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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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An increase of TMRE fluorescence signals depolarization of Δψm in a single cardiomyocyte. (a) High-resolution image taken  from a cardiomyocyte loaded with TMRE using a confocal imaging system (excitation at 543 nm). The discrete localization of signal to  mitochondria and the distribution of mitochondria in bands running along the longitudinal axis of the cell is clear. The asterisks in this  and the following panels indicate the position of the nucleus. (b) CCD image of a similar cell. The longitudinal distribution of mitochondria is still evident, despite the loss of resolution. c and d show images of a TMRE-loaded cell before (c) and after (d) application of the  uncoupler, FCCP (1 μM). The plot shown in e (a surface plot) and the corresponding “line image” shown in f were obtained from the pixel  values extracted from the image series along the line drawn along the axis of the cell as shown in c and d, and they illustrate the evolution of the response to FCCP with time. In response to FCCP, the TMRE signal increased approximately threefold in this cell.
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Figure 1: An increase of TMRE fluorescence signals depolarization of Δψm in a single cardiomyocyte. (a) High-resolution image taken from a cardiomyocyte loaded with TMRE using a confocal imaging system (excitation at 543 nm). The discrete localization of signal to mitochondria and the distribution of mitochondria in bands running along the longitudinal axis of the cell is clear. The asterisks in this and the following panels indicate the position of the nucleus. (b) CCD image of a similar cell. The longitudinal distribution of mitochondria is still evident, despite the loss of resolution. c and d show images of a TMRE-loaded cell before (c) and after (d) application of the uncoupler, FCCP (1 μM). The plot shown in e (a surface plot) and the corresponding “line image” shown in f were obtained from the pixel values extracted from the image series along the line drawn along the axis of the cell as shown in c and d, and they illustrate the evolution of the response to FCCP with time. In response to FCCP, the TMRE signal increased approximately threefold in this cell.

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)

An increase of TMRE fluorescence signals depolarization of Δψm in a single cardiomyocyte. (a) High-resolution image taken  from a cardiomyocyte loaded with TMRE using a confocal imaging system (excitation at 543 nm). The discrete localization of signal to  mitochondria and the distribution of mitochondria in bands running along the longitudinal axis of the cell is clear. The asterisks in this  and the following panels indicate the position of the nucleus. (b) CCD image of a similar cell. The longitudinal distribution of mitochondria is still evident, despite the loss of resolution. c and d show images of a TMRE-loaded cell before (c) and after (d) application of the  uncoupler, FCCP (1 μM). The plot shown in e (a surface plot) and the corresponding “line image” shown in f were obtained from the pixel  values extracted from the image series along the line drawn along the axis of the cell as shown in c and d, and they illustrate the evolution of the response to FCCP with time. In response to FCCP, the TMRE signal increased approximately threefold in this cell.
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Related In: Results  -  Collection

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

Figure 1: An increase of TMRE fluorescence signals depolarization of Δψm in a single cardiomyocyte. (a) High-resolution image taken from a cardiomyocyte loaded with TMRE using a confocal imaging system (excitation at 543 nm). The discrete localization of signal to mitochondria and the distribution of mitochondria in bands running along the longitudinal axis of the cell is clear. The asterisks in this and the following panels indicate the position of the nucleus. (b) CCD image of a similar cell. The longitudinal distribution of mitochondria is still evident, despite the loss of resolution. c and d show images of a TMRE-loaded cell before (c) and after (d) application of the uncoupler, FCCP (1 μM). The plot shown in e (a surface plot) and the corresponding “line image” shown in f were obtained from the pixel values extracted from the image series along the line drawn along the axis of the cell as shown in c and d, and they illustrate the evolution of the response to FCCP with time. In response to FCCP, the TMRE signal increased approximately threefold in this cell.
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