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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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Contractile waves  are associated with waves of  mitochondrial depolarization.  (a) A spontaneous contractile wave propagating across  the cell (inset) was associated  with a propagating wave of  mitochondrial depolarization  that coincided with the contraction. The intensity of signal at the areas indicated on  the image are shown as a  function of time. Note that  the decrease in signal at the  cell edges (i and ii) due to the  contraction coincided well  with the increased signal  seen at the corresponding  part of the cell. In addition,  at position a, a doublet of  brief transient events were  seen later in the image sequence. It is noteworthy that  these had a similar amplitude  as the transient associated  with the contraction. In b, a  line image is shown to further illustrate the spatial  characteristics of the propagation of the wave of mitochondrial depolarization, seen  here as an increase in signal  that moves diagonally across  the image, starting at one  edge of the cell (the upper  boundary of this image) and  moving towards the other  (at the lower edge of this sequence). The transient events  seen later (position a) are indicated here as well (asterisks). In c, a plot of intensity  with time is shown to illustrate the time course of the  mitochondrial response to  depolarization of a cell with  50 mM KCl (applied for the  period indicated by the white  bar). The depolarization  caused a series of diminishing  twitches, each of which was  associated with mitochondrial  depolarizations. Note also  that the largest twitch was associated with the largest increase in TMRE signal.
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Figure 6: Contractile waves are associated with waves of mitochondrial depolarization. (a) A spontaneous contractile wave propagating across the cell (inset) was associated with a propagating wave of mitochondrial depolarization that coincided with the contraction. The intensity of signal at the areas indicated on the image are shown as a function of time. Note that the decrease in signal at the cell edges (i and ii) due to the contraction coincided well with the increased signal seen at the corresponding part of the cell. In addition, at position a, a doublet of brief transient events were seen later in the image sequence. It is noteworthy that these had a similar amplitude as the transient associated with the contraction. In b, a line image is shown to further illustrate the spatial characteristics of the propagation of the wave of mitochondrial depolarization, seen here as an increase in signal that moves diagonally across the image, starting at one edge of the cell (the upper boundary of this image) and moving towards the other (at the lower edge of this sequence). The transient events seen later (position a) are indicated here as well (asterisks). In c, a plot of intensity with time is shown to illustrate the time course of the mitochondrial response to depolarization of a cell with 50 mM KCl (applied for the period indicated by the white bar). The depolarization caused a series of diminishing twitches, each of which was associated with mitochondrial depolarizations. Note also that the largest twitch was associated with the largest increase in TMRE signal.

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)

Contractile waves  are associated with waves of  mitochondrial depolarization.  (a) A spontaneous contractile wave propagating across  the cell (inset) was associated  with a propagating wave of  mitochondrial depolarization  that coincided with the contraction. The intensity of signal at the areas indicated on  the image are shown as a  function of time. Note that  the decrease in signal at the  cell edges (i and ii) due to the  contraction coincided well  with the increased signal  seen at the corresponding  part of the cell. In addition,  at position a, a doublet of  brief transient events were  seen later in the image sequence. It is noteworthy that  these had a similar amplitude  as the transient associated  with the contraction. In b, a  line image is shown to further illustrate the spatial  characteristics of the propagation of the wave of mitochondrial depolarization, seen  here as an increase in signal  that moves diagonally across  the image, starting at one  edge of the cell (the upper  boundary of this image) and  moving towards the other  (at the lower edge of this sequence). The transient events  seen later (position a) are indicated here as well (asterisks). In c, a plot of intensity  with time is shown to illustrate the time course of the  mitochondrial response to  depolarization of a cell with  50 mM KCl (applied for the  period indicated by the white  bar). The depolarization  caused a series of diminishing  twitches, each of which was  associated with mitochondrial  depolarizations. Note also  that the largest twitch was associated with the largest increase in TMRE signal.
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Related In: Results  -  Collection

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

Figure 6: Contractile waves are associated with waves of mitochondrial depolarization. (a) A spontaneous contractile wave propagating across the cell (inset) was associated with a propagating wave of mitochondrial depolarization that coincided with the contraction. The intensity of signal at the areas indicated on the image are shown as a function of time. Note that the decrease in signal at the cell edges (i and ii) due to the contraction coincided well with the increased signal seen at the corresponding part of the cell. In addition, at position a, a doublet of brief transient events were seen later in the image sequence. It is noteworthy that these had a similar amplitude as the transient associated with the contraction. In b, a line image is shown to further illustrate the spatial characteristics of the propagation of the wave of mitochondrial depolarization, seen here as an increase in signal that moves diagonally across the image, starting at one edge of the cell (the upper boundary of this image) and moving towards the other (at the lower edge of this sequence). The transient events seen later (position a) are indicated here as well (asterisks). In c, a plot of intensity with time is shown to illustrate the time course of the mitochondrial response to depolarization of a cell with 50 mM KCl (applied for the period indicated by the white bar). The depolarization caused a series of diminishing twitches, each of which was associated with mitochondrial depolarizations. Note also that the largest twitch was associated with the largest increase in TMRE signal.
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