Mitochondrial instability during regional ischemia-reperfusion underlies arrhythmias in monolayers of cardiomyocytes.
ABSTRACT: Regional depolarization of the mitochondrial network can alter cellular electrical excitability and increase the propensity for reentry, in part, through the opening of sarcolemmal KATP channels. Mitochondrial inner membrane potential (??m) instability or oscillation can be induced in myocytes by exposure to reactive oxygen species (ROS), laser excitation, or glutathione depletion, and is thought to be a major factor in arrhythmogenesis during ischemia-reperfusion. Nevertheless, the correlation between ??m recovery kinetics and reperfusion-induced arrhythmias has been difficult to demonstrate experimentally. Here, we investigate the relationship between subcellular changes in ??m, cellular glutathione redox potential, electrical excitability, and wave propagation during coverslip-induced ischemia-reperfusion (IR) in neonatal rat ventricular myocyte (NRVM) monolayers. Ischemia led to decreased action potential amplitude and duration followed by electrical inexcitability after ~15min of ischemia. ??m depolarization occurred in two phases during ischemia: in phase 1 (<30min ischemia), mitochondrial clusters within individual NRVMs depolarized, while phase 2 ??m depolarization (30-60min) was characterized by global functional collapse of the mitochondrial network across the whole ischemic region of the monolayer, typically involving a propagating metabolic wave. Oxidation of the glutathione (GSSG:GSH) redox potential occurred during ischemia, followed by recovery upon reperfusion (i.e., lifting the coverslip). ??m recovered in the mitochondria of individual myocytes quite rapidly upon reperfusion (<5min), but was highly unstable, characterized by subcellular oscillations or flickering of clusters of mitochondria in NRVMs across the reperfused region. Electrical excitability also recovered in a heterogeneous manner, providing an arrhythmogenic substrate which led to formation of sustained reentry. Treatment with 4'-chlorodiazepam, a peripheral benzodiazepine receptor ligand, prevented ??m oscillation, improved GSH recovery rate, and prevented reentry during reperfusion, indicating that stabilization of mitochondrial network dynamics is important for preventing post-ischemic arrhythmias. This article is part of a Special Issue entitled "Mitochondria: From Basic Mitochondrial Biology to Cardiovascular Disease".
Project description:Electrophysiological changes promoting arrhythmias during acute regional ischemia/reperfusion are challenging to study in intact cardiac tissue because of complex 3-dimensional myocardial and vascular geometry. We characterized electrophysiological alterations and arrhythmias during regional ischemia/reperfusion in a simpler 2-dimensional geometry of cultured neonatal rat ventricular myocyte monolayers.Optical mapping of intracellular Ca (Ca(i)) and voltage was performed with the use of Rhod 2-AM and Rh-237, respectively. Regional ischemia was mimicked by covering the central portion of monolayer with a glass coverslip, and reperfusion was mimicked by removing the coverslip. Monolayers were stained with fluorescent antibodies to detect total and dephosphorylated connexin-43 at various time points. During coverslip ischemia, action potential duration shortened, Ca(i) transient duration was prolonged, and local conduction velocity (CV) slowed progressively, with loss of excitability after 10.6 +/- 3.6 minutes. CV slowing was accompanied by connexin-43 dephosphorylation. During ischemia, spontaneous reentry occurred in 5 of 11 monolayers, initiated by extrasystoles arising from the border zone or unidirectional conduction block of paced beats. On reperfusion, excitability recovered within 1.0 +/- 0.8 minutes, but CV remained depressed for 9.0 +/- 3.0 minutes, promoting reentry in the reperfused zone. As connexin-43 phosphorylation recovered in the reperfused zone, CV normalized, and arrhythmias resolved.Acute regional ischemia/reperfusion in neonatal rat ventricular myocyte monolayers recapitulates electrophysiological alterations and arrhythmias similar to those observed during acute coronary occlusion/reperfusion in intact hearts. During early reperfusion, slow recovery from connexin-43 dephosphorylation leads to persistent CV slowing, creating a highly arrhythmogenic substrate.
Project description:NRVMs were subjected to varying durations of ischemia or ischemia+reperfusion using coverslip hypoxia. Expression profiling was used to identify genes that are differentially regulated in either event. We used microarrays to detail the global program of gene expression underlying ischemia and reperfusion using Coverslip Hypoxia and identified distinct classes of genes regulated during these processes. Experiment Overall Design: RNA was extracted from NRVMs subjected to varying durations of ischemia or ischemia+reperfusion and hybridized to rat genome Affymetrix arrays.
Project description:Myocardial ischemia-reperfusion (IR) injury represents a constellation of pathological processes that occur when ischemic myocardium experiences a restoration of perfusion. Reentrant arrhythmias, which represent a particularly lethal manifestation of IR injury, can result when ischemic tissue exhibits decreased excitability and/or changes of action potential duration (APD), conditions that precipitate unidirectional conduction block. Many of the cellular components that are involved with IR injury are modulated by pH and/or phosphometabolites such as ATP and phosphocreatine (PCr), all of which can be manipulated in vivo and potentially in the clinical setting. Using a mathematical model of the cardiomyocyte that we previously developed to study ischemia and reperfusion, we performed a series of simulations with the aim of determining whether pH- or phosphometabolite-related processes play a more significant role in generating changes in excitability and action potential morphology that are associated with the development of reentry. In our simulations, persistent shortening of APD, action potential amplitude (APA), and depolarization of the resting membrane potential were more severe when ATP and PCr availability were suppressed during reperfusion than when extracellular pH recovery was inhibited. Reduced phosphometabolite availability and pH recovery affected multiple ion channels and exchangers. Some of these effects were the result of direct modulation by phosphometabolites and/or acidosis, while others resulted from elevated sodium and calcium loads during reperfusion. In addition, increasing ATP and PCr availability during reperfusion was more beneficial in terms of increasing APD and APA than was increasing the amount of pH recovery. Together, these results suggest that therapies directed at increasing ATP and/or PCr availability during reperfusion may be more beneficial than perturbing pH recovery with regard to mitigating action potential changes that increase the likelihood of reentrant arrhythmias.
Project description:Recovery of the mitochondrial inner membrane potential (DeltaPsi(m)) is a key determinant of postischemic functional recovery of the heart. Mitochondrial ROS-induced ROS release causes the collapse of DeltaPsi(m) and the destabilization of the action potential (AP) through a mechanism involving a mitochondrial inner membrane anion channel (IMAC) modulated by the mitochondrial benzodiazepine receptor (mBzR). Here, we test the hypothesis that this mechanism contributes to spatiotemporal heterogeneity of DeltaPsi(m) during ischemia-reperfusion (IR), thereby promoting abnormal electrical activation and arrhythmias in the whole heart. High-resolution optical AP mapping was performed in perfused guinea pig hearts subjected to 30 minutes of global ischemia followed by reperfusion. Typical electrophysiological responses, including progressive AP shortening followed by membrane inexcitablity in ischemia and ventricular fibrillation upon reperfusion, were observed in control hearts. These responses were reduced or eliminated by treatment with the mBzR antagonist 4'-chlorodiazepam (4'-Cl-DZP), which blocks depolarization of DeltaPsi(m). When applied throughout the IR protocol, 4'-Cl-DZP blunted AP shortening and prevented reperfusion arrhythmias. Inhibition of ventricular fibrillation was also achieved by bolus infusion of 4'-Cl-DZP just before reperfusion. Conversely, treatment with an agonist of the mBzR that promotes DeltaPsi(m) depolarization exacerbated IR-induced electrophysiological changes and failed to prevent arrhythmias. The effects of these compounds were consistent with their actions on IMAC and DeltaPsi(m). These findings directly link instability of DeltaPsi(m) to the heterogeneous electrophysiological substrate of the postischemic heart and highlight the mitochondrial membrane as a new therapeutic target for arrhythmia prevention in ischemic heart disease.
Project description: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).
Project description:Ischemia-induced shortening of the cardiac action potential and its heterogeneous recovery upon reperfusion are thought to set the stage for reentrant arrhythmias and sudden cardiac death. We have recently reported that the collapse of mitochondrial membrane potential (DeltaPsi(m)) through a mechanism triggered by reactive oxygen species (ROS), coupled to the opening of sarcolemmal ATP-sensitive potassium (K(ATP)) channels, contributes to electrical dysfunction during ischemia-reperfusion. Here we present a computational model of excitation-contraction coupling linked to mitochondrial bioenergetics that incorporates mitochondrial ROS-induced ROS release with coupling between the mitochondrial energy state and electrical excitability mediated by the sarcolemmal K(ATP) current (I(K,ATP)). Whole-cell model simulations demonstrate that increasing the fraction of oxygen diverted from the respiratory chain to ROS production triggers limit-cycle oscillations of DeltaPsi(m), redox potential, and mitochondrial respiration through the activation of a ROS-sensitive inner membrane anion channel. The periods of transient mitochondrial uncoupling decrease the cytosolic ATP/ADP ratio and activate I(K,ATP), consequently shortening the cellular action potential duration and ultimately suppressing electrical excitability. The model simulates emergent behavior observed in cardiomyocytes subjected to metabolic stress and provides a new tool for examining how alterations in mitochondrial oxidative phosphorylation will impact the electrophysiological, contractile, and Ca(2+) handling properties of the cardiac cell. Moreover, the model is an important step toward building multiscale models that will permit investigation of the role of spatiotemporal heterogeneity of mitochondrial metabolism in the mechanisms of arrhythmogenesis and contractile dysfunction in cardiac muscle.
Project description:Instability of the inner mitochondrial membrane potential (??m) has been implicated in electrical dysfunction, including arrhythmogenesis during ischemia-reperfusion. Monitoring ??m has led to conflicting results, where depolarization has been reported as sporadic and as a propagating wave. The present study was designed to resolve the aforementioned difference and determine the unknown relationship between ??m and electrophysiology. We developed a novel imaging modality for simultaneous optical mapping of ??m and transmembrane potential (Vm). Optical mapping was performed using potentiometric dyes on preparations from 4 mouse hearts, 14 rabbit hearts, and 7 human hearts. Our data showed that during ischemia, ??m depolarization is sporadic and changes asynchronously with electrophysiological changes. Spatially, ??m depolarization was associated with action potential duration shortening but not conduction slowing. Analysis of focal activity indicated that ??m is not different within the myocardium where the focus originates compared with normal ventricular tissue. Overall, our data suggest that during ischemia, mitochondria maintain their function at the expense of sarcolemmal electrophysiology, but ??m depolarization does not have a direct association to ischemia-induced arrhythmias.
Project description:Hepatic ischemia-reperfusion is a major clinical problem with limited treatment options. The pathophysiology of hepatic ischemia-reperfusion is characterized by mitochondrial dysfunction and cellular energy deficits. Sirtuin 1 is an energy-sensing enzyme known to modulate mitochondrial biogenesis. We hypothesized that pharmacologic activation of sirtuin 1 is protective after hepatic ischemia-reperfusion injury.Animal study.University-based experimental laboratory.Wild-type C57BL/6 mice.C57BL/6 mice were subjected to 60-minute partial hepatic ischemia-reperfusion and posttreated with sirtuin 1 activator, SRT1720 (20?mg/kg), or vehicle. Blood and liver were collected at 24 hours after ischemia-reperfusion for analyses of hepatic injury, adenosine triphosphate levels, mitochondrial mass, autophagy, inflammation, and oxidative stress. H4IIE hepatoma cells and rat primary hepatocytes were incubated with oxyrase to induce hypoxia followed by reoxygenation in the presence or absence of SRT1720 for assessment of mitochondrial mass, mitochondrial membrane potential, and autophagy.SRT1720 restored the reduction in mitochondrial mass, enhanced autophagy, and preserved adenosine triphosphate levels in the liver after ischemia-reperfusion, which was associated with a decrease in ischemia-reperfusion-induced hepatic injury, apoptosis, and necrosis. Ischemia-reperfusion-induced inflammation was also significantly reduced by SRT1720 as measured by systemic and hepatic cytokine and chemokine levels, as well as a decrease in neutrophil infiltration to the liver. Furthermore, oxidative stress was markedly attenuated in the SRT1720-treated mice compared with the vehicle. SRT1720 treatment increased adenosine triphosphate levels and survival of cultured hepatocytes after hypoxia-reoxygenation. SRT1720 not only increased the mitochondrial mass but also increased mitochondrial membrane potential per cell in cultured hepatocytes after hypoxia-reoxygenation. Moreover, SRT1720 prevented the hypoxia-reoxygenation-induced mitochondrial depolarization and resulted in an enhancement of autophagy in cultured hepatocytes after hypoxia-reoxygenation.Pharmacologic stimulation of sirtuin 1 attenuates liver injury after hepatic ischemia-reperfusion by restoring mitochondrial mass and membrane potential, which is associated with the enhancement of autophagy.
Project description:MicroRNAs, a class of small and non-encoding RNAs that transcriptionally or post-transcriptionally modulate the expression of their target genes, has been implicated as critical regulatory molecules in many cardiovascular diseases, including ischemia/reperfusion induced cardiac injury. Here, we report microRNA-145, a tumor suppressor miRNA, can protect cardiomyocytes from hydrogen peroxide H?O?-induced apoptosis through targeting the mitochondrial pathway. Quantitative real-time PCR (qPCR) demonstrated that the expression of miR-145 in either ischemia/reperfused mice myocardial tissues or H?O?-treated neonatal rat ventricle myocytes (NRVMs) was markedly down-regulated. Over-expression of miR-145 significantly inhibited the H?O?-induced cellular apoptosis, ROS production, mitochondrial structure disruption as well as the activation of key signaling proteins in mitochondrial apoptotic pathway. These protective effects of miR-145 were abrogated by over-expression of Bnip3, an initiation factor of the mitochondrial apoptotic pathway in cardiomyocytes. Finally, we utilized both luciferase reporter assay and western blot analysis to identify Bnip3 as a direct target of miR-145. Our results suggest miR-145 plays an important role in regulating mitochondrial apoptotic pathway in heart challenged with oxidative stress. MiR-145 may represent a potential therapeutic target for treatment of oxidative stress-associated cardiovascular diseases, such as myocardial ischemia/reperfusion injury.