Limits...
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.

Show MeSH

Related in: MedlinePlus

Characteristics of  transient depolarizations of  single mitochondria. a, i–iv,  shows examples of changes  in signal measured over  clearly identifiable single mitochondria measured as a  function of time. Mitochondria were identified as single  objects by thresholding the  images, which clearly revealed distinct rod-shaped  objects. In each case, the recovery phase of the transient  depolarizations was fitted  well by a single exponential  decay function with time  constants indicated. Note  particularly the example  shown in iv, in which two  clearly distinct events were  identified, each with a similar  time course, suggesting complete repolarization after the  first event. Surface plots (b, i  and c, i) and their equivalent  line images (b, ii and c, ii) are  shown; the intensity profile  along a line selected to follow the long axis of a single  identifiable mitochondrion  is shown as a function of  time. The band of increased  intensity in the middle of the  image field is the increased  intensity seen over the mitochondrion, and the transient  events can clearly be seen to  involve a spread of indicator  beyond the boundaries of the  mitochondrion. Again the  example shown in c illustrates two consecutive events  occurring within a single mitochondrion with time.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2132882&req=5

Figure 4: Characteristics of transient depolarizations of single mitochondria. a, i–iv, shows examples of changes in signal measured over clearly identifiable single mitochondria measured as a function of time. Mitochondria were identified as single objects by thresholding the images, which clearly revealed distinct rod-shaped objects. In each case, the recovery phase of the transient depolarizations was fitted well by a single exponential decay function with time constants indicated. Note particularly the example shown in iv, in which two clearly distinct events were identified, each with a similar time course, suggesting complete repolarization after the first event. Surface plots (b, i and c, i) and their equivalent line images (b, ii and c, ii) are shown; the intensity profile along a line selected to follow the long axis of a single identifiable mitochondrion is shown as a function of time. The band of increased intensity in the middle of the image field is the increased intensity seen over the mitochondrion, and the transient events can clearly be seen to involve a spread of indicator beyond the boundaries of the mitochondrion. Again the example shown in c illustrates two consecutive events occurring within a single mitochondrion with time.

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)

Characteristics of  transient depolarizations of  single mitochondria. a, i–iv,  shows examples of changes  in signal measured over  clearly identifiable single mitochondria measured as a  function of time. Mitochondria were identified as single  objects by thresholding the  images, which clearly revealed distinct rod-shaped  objects. In each case, the recovery phase of the transient  depolarizations was fitted  well by a single exponential  decay function with time  constants indicated. Note  particularly the example  shown in iv, in which two  clearly distinct events were  identified, each with a similar  time course, suggesting complete repolarization after the  first event. Surface plots (b, i  and c, i) and their equivalent  line images (b, ii and c, ii) are  shown; the intensity profile  along a line selected to follow the long axis of a single  identifiable mitochondrion  is shown as a function of  time. The band of increased  intensity in the middle of the  image field is the increased  intensity seen over the mitochondrion, and the transient  events can clearly be seen to  involve a spread of indicator  beyond the boundaries of the  mitochondrion. Again the  example shown in c illustrates two consecutive events  occurring within a single mitochondrion with time.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Characteristics of transient depolarizations of single mitochondria. a, i–iv, shows examples of changes in signal measured over clearly identifiable single mitochondria measured as a function of time. Mitochondria were identified as single objects by thresholding the images, which clearly revealed distinct rod-shaped objects. In each case, the recovery phase of the transient depolarizations was fitted well by a single exponential decay function with time constants indicated. Note particularly the example shown in iv, in which two clearly distinct events were identified, each with a similar time course, suggesting complete repolarization after the first event. Surface plots (b, i and c, i) and their equivalent line images (b, ii and c, ii) are shown; the intensity profile along a line selected to follow the long axis of a single identifiable mitochondrion is shown as a function of time. The band of increased intensity in the middle of the image field is the increased intensity seen over the mitochondrion, and the transient events can clearly be seen to involve a spread of indicator beyond the boundaries of the mitochondrion. Again the example shown in c illustrates two consecutive events occurring within a single mitochondrion with time.
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