3 at 37°C, the fluorophore was loaded extensively into the cytosol and nucleus, but poorly into mitochondria, and Fluo 3 fluorescence transients after field stimulation were confined to the cytosol and nucleus. In contrast, after loading at 4°C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 μM) increased the magnitude of Ca²⁺ transients and their subsequent rate decay, an effect more marked in the cytosol and nucleus than in the mitochondria. As pacing frequency was increased from 0.5 to 2 Hz, diastolic mitochondrial Ca²⁺ rose markedly in the absence but not in the presence of isoproterenol. Resting Ca²⁺ estimated by Indo 1 ratio imaging using UV/visible laser scanning confocal microscopy was about 200 nM in all compartments. During field stimulation, Ca²⁺ transiently increased to 671, 522, and 487 nM in cytosol, interfibrillar mitochondria, and perinuclear mitochondria, respectively. Isoproterenol increased these respective peak values to 1280, 750, and 573 nM. These results were consistent with those obtained in Fluo 3 experiments. We conclude that rapid mitochondrial Ca²⁺ transients occur during excitation-contraction coupling in adult rabbit cardiac myocytes, which may be important in matching mitochondrial metabolism to myocardial ATP demand during changes in cardiac output.