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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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Transient mitochondrial depolarizations require mitochondrial calcium  uptake. (a) After microinjection of a cell with DAPPAC  and fura-2–free acid, a challenge with caffeine (10 mM)  raised [Ca2+]i and caused a  twitch, showing that the SR  calcium release mechanism  was operational. Nevertheless, the mitochondrial  flicker was almost completely suppressed, as shown  in the surface and line images  shown in c (upper and lower  traces, respectively), and  compared with a control obtained in the same preparation (b, upper and lower panels). To illustrate the time  course of cell shortening, a  binary image of the cell was  created. The intensity was  measured in a region that included the edge of the cell.  Since the signal is simply a  function of the number of  pixels within the region set to  unity, cell shortening reduced the signal, illustrating  the time course of the twitch.
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Figure 8: Transient mitochondrial depolarizations require mitochondrial calcium uptake. (a) After microinjection of a cell with DAPPAC and fura-2–free acid, a challenge with caffeine (10 mM) raised [Ca2+]i and caused a twitch, showing that the SR calcium release mechanism was operational. Nevertheless, the mitochondrial flicker was almost completely suppressed, as shown in the surface and line images shown in c (upper and lower traces, respectively), and compared with a control obtained in the same preparation (b, upper and lower panels). To illustrate the time course of cell shortening, a binary image of the cell was created. The intensity was measured in a region that included the edge of the cell. Since the signal is simply a function of the number of pixels within the region set to unity, cell shortening reduced the signal, illustrating the time course of the twitch.

Mentions: As a device to represent changing intensities in space and time, we have chosen to present data from image sequences as “line images,” in which each line of the image gives the color-coded fluorescence intensity profile along a chosen line drawn along the length of the cell (see Figs. 1 and 4–8), the post-hoc equivalent of the line scan used for confocal imaging. The images shown were constructed as follows: A line was selected along the long axis of the cell. (Clearly this will exclude some events and will only illustrate events occurring along the chosen line.) The Kinetic Imaging software allows the creation of ASCII files consisting of the pixel values along that line for the full image sequence, typically between 30 and 60 image frames. The ASCII matrices were then read into MatLab (The Mathworks, Inc., Natick, MA), which was used to create the surface plots shown. We have also used as a convention a color look up table that runs from black through red and orange to yellow and white with increasing values for otherwise unprocessed image data, while for images that have been thresholded and ratioed, we have applied a look up table that runs through the spectrum, with blue as the lowest value and red as the highest.


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

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

Transient mitochondrial depolarizations require mitochondrial calcium  uptake. (a) After microinjection of a cell with DAPPAC  and fura-2–free acid, a challenge with caffeine (10 mM)  raised [Ca2+]i and caused a  twitch, showing that the SR  calcium release mechanism  was operational. Nevertheless, the mitochondrial  flicker was almost completely suppressed, as shown  in the surface and line images  shown in c (upper and lower  traces, respectively), and  compared with a control obtained in the same preparation (b, upper and lower panels). To illustrate the time  course of cell shortening, a  binary image of the cell was  created. The intensity was  measured in a region that included the edge of the cell.  Since the signal is simply a  function of the number of  pixels within the region set to  unity, cell shortening reduced the signal, illustrating  the time course of the twitch.
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Related In: Results  -  Collection

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Figure 8: Transient mitochondrial depolarizations require mitochondrial calcium uptake. (a) After microinjection of a cell with DAPPAC and fura-2–free acid, a challenge with caffeine (10 mM) raised [Ca2+]i and caused a twitch, showing that the SR calcium release mechanism was operational. Nevertheless, the mitochondrial flicker was almost completely suppressed, as shown in the surface and line images shown in c (upper and lower traces, respectively), and compared with a control obtained in the same preparation (b, upper and lower panels). To illustrate the time course of cell shortening, a binary image of the cell was created. The intensity was measured in a region that included the edge of the cell. Since the signal is simply a function of the number of pixels within the region set to unity, cell shortening reduced the signal, illustrating the time course of the twitch.
Mentions: As a device to represent changing intensities in space and time, we have chosen to present data from image sequences as “line images,” in which each line of the image gives the color-coded fluorescence intensity profile along a chosen line drawn along the length of the cell (see Figs. 1 and 4–8), the post-hoc equivalent of the line scan used for confocal imaging. The images shown were constructed as follows: A line was selected along the long axis of the cell. (Clearly this will exclude some events and will only illustrate events occurring along the chosen line.) The Kinetic Imaging software allows the creation of ASCII files consisting of the pixel values along that line for the full image sequence, typically between 30 and 60 image frames. The ASCII matrices were then read into MatLab (The Mathworks, Inc., Natick, MA), which was used to create the surface plots shown. We have also used as a convention a color look up table that runs from black through red and orange to yellow and white with increasing values for otherwise unprocessed image data, while for images that have been thresholded and ratioed, we have applied a look up table that runs through the spectrum, with blue as the lowest value and red as the highest.

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