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Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes.

Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ - J. Exp. Med. (2000)

Bottom Line: We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi.The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione.The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cardiovascular Sciences, Gerontology Research Center, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6825, USA.

ABSTRACT
We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

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Scavenging the ROS trigger or exposure to exogenous NO inhibits the MPT. Cells loaded with 125 nM TMRM and confocal line-scan–imaged at 2 Hz. (A) Mean time to MPT induction in control versus pretreated cells as indicated: Trolox (2 mM); SNAP (100 μM); L-NAME (1 mM). Data represent the average from 8–10 cells in each group. (B) Evidence of endogenous production of NO by mitochondria after MPT induction and inhibition by L-NAME (4 mM). Myocytes were loaded with 125 nM TMRM (red) and 10 mM DAF-2 (green) and line scanned at 100 Hz.
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Figure 7: Scavenging the ROS trigger or exposure to exogenous NO inhibits the MPT. Cells loaded with 125 nM TMRM and confocal line-scan–imaged at 2 Hz. (A) Mean time to MPT induction in control versus pretreated cells as indicated: Trolox (2 mM); SNAP (100 μM); L-NAME (1 mM). Data represent the average from 8–10 cells in each group. (B) Evidence of endogenous production of NO by mitochondria after MPT induction and inhibition by L-NAME (4 mM). Myocytes were loaded with 125 nM TMRM (red) and 10 mM DAF-2 (green) and line scanned at 100 Hz.

Mentions: To confirm that ROS triggered both MPT and ROS release, experiments were performed in the presence of 2 mM Trolox. Trolox treatment was found to prevent ΔΨ loss during laser line-scan imaging compared with control cells (Fig. 7 A). It is interesting to note that cells pretreated with the membrane-permeant superoxide scavenger Tiron (1–2 mM) or the superoxide dismutase (SOD) mimetics MnTBAP or MnTMPyP (500 μM each) failed to alter the ability to induce the MPT (not shown). Based on the fact that Trolox, but not the ·O2− scavengers, prevented the MPT and RIRR, it is tempting to speculate that the important ROS species from the standpoint of MPT induction/RIRR is likely to be peroxide rather than ·O2−.


Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes.

Zorov DB, Filburn CR, Klotz LO, Zweier JL, Sollott SJ - J. Exp. Med. (2000)

Scavenging the ROS trigger or exposure to exogenous NO inhibits the MPT. Cells loaded with 125 nM TMRM and confocal line-scan–imaged at 2 Hz. (A) Mean time to MPT induction in control versus pretreated cells as indicated: Trolox (2 mM); SNAP (100 μM); L-NAME (1 mM). Data represent the average from 8–10 cells in each group. (B) Evidence of endogenous production of NO by mitochondria after MPT induction and inhibition by L-NAME (4 mM). Myocytes were loaded with 125 nM TMRM (red) and 10 mM DAF-2 (green) and line scanned at 100 Hz.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193314&req=5

Figure 7: Scavenging the ROS trigger or exposure to exogenous NO inhibits the MPT. Cells loaded with 125 nM TMRM and confocal line-scan–imaged at 2 Hz. (A) Mean time to MPT induction in control versus pretreated cells as indicated: Trolox (2 mM); SNAP (100 μM); L-NAME (1 mM). Data represent the average from 8–10 cells in each group. (B) Evidence of endogenous production of NO by mitochondria after MPT induction and inhibition by L-NAME (4 mM). Myocytes were loaded with 125 nM TMRM (red) and 10 mM DAF-2 (green) and line scanned at 100 Hz.
Mentions: To confirm that ROS triggered both MPT and ROS release, experiments were performed in the presence of 2 mM Trolox. Trolox treatment was found to prevent ΔΨ loss during laser line-scan imaging compared with control cells (Fig. 7 A). It is interesting to note that cells pretreated with the membrane-permeant superoxide scavenger Tiron (1–2 mM) or the superoxide dismutase (SOD) mimetics MnTBAP or MnTMPyP (500 μM each) failed to alter the ability to induce the MPT (not shown). Based on the fact that Trolox, but not the ·O2− scavengers, prevented the MPT and RIRR, it is tempting to speculate that the important ROS species from the standpoint of MPT induction/RIRR is likely to be peroxide rather than ·O2−.

Bottom Line: We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi.The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione.The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cardiovascular Sciences, Gerontology Research Center, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224-6825, USA.

ABSTRACT
We sought to understand the relationship between reactive oxygen species (ROS) and the mitochondrial permeability transition (MPT) in cardiac myocytes based on the observation of increased ROS production at sites of spontaneously deenergized mitochondria. We devised a new model enabling incremental ROS accumulation in individual mitochondria in isolated cardiac myocytes via photoactivation of tetramethylrhodamine derivatives, which also served to report the mitochondrial transmembrane potential, DeltaPsi. This ROS accumulation reproducibly triggered abrupt (and sometimes reversible) mitochondrial depolarization. This phenomenon was ascribed to MPT induction because (a) bongkrekic acid prevented it and (b) mitochondria became permeable for calcein ( approximately 620 daltons) concurrently with depolarization. These photodynamically produced "triggering" ROS caused the MPT induction, as the ROS scavenger Trolox prevented it. The time required for triggering ROS to induce the MPT was dependent on intrinsic cellular ROS-scavenging redox mechanisms, particularly glutathione. MPT induction caused by triggering ROS coincided with a burst of mitochondrial ROS generation, as measured by dichlorofluorescein fluorescence, which we have termed mitochondrial "ROS-induced ROS release" (RIRR). This MPT induction/RIRR phenomenon in cardiac myocytes often occurred synchronously and reversibly among long chains of adjacent mitochondria demonstrating apparent cooperativity. The observed link between MPT and RIRR could be a fundamental phenomenon in mitochondrial and cell biology.

Show MeSH
Related in: MedlinePlus