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Glutathione oxidation as a trigger of mitochondrial depolarization and oscillation in intact hearts.

ABSTRACT: Depolarization of the mitochondrial inner membrane potential (DeltaPsi(m)) associated with oxidative stress is thought to be a critical factor in cardiac dysfunction and cell injury following ischemia-reperfusion or exposure to cardiotoxic agents. In isolated cardiomyocytes, mitochondrially-generated reactive oxygen species (ROS) can readily trigger cell-wide collapse or oscillations of DeltaPsi(m) but it is not known whether these phenomena scale to the level of the whole heart. Here we utilize two-photon laser scanning fluorescence microscopy to track DeltaPsi(m), ROS, and reduced glutathione (GSH) levels in intact perfused guinea-pig hearts subjected to simulated ischemia reperfusion or GSH depletion with the thiol oxidizing agent diamide. Exposure to oxidative stress by either method provoked heterogeneous DeltaPsi(m) depolarization and occasional oscillation in clusters of myocytes in the epicardium in association with increased mitochondrial ROS production. Furthermore, the whole-heart oxidative stress dramatically increased the sensitivity of seemingly quiescent cells to DeltaPsi(m) depolarization induced by a localized laser flash. These effects were directly correlated with depletion of the intracellular GSH pool. Unexpectedly, hearts perfused with nominally Ca2+-free solution or those switched from 0.5 mM Ca2+ to nominally Ca2+-free solution also displayed heterogeneous DeltaPsi(m) depolarization and oscillation, in parallel with net oxidation of the GSH pool. The findings demonstrate that metabolic heterogeneity initiated by mitochondrial ROS-induced ROS release is present in the intact heart, and that the redox state of the glutathione pool is a key determinant of loss of DeltaPsi(m).

SUBMITTER: Slodzinski MK

PROVIDER: S-EPMC2604133 | biostudies-literature | 2008 Nov

REPOSITORIES: biostudies-literature

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Glutathione oxidation as a trigger of mitochondrial depolarization and oscillation in intact hearts.

Slodzinski Martin K MK Aon Miguel A MA O'Rourke Brian B

Journal of molecular and cellular cardiology 20080807 5

Depolarization of the mitochondrial inner membrane potential (DeltaPsi(m)) associated with oxidative stress is thought to be a critical factor in cardiac dysfunction and cell injury following ischemia-reperfusion or exposure to cardiotoxic agents. In isolated cardiomyocytes, mitochondrially-generated reactive oxygen species (ROS) can readily trigger cell-wide collapse or oscillations of DeltaPsi(m) but it is not known whether these phenomena scale to the level of the whole heart. Here we utilize ...[more]

PMID: 18760283

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Project description:Cardiovascular diseases (CVDs) are the leading cause of death worldwide, and the prevalence of CVDs increases markedly with age. Due to the high energetic demand, the heart is highly sensitive to mitochondrial dysfunction. The complexity of the cardiac mitochondrial proteome hinders the development of effective strategies that target mitochondrial dysfunction in CVDs. Mammalian mitochondria are composed of over 1000 proteins, most of which can undergo post-translational protein modifications (PTMs). Top-down proteomics is a powerful technique for characterizing and quantifying all protein sequence variations and PTMs. However, there are still knowledge gaps in the study of age-related mitochondrial proteoform changes using this technique. In this study, we used top-down proteomics to identify intact mitochondrial proteoforms in young and old hearts and determined changes in protein abundance and PTMs in cardiac aging.MethodsIntact mitochondria were isolated from the hearts of young (4-month-old) and old (24-25-month-old) mice. The mitochondria were lysed, and mitochondrial lysates were subjected to denaturation, reduction, and alkylation. For quantitative top-down analysis, there were 12 runs in total arising from 3 biological replicates in two conditions, with technical duplicates for each sample. The collected top-down datasets were deconvoluted and quantified, and then the proteoforms were identified.ResultsFrom a total of 12 LC-MS/MS runs, we identified 134 unique mitochondrial proteins in the different sub-mitochondrial compartments (OMM, IMS, IMM, matrix). 823 unique proteoforms in different mass ranges were identified. Compared to cardiac mitochondria of young mice, 7 proteoforms exhibited increased abundance and 13 proteoforms exhibited decreased abundance in cardiac mitochondria of old mice. Our analysis also detected PTMs of mitochondrial proteoforms, including N-terminal acetylation, lysine succinylation, lysine acetylation, oxidation, and phosphorylation.ConclusionBy combining mitochondrial protein enrichment using mitochondrial fractionation with quantitative top-down analysis using ultrahigh-pressure liquid chromatography (UPLC)-MS and label-free quantitation, we successfully identified and quantified intact proteoforms in the complex mitochondrial proteome. Using this approach, we detected age-related changes in abundance and PTMs of mitochondrial proteoforms in the heart.

Project description:Key pointsAlthough optogenetics has clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies lack the capability to react acutely to ongoing cardiac wave dynamics. Here, we developed an all-optical platform to monitor and control electrical activity in real-time. The methodology was applied to restore normal electrical activity after atrioventricular block and to manipulate the intraventricular propagation of the electrical wavefront. The closed-loop approach was also applied to simulate a re-entrant circuit across the ventricle. The development of this innovative optical methodology provides the first proof-of-concept that a real-time all-optical stimulation can control cardiac rhythm in normal and abnormal conditions.AbstractOptogenetics has provided new insights in cardiovascular research, leading to new methods for cardiac pacing, resynchronization therapy and cardioversion. Although these interventions have clearly demonstrated the feasibility of cardiac manipulation, current optical stimulation strategies do not take into account cardiac wave dynamics in real time. Here, we developed an all-optical platform complemented by integrated, newly developed software to monitor and control electrical activity in intact mouse hearts. The system combined a wide-field mesoscope with a digital projector for optogenetic activation. Cardiac functionality could be manipulated either in free-run mode with submillisecond temporal resolution or in a closed-loop fashion: a tailored hardware and software platform allowed real-time intervention capable of reacting within 2 ms. The methodology was applied to restore normal electrical activity after atrioventricular block, by triggering the ventricle in response to optically mapped atrial activity with appropriate timing. Real-time intraventricular manipulation of the propagating electrical wavefront was also demonstrated, opening the prospect for real-time resynchronization therapy and cardiac defibrillation. Furthermore, the closed-loop approach was applied to simulate a re-entrant circuit across the ventricle demonstrating the capability of our system to manipulate heart conduction with high versatility even in arrhythmogenic conditions. The development of this innovative optical methodology provides the first proof-of-concept that a real-time optically based stimulation can control cardiac rhythm in normal and abnormal conditions, promising a new approach for the investigation of the (patho)physiology of the heart.

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Glutathione oxidation as a trigger of mitochondrial depolarization and oscillation in intact hearts.

Publications

Glutathione oxidation as a trigger of mitochondrial depolarization and oscillation in intact hearts.

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