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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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Related in: MedlinePlus

Localized transient depolarization of Δψm  in processed image sequences. (a) A series of six  images taken from a time series obtained over ∼65 s is  shown with the time of acquisition of each within the series as indicated. These images were processed as  described and ratioed against  a virtual image constructed  from the darkest value for  each pixel throughout the  time series. b and c show the  surface and line images, respectively, for this cell, showing changes in intensity along  the axis of the cell with time.  Note that such processing of  course excludes the small  events seen close to the edge  of these cells at 38 and 45 s.
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Figure 5: Localized transient depolarization of Δψm in processed image sequences. (a) A series of six images taken from a time series obtained over ∼65 s is shown with the time of acquisition of each within the series as indicated. These images were processed as described and ratioed against a virtual image constructed from the darkest value for each pixel throughout the time series. b and c show the surface and line images, respectively, for this cell, showing changes in intensity along the axis of the cell with time. Note that such processing of course excludes the small events seen close to the edge of these cells at 38 and 45 s.

Mentions: Perhaps a more convincing functional indication of reversibility is the observation of events seen repeatedly over the same single mitochondrion; these must of necessity involve a true repolarization after each event if a further event is to occur. Such repeated events were routinely seen and are illustrated specifically in Fig. 4, a (iv) and c (see below), but they are also apparent in a number of other image sequences (Figs. 2, 5, 6, and 7).


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

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

Localized transient depolarization of Δψm  in processed image sequences. (a) A series of six  images taken from a time series obtained over ∼65 s is  shown with the time of acquisition of each within the series as indicated. These images were processed as  described and ratioed against  a virtual image constructed  from the darkest value for  each pixel throughout the  time series. b and c show the  surface and line images, respectively, for this cell, showing changes in intensity along  the axis of the cell with time.  Note that such processing of  course excludes the small  events seen close to the edge  of these cells at 38 and 45 s.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Localized transient depolarization of Δψm in processed image sequences. (a) A series of six images taken from a time series obtained over ∼65 s is shown with the time of acquisition of each within the series as indicated. These images were processed as described and ratioed against a virtual image constructed from the darkest value for each pixel throughout the time series. b and c show the surface and line images, respectively, for this cell, showing changes in intensity along the axis of the cell with time. Note that such processing of course excludes the small events seen close to the edge of these cells at 38 and 45 s.
Mentions: Perhaps a more convincing functional indication of reversibility is the observation of events seen repeatedly over the same single mitochondrion; these must of necessity involve a true repolarization after each event if a further event is to occur. Such repeated events were routinely seen and are illustrated specifically in Fig. 4, a (iv) and c (see below), but they are also apparent in a number of other image sequences (Figs. 2, 5, 6, and 7).

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