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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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Localized transient depolarization of Δψm.  a, i and ii, shows a selection  of images extracted from image series obtained from two  cells over a period of ∼60 s  and illustrates the transient  localized depolarizations of  mitochondria or groups of  mitochondria throughout the  cell with time. Each arrowhead points to a region of the  image that shows a marked  transient increase in intensity, and the times of the individual images are indicated.  In b, i and ii, the differential images corresponding to  those selected in a, i and ii,  are shown, revealing the distribution over which the signal has changed between  image frames. The black calibration bars to the right of  each group of images represent 20 μm. The traces below  (c) illustrate the change in intensity with time for events  identified in each of four  cells. In each case, an area or  areas of just a few pixels  were selected to limit the collection of signal to a small  volume of the cell. In c, i,  three widely separated areas  within a cell were chosen,  over which single events occurred during the sampling  period. The record in ii  shows a single brief event  highly localized to a small  part of a cell superimposed  on a very quiet baseline. In  iii, an example has been  selected in which several  events occurred repeatedly  at a single point within the  acquisition period, and in iv,  FCCP was applied at the end  of the acquisition time to  demonstrate the full range of  TMRE signal with complete  dissipation of the mitochondrial potential in comparison  with the small transient event  that preceded it.
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Figure 2: Localized transient depolarization of Δψm. a, i and ii, shows a selection of images extracted from image series obtained from two cells over a period of ∼60 s and illustrates the transient localized depolarizations of mitochondria or groups of mitochondria throughout the cell with time. Each arrowhead points to a region of the image that shows a marked transient increase in intensity, and the times of the individual images are indicated. In b, i and ii, the differential images corresponding to those selected in a, i and ii, are shown, revealing the distribution over which the signal has changed between image frames. The black calibration bars to the right of each group of images represent 20 μm. The traces below (c) illustrate the change in intensity with time for events identified in each of four cells. In each case, an area or areas of just a few pixels were selected to limit the collection of signal to a small volume of the cell. In c, i, three widely separated areas within a cell were chosen, over which single events occurred during the sampling period. The record in ii shows a single brief event highly localized to a small part of a cell superimposed on a very quiet baseline. In iii, an example has been selected in which several events occurred repeatedly at a single point within the acquisition period, and in iv, FCCP was applied at the end of the acquisition time to demonstrate the full range of TMRE signal with complete dissipation of the mitochondrial potential in comparison with the small transient event that preceded it.

Mentions: As an initial experimental protocol, a series of images were acquired in sequence, typically giving an acquisition rate of 1–2 images per second. The transient reversible changes in Δψm localized to small discrete areas of the cells seen in such an image series are illustrated in Fig. 2 a, i and ii (arrows). The events occurred as small increases in signal over a relatively bright baseline signal, with an increase of ∼10–20% for most events. Small events and their distribution were more readily revealed by image processing, in which a running differential of the image series was constructed. In this mode, the preceding image of the series was subtracted from each image, revealing only those pixels in which the signal had changed. Fig. 2 b, i and ii, illustrates the distribution of the flicker events revealed from the differentials of the raw signals illustrated in Fig. 2 a. Individual events could be extremely localized and could involve greater volumes of the cell, and several events could occur simultaneously in different parts of the cell. The changes in signal with time are shown in Fig. 2 c, in which the intensity over small groups of pixels is plotted as a function of time for four different cells. Note the apparent independence of individual transient events in different parts of the cell with time (Fig. 2 c, i). Fig. 2 c, ii, illustrates an isolated brief event to show its occurrence in relation to an otherwise very quiet and stable baseline. The recurrent appearance of brief transients over the same point in the cell (Fig. 2 c, iii) suggests that it is unlikely that the transient events could in fact reflect complete dissipation of potential in single mitochondria (and see below). A further observation that strengthens this view is the relatively small amplitude of the signal compared with that seen with complete dissipation of Δψm (demonstrated after the application of FCCP). This is illustrated in Fig. 2 c, iv, which demonstrates that after a transient depolarization, the signal could still be dramatically increased by the uncoupler.


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.  a, i and ii, shows a selection  of images extracted from image series obtained from two  cells over a period of ∼60 s  and illustrates the transient  localized depolarizations of  mitochondria or groups of  mitochondria throughout the  cell with time. Each arrowhead points to a region of the  image that shows a marked  transient increase in intensity, and the times of the individual images are indicated.  In b, i and ii, the differential images corresponding to  those selected in a, i and ii,  are shown, revealing the distribution over which the signal has changed between  image frames. The black calibration bars to the right of  each group of images represent 20 μm. The traces below  (c) illustrate the change in intensity with time for events  identified in each of four  cells. In each case, an area or  areas of just a few pixels  were selected to limit the collection of signal to a small  volume of the cell. In c, i,  three widely separated areas  within a cell were chosen,  over which single events occurred during the sampling  period. The record in ii  shows a single brief event  highly localized to a small  part of a cell superimposed  on a very quiet baseline. In  iii, an example has been  selected in which several  events occurred repeatedly  at a single point within the  acquisition period, and in iv,  FCCP was applied at the end  of the acquisition time to  demonstrate the full range of  TMRE signal with complete  dissipation of the mitochondrial potential in comparison  with the small transient event  that preceded it.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2132882&req=5

Figure 2: Localized transient depolarization of Δψm. a, i and ii, shows a selection of images extracted from image series obtained from two cells over a period of ∼60 s and illustrates the transient localized depolarizations of mitochondria or groups of mitochondria throughout the cell with time. Each arrowhead points to a region of the image that shows a marked transient increase in intensity, and the times of the individual images are indicated. In b, i and ii, the differential images corresponding to those selected in a, i and ii, are shown, revealing the distribution over which the signal has changed between image frames. The black calibration bars to the right of each group of images represent 20 μm. The traces below (c) illustrate the change in intensity with time for events identified in each of four cells. In each case, an area or areas of just a few pixels were selected to limit the collection of signal to a small volume of the cell. In c, i, three widely separated areas within a cell were chosen, over which single events occurred during the sampling period. The record in ii shows a single brief event highly localized to a small part of a cell superimposed on a very quiet baseline. In iii, an example has been selected in which several events occurred repeatedly at a single point within the acquisition period, and in iv, FCCP was applied at the end of the acquisition time to demonstrate the full range of TMRE signal with complete dissipation of the mitochondrial potential in comparison with the small transient event that preceded it.
Mentions: As an initial experimental protocol, a series of images were acquired in sequence, typically giving an acquisition rate of 1–2 images per second. The transient reversible changes in Δψm localized to small discrete areas of the cells seen in such an image series are illustrated in Fig. 2 a, i and ii (arrows). The events occurred as small increases in signal over a relatively bright baseline signal, with an increase of ∼10–20% for most events. Small events and their distribution were more readily revealed by image processing, in which a running differential of the image series was constructed. In this mode, the preceding image of the series was subtracted from each image, revealing only those pixels in which the signal had changed. Fig. 2 b, i and ii, illustrates the distribution of the flicker events revealed from the differentials of the raw signals illustrated in Fig. 2 a. Individual events could be extremely localized and could involve greater volumes of the cell, and several events could occur simultaneously in different parts of the cell. The changes in signal with time are shown in Fig. 2 c, in which the intensity over small groups of pixels is plotted as a function of time for four different cells. Note the apparent independence of individual transient events in different parts of the cell with time (Fig. 2 c, i). Fig. 2 c, ii, illustrates an isolated brief event to show its occurrence in relation to an otherwise very quiet and stable baseline. The recurrent appearance of brief transients over the same point in the cell (Fig. 2 c, iii) suggests that it is unlikely that the transient events could in fact reflect complete dissipation of potential in single mitochondria (and see below). A further observation that strengthens this view is the relatively small amplitude of the signal compared with that seen with complete dissipation of Δψm (demonstrated after the application of FCCP). This is illustrated in Fig. 2 c, iv, which demonstrates that after a transient depolarization, the signal could still be dramatically increased by the uncoupler.

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