Untitled

Abstract

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 Fluo 3 at 37 degrees 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 degrees C, Fluo 3 also entered mitochondria, and large transients of mitochondrial Fluo 3 fluorescence then occurred after stimulation. Isoproterenol (1 microM) increased the magnitude of Ca2+ transients and their subsequent rate of decay, an effect more marked in the cytosol and nucleus than in 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.

A Minimal Model for the Mitochondrial Rapid Mode of Ca2+ Uptake Mechanism

“…Thus, it seems that the RaM mechanism may be a component of the physiological response to upstream Ca 2+ signaling. Supporting this view, many groups have observed mitochondria in situ exhibit RaM-like behavior when exposed to nM levels of Ca 2+ [15], [24], [33], [34].…”

Mitochondria in cardiomyocyte Ca2+ signaling

“…Similarly, Ohata et al (1998) suggested that mitochondrial accumulation of Ca 2+ released from the cardiac SR could be mediated by the structural association between IMFM and the SR. The details of ultrastructural links, however, remain under investigation.…”

“…Cardiac IMFMs have been isolated from a number of animal species (Hoppel et al, 1982; Matlib et al, 1978; McMillin-Wood et al, 1980; Ohata et al, 1998; Palmer et al, 1977; Weinstein et al, 1985, 1986). The abundance of fibrillar material in the heart, coupled with the tight packing of this mitochondria between the Z-disks make isolation of IMFMs very difficult.…”

Mitochondrial Ca2+ uptake: Tortoise or hare?

“…The challenge with these methods is to avoid contamination from dye in the cytosolic component when intact myocytes are employed. Several papers from the Lemasters group tackled this issue by using confocal microscopy in rabbit cardiac myocytes loaded with either fluo-3/AM[24, 25], rhod-2/AM-[26, 27] or indo-1/AM[25]. They argued that the high spatial resolution of the confocal permitted them to distinguish cytosolic and mitochondrial signals and they found that Ca 2+ signals arising from mitochondria (i.e., colocalizing with the ΔΨ m probe TMRE) had rapid kinetics almost identical to the cytosolic Ca 2+ transient (although the time resolution of the recordings was not sufficient to analyze the rising phase of the transients in detail).…”

“…They argued that the high spatial resolution of the confocal permitted them to distinguish cytosolic and mitochondrial signals and they found that Ca 2+ signals arising from mitochondria (i.e., colocalizing with the ΔΨ m probe TMRE) had rapid kinetics almost identical to the cytosolic Ca 2+ transient (although the time resolution of the recordings was not sufficient to analyze the rising phase of the transients in detail). β-Adrenergic stimulation increased amplitudes of both the cytosolic and mitochondrial Ca 2+ transients[24, 25]. Although it has been argued that this experimental design inadequately excludes contamination of the fluorescence signal from the cytosolic compartment[11], it was shown that the mCU-inhibitor ruthenium red preferentially inhibited the mitochondrial Ca 2+ signal without altering the cytoplasmic transient[26].…”