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Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes.

Duchen MR, Leyssens A, Crompton M - J. Cell Biol. (1998)

Bottom Line: Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria.Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 microM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter.These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University College London, London WC1E 6BT, United Kingdom. m.duchen@ucl.ac.uk

ABSTRACT
Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call "flicker." These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K+, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 microM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.

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Resolution of transient depolarizations to single mitochondria. In a, a confocal image of an adult  myocyte reveals the organization of mitochondria in  longitudinal bands. A single  mitochondrion showed a  transient increase in intensity, and the differential image is shown in iii. b shows an  increased magnification of  the area of the cell for the  three images shown above.  In c, a series of images has  been extracted from a sequence obtained from the  neonatal myocytes in culture  to illustrate the occurrence of  transient events restricted to  single identifiable mitochondria (arrows).
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Figure 3: Resolution of transient depolarizations to single mitochondria. In a, a confocal image of an adult myocyte reveals the organization of mitochondria in longitudinal bands. A single mitochondrion showed a transient increase in intensity, and the differential image is shown in iii. b shows an increased magnification of the area of the cell for the three images shown above. In c, a series of images has been extracted from a sequence obtained from the neonatal myocytes in culture to illustrate the occurrence of transient events restricted to single identifiable mitochondria (arrows).

Mentions: In single intact cardiomyocytes, TMRE partitions between the cytosol and mitochondria with a Nernstian distribution and can be readily seen accumulated within energized mitochondria. In a high-resolution confocal image of a cardiomyocyte loaded with TMRE (Fig. 1 a, see also Fig. 3, a and b), the mitochondria could be clearly identified, running parallel to the longitudinal axis of the cell. In an image taken with our cooled CCD system (Fig. 1 b), individual mitochondria were no longer clearly resolved, but their distribution in bands running along the length of the cell was still clearly identified, and single-stained mitochondria can be resolved at the edge of the cell. The nuclei, effectively mitochondrion-free zones, showed only minimal fluorescence (Fig. 1, c, e, and f). Under the loading conditions used in this study, the intramitochondrial accumulation of TMRE causes autoquenching of fluorescence, much as seen with rhodamine 123 (Emaus et al., 1986; Duchen, 1992; Duchen and Biscoe, 1992; Bunting et al., 1993) and TMRM (Ichas et al., 1997). Thus, TMRE fluorescence in cells routinely increased with a wide range of manipulations expected to depolarize Δψm, thus releasing the dye into the cytosol with effective dequench of the fluorescence. This is illustrated in Fig. 1, c and d, which shows images of a cardiomyocyte before and after application of the uncoupler, FCCP (1 μM). The collapse of Δψm after the addition of the protonophore was signaled by a large increase in TMRE fluorescence. These images were extracted from a series of 50 images, and the sequential change in signal, measured along a line selected along the longitudinal axis of the cell as indicated, is shown in Fig. 1, e and f. This series of images also illustrates how many of the later figures were constructed. In response to FCCP, the signal increased by a mean of 262 ± 18% (SD; n = 17).


Transient mitochondrial depolarizations reflect focal sarcoplasmic reticular calcium release in single rat cardiomyocytes.

Duchen MR, Leyssens A, Crompton M - J. Cell Biol. (1998)

Resolution of transient depolarizations to single mitochondria. In a, a confocal image of an adult  myocyte reveals the organization of mitochondria in  longitudinal bands. A single  mitochondrion showed a  transient increase in intensity, and the differential image is shown in iii. b shows an  increased magnification of  the area of the cell for the  three images shown above.  In c, a series of images has  been extracted from a sequence obtained from the  neonatal myocytes in culture  to illustrate the occurrence of  transient events restricted to  single identifiable mitochondria (arrows).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132882&req=5

Figure 3: Resolution of transient depolarizations to single mitochondria. In a, a confocal image of an adult myocyte reveals the organization of mitochondria in longitudinal bands. A single mitochondrion showed a transient increase in intensity, and the differential image is shown in iii. b shows an increased magnification of the area of the cell for the three images shown above. In c, a series of images has been extracted from a sequence obtained from the neonatal myocytes in culture to illustrate the occurrence of transient events restricted to single identifiable mitochondria (arrows).
Mentions: In single intact cardiomyocytes, TMRE partitions between the cytosol and mitochondria with a Nernstian distribution and can be readily seen accumulated within energized mitochondria. In a high-resolution confocal image of a cardiomyocyte loaded with TMRE (Fig. 1 a, see also Fig. 3, a and b), the mitochondria could be clearly identified, running parallel to the longitudinal axis of the cell. In an image taken with our cooled CCD system (Fig. 1 b), individual mitochondria were no longer clearly resolved, but their distribution in bands running along the length of the cell was still clearly identified, and single-stained mitochondria can be resolved at the edge of the cell. The nuclei, effectively mitochondrion-free zones, showed only minimal fluorescence (Fig. 1, c, e, and f). Under the loading conditions used in this study, the intramitochondrial accumulation of TMRE causes autoquenching of fluorescence, much as seen with rhodamine 123 (Emaus et al., 1986; Duchen, 1992; Duchen and Biscoe, 1992; Bunting et al., 1993) and TMRM (Ichas et al., 1997). Thus, TMRE fluorescence in cells routinely increased with a wide range of manipulations expected to depolarize Δψm, thus releasing the dye into the cytosol with effective dequench of the fluorescence. This is illustrated in Fig. 1, c and d, which shows images of a cardiomyocyte before and after application of the uncoupler, FCCP (1 μM). The collapse of Δψm after the addition of the protonophore was signaled by a large increase in TMRE fluorescence. These images were extracted from a series of 50 images, and the sequential change in signal, measured along a line selected along the longitudinal axis of the cell as indicated, is shown in Fig. 1, e and f. This series of images also illustrates how many of the later figures were constructed. In response to FCCP, the signal increased by a mean of 262 ± 18% (SD; n = 17).

Bottom Line: Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria.Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 microM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter.These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University College London, London WC1E 6BT, United Kingdom. m.duchen@ucl.ac.uk

ABSTRACT
Digital imaging of mitochondrial potential in single rat cardiomyocytes revealed transient depolarizations of mitochondria discretely localized within the cell, a phenomenon that we shall call "flicker." These events were usually highly localized and could be restricted to single mitochondria, but they could also be more widely distributed within the cell. Contractile waves, either spontaneous or in response to depolarization with 50 mM K+, were associated with propagating waves of mitochondrial depolarization, suggesting that propagating calcium waves are associated with mitochondrial calcium uptake and consequent depolarization. Here we demonstrate that the mitochondrial flicker was directly related to the focal release of calcium from sarcoplasmic reticular (SR) calcium stores and consequent uptake of calcium by local mitochondria. Thus, the events were dramatically reduced by (a) depletion of SR calcium stores after long-term incubation in EGTA or thapsigargin (500 nM); (b) buffering intracellular calcium using BAPTA-AM loading; (c) blockade of SR calcium release with ryanodine (30 microM); and (d) blockade of mitochondrial calcium uptake by microinjection of diaminopentane pentammine cobalt (DAPPAC), a novel inhibitor of the mitochondrial calcium uniporter. These observations demonstrate that focal SR calcium release results in calcium microdomains sufficient to promote local mitochondrial calcium uptake, suggesting a tight coupling of calcium signaling between SR release sites and nearby mitochondria.

Show MeSH
Related in: MedlinePlus