Effects of pacing and isoproterenol on mitochondrial Ca2+ transients in adult rabbit cardiac myocytes during the contractile cycle

Abstract

Using laser scanning confocal microscopy, we have developed methods to measure mitochondrial free Ca2+ in cultured adult rabbit cardiac myocytes during excitation-contraction. Here, we examined the effect of isoproterenol and the rate of electrical pacing on the magnitude and duration of mitochondrial Ca transients. The mitochondria and cytosol of myocytes were loaded with the Ca2+-indicating fluorophore, Fluo-3-AM, at 4°C. Subsequently, the mitochondria were labeled with tetramethylrhodamine methylester (TMRM). In line scans, red TMRM fluorescence and green Fluo-3 fluorescence were simultaneously imaged by laser scanning confocal microscopy. Large transients of Fluo-3 fluorescence after electrical stimulation were observed both in cytosol and mitochondria (Figures 1). Isoproterenol (1 μM) increased the magnitude of Ca2+ transients caused by field stimulation and their subsequent rate of decay (Figure 2). This effect of isoproterenol was more marked in the cytosol than in mitochondria. In particular, the effect of isoproterenol on Ca2+ transients was least in perinuclear mitochondria.