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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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Induction of Ca2+ sparks after the MPT. Cell is dual-loaded with 125 nM TMRM (ΔΨ) and fluo-3 (Ca2+) and line-scan imaged at 230 Hz. Representative example showing the dissipation of TMRM fluorescence from a single mitochondrion and a cluster of Ca2+ sparks in the immediate vicinity, within seconds of MPT induction. Inset: comparison of Ca2+ spark rate in proximity of MPT occurrence (i.e., within the sarcomere containing the involved mitochondria and within 3 s after MPT occurrence; n = 90 sparks) versus that at background (n = 150 sparks; P < 0.05).
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Figure 9: Induction of Ca2+ sparks after the MPT. Cell is dual-loaded with 125 nM TMRM (ΔΨ) and fluo-3 (Ca2+) and line-scan imaged at 230 Hz. Representative example showing the dissipation of TMRM fluorescence from a single mitochondrion and a cluster of Ca2+ sparks in the immediate vicinity, within seconds of MPT induction. Inset: comparison of Ca2+ spark rate in proximity of MPT occurrence (i.e., within the sarcomere containing the involved mitochondria and within 3 s after MPT occurrence; n = 90 sparks) versus that at background (n = 150 sparks; P < 0.05).

Mentions: This newly described RIRR phenomenon, though only seconds in duration, produces an apparently significant burst of local ROS that could have immediate consequences for the local cellular homeostasis. In particular, since oxidative and nitrosative stress can modulate the spontaneous activity of the ryanodine receptor (see reference 21), we examined the nature of Ca2+ spark activity in the “wake” of MPT induction. As shown in Fig. 9 in a representative cell dual-loaded with TMRM and the Ca2+-sensitive dye fluo-3, there is frequently a period of significantly increased Ca2+ spark frequency at the z-lines (i.e., the site of the T tubule–sarcoplasmic reticulum junction) in the immediate vicinity of the mitochondrion shortly after MPT induction. Ordinarily, the spontaneous background (stochastic) event rate is about two to three sparks per 100 μm·s. However, in proximity of the MPT (defined as within the sarcomere containing the involved mitochondria and within 3 s after MPT occurrence), the event rate approximately doubles to about five to six per 100 μm·s (P < 0.05; Fig. 9, inset). While ordinary background Ca2+ sparks are typically single events, the MPT-associated ones frequently occur as clusters, as seen in Fig. 9.


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)

Induction of Ca2+ sparks after the MPT. Cell is dual-loaded with 125 nM TMRM (ΔΨ) and fluo-3 (Ca2+) and line-scan imaged at 230 Hz. Representative example showing the dissipation of TMRM fluorescence from a single mitochondrion and a cluster of Ca2+ sparks in the immediate vicinity, within seconds of MPT induction. Inset: comparison of Ca2+ spark rate in proximity of MPT occurrence (i.e., within the sarcomere containing the involved mitochondria and within 3 s after MPT occurrence; n = 90 sparks) versus that at background (n = 150 sparks; P < 0.05).
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Related In: Results  -  Collection

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Figure 9: Induction of Ca2+ sparks after the MPT. Cell is dual-loaded with 125 nM TMRM (ΔΨ) and fluo-3 (Ca2+) and line-scan imaged at 230 Hz. Representative example showing the dissipation of TMRM fluorescence from a single mitochondrion and a cluster of Ca2+ sparks in the immediate vicinity, within seconds of MPT induction. Inset: comparison of Ca2+ spark rate in proximity of MPT occurrence (i.e., within the sarcomere containing the involved mitochondria and within 3 s after MPT occurrence; n = 90 sparks) versus that at background (n = 150 sparks; P < 0.05).
Mentions: This newly described RIRR phenomenon, though only seconds in duration, produces an apparently significant burst of local ROS that could have immediate consequences for the local cellular homeostasis. In particular, since oxidative and nitrosative stress can modulate the spontaneous activity of the ryanodine receptor (see reference 21), we examined the nature of Ca2+ spark activity in the “wake” of MPT induction. As shown in Fig. 9 in a representative cell dual-loaded with TMRM and the Ca2+-sensitive dye fluo-3, there is frequently a period of significantly increased Ca2+ spark frequency at the z-lines (i.e., the site of the T tubule–sarcoplasmic reticulum junction) in the immediate vicinity of the mitochondrion shortly after MPT induction. Ordinarily, the spontaneous background (stochastic) event rate is about two to three sparks per 100 μm·s. However, in proximity of the MPT (defined as within the sarcomere containing the involved mitochondria and within 3 s after MPT occurrence), the event rate approximately doubles to about five to six per 100 μm·s (P < 0.05; Fig. 9, inset). While ordinary background Ca2+ sparks are typically single events, the MPT-associated ones frequently occur as clusters, as seen in Fig. 9.

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