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Atrial fibrillation (AF) is the most common arrhythmia and is associated with increased morbidity and mortality. The mechanisms underlying the transition from paroxysmal to persistent AF are still a matter of debate. Recent studies indicate a potential role for metabolic remodelling in the AF stabilization process and increased levels of a specific metabolic substrate, succinate, have been found in blood plasma of AF patients. While succinate is known to increase reactive oxygen species (ROS) production, the role of the succinate receptor (GPR91) in atrial function is unknown. To assess the impact of the succinate pathway on AF vulnerability in a sheep model. GPR91 expression levels were quantified by Western Blot (WB) in a previously described sheep model of persistent AF induced by burst-pacing. Optical experiments were subsequently performed in ex vivo right atria (RA) from control sheep (n = 5). RA were perfused by Tyrode solution, then supplemented with the cis-epoxysuccinic acid (cESA), a specific activator of GPR91 (150 μM) to study its involvement in atrial electrophysiology and AF. Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5 Hz pacing frequency. We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30 Hz) to assess ex vivo AF vulnerability. Finally, we investigated these properties in a human RA from an AF patient. WB experiments revealed an increase in GPR91 expression in AF sheep compared to sham sheep. Interestingly, in resistant sheep (no AF after 90 days of burst pacing), expression levels were decreased compared to sham. During cESA perfusion in control sheep RA, sinus rhythm (119 vs. 95 bpm; P = 0.03) and ERP (202 vs. 178 ms; P = 0.36) were decreased when compared to baseline. We also observed a decrease in APD80 (199 vs. 177 ms; P = 0.009) and in CV (127 vs. 115 cm/sec; P = 0.01). These electrophysiological perturbations led to an increase in AF vulnerability by burst pacing (0% vs. 60%) and an increase in spontaneous arrhythmias. These results were corroborated in the human RA were cESA decreased APD80 (265 vs. 241 ms), ERP (240 vs. 220 ms), and CV (119 vs. 94 cm/sec) and increased arrhythmia vulnerability. For the first time, we demonstrated that GPR91 is expressed in atrial tissue and that its activation through succinate can play a major role in AF mechanisms. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most sustained arrhythmia and increases morbidity and mortality. However, development and maintenance of AF is a still poorly understood process. Metabolic remodelling could induce transition from paroxysmal to persistent AF. Succinate levels are increased in AF patients indicating a potential role for metabolic remodelling. To assess the impact of the succinate pathway on the electrophysiological properties of right atria (RA) in persistent AF sheep model. Optical experiments were performed in ex vivo RA from a burst pacing AF sheep model. RA were perfused by Tyrode solution with glucose (5.6 mM), subsequently replaced by succinate (10 mM) to increase mitochondrial reactive oxygen species (ROS) production which are known to modulate excitation-contraction coupling (ECC) and study metabolic remodelling. An activator of the succinate β-adrenergic receptor (GPR91), cis-epoxysuccinic acid (300 μM), was used to study the GPR91 involvement in atrial electrophysiology and AF. Action potential duration at 80% of repolarization (APD80) were assessed from 2 to 5 Hz pacing frequency and during sinus rhythm (SR). We used an S1S2 pacing protocol to determine effective refractory period (ERP) and a burst pacing protocol (30 Hz) to assess ex vivo AF vulnerability. Finally, an organ donation program allowed us to investigate these properties in a human RA from an AF patient. During succinate perfusion SR is decreased in Sham (1,5 vs 1,2 Hz; P = 0,03), AF (1,5 vs 1 Hz; P = 0,003) and resistant (1,3 vs 1 Hz; P = 0,01) sheep, ERP is increased in AF sheep (184 vs 344 ms; P = 0,0007) and APD80 is increased in Sham (217 vs 264 ms; P = 0,0004) and AF sheep (195 vs 275 ms; P = 0,003). We also observed an increase in amplitude alternans which can induce re-entries and therefore be pro-arrhythmic. These results seems to be confirmed on human AF RA by increasing APD80 (257 vs 309 ms), ERP (260 vs 330 ms) and spontaneous arrhythmias. GPR91 activation led to a slowing of SR (1,7 vs 1,3 Hz; P = 0,03), a shortening of APD80 (200 vs 170 ms; P = 0,007) and an increase in spontaneous arrhythmias. A dysregulation of ECC could explain our results consistently with the ROS involvement and metabolic remodelling in AF. For the first time, we also demonstrated that GPR91 pathway is involved in atrial electrophysiology and could be involved in AF mechanisms. [ABSTRACT FROM AUTHOR]

Atrial Fibrillation (AF) stabilization depends on remodelling processes affecting the electrophysiological, metabolic, and structural properties of the atrial muscle. Develop an AF Sheep model to study the electrophysiogical, structural and bioenergetic remodelling of the cardiac tissue. An algorithm controlled pacemaker stimulating repeatedly the right atria at 10 Hz during 30 sec followed by 5 sec of sinus rhythm (SR) detection was used to induce AF. Its development was monitored by telemetry. Electrophysiological remodelling was studied in vivo by contact mapping and ex vivo by optical mapping. Structural remodelling was studied at the organ level using computed tomography (μCT) and at the mitochondrial level by electronic microscopy. Bioenergetic remodelling was evaluated on isolated mitochondria. AF sheep were kept 75 days before being studied. AF induction rate was 80%. AF developed and stabilized after 25 and 48 days, respectively. Animals remained less than 0.02 ± 0.03% in SR once AF was installed. In vivo contact mapping revealed a decreased voltage in AF atria compared to Sham. The highest AF frequencies were localised in the pulmonary veins. When compared to Sham, AF induced: (1) a 50% decrease of the atrial effective refractory periods; (2) a left atrial decrease in action potential duration; (3) an increase in atrial volumes; (4) Fat infiltration; (5) a decrease in mitochondrial cristae density; (6) a decrease in ATP synthesis rate; (7) an increased oxidation of the mitochondrial NAD(P)H pool; (9) an increased mitochondrial H2O2 emission, independently of the respiratory substrates used to fuel the electron transport chain. The AF Sheep model developed is characterized by occurrence of remodelling processes at different levels. Further studies will establish the time course of development of remodelling processes in order to improve AF therapeutics strategies by targeting atrial remodelling. [ABSTRACT FROM AUTHOR]

Introduction Cardiac arrhythmias are an important cause of morbidity and mortality. Optical mapping using voltage-sensitive dyes (VSDs) allows to study these disorders in isolated perfused tissues. Until recently, this technique remained limited to the myocardial surface due to the short excitation wavelengths of common VSDs. Using red excitation light allows to by-pass this problem, but availability of such dyes remains limited. Objective To develop new families of red-shifted VSDs and to characterize both their spectral and potentiometric properties. Method Three families of VSDs were designed. The spectral properties of each VSD was assessed by spectrofluorometry in Langendorff perfused rat hearts. The potentiometric properties were assessed through optical mapping in Langendorff perfused rat hearts. For spectrofluorometry, hearts were freely beating and systolic and diastolic pressures were continuously monitored. In optical mapping experiments hearts were mechanically arrested by supplementation of blebbistatin (10 μM). Results Out of the three families, one showed a strong shift in excitation and emission wavelengths towards the near-infrared. Within this family, three VSDs, with variations in the hydrophilic tail and charged carrier, were tested. Results showed that these VSDs have peak excitation above 500 nm and peak emission around 730 nm. Although two of these dyes showed a low potentiometric sensitivity (<1%), one VSD had a ΔF/F similar to conventional dyes (>5%) with a stable signal for over 2 hours after a bolus injection in the hearts. In mechanically arrested hearts, the VSDs had no impact on electrophysiological parameters. However, in beating hearts, the VSDs significantly increased diastolic pressure. Conclusion We have developed new red-shifted VSDs and validate their use in cardiac applications, which might allow optimizing optical mapping of cardiac arrhythmia dynamics in deeper tissue layers and/or in blood-perfused tissues. [ABSTRACT FROM AUTHOR]

Background The hexosamine biosynthetic pathway (HBP) is heightened in cardiomyopathies. We aimed at understanding how over-activation of HBP impacts on cardiac electrophysiology, and acts as a trigger or a substrate of cardiac arrhythmias. Methods Overactivation of the HBP by injection of glucosamine (GlcN 270mg/kg) was assessed under basal conditions and during beta-adrenergic stress on the myocardial energetic status (phosphocreatine to ATP ratio) of rats by 31 P nuclear magnetic resonance spectroscopy. Rat hearts were perfused ex vivo to evaluate the effects of the HBP activation (physiologically: glutamine 0.5mM – gln; overactivated: GlcN 1mM) on: cardiac function, MVO 2 , and arrhythmia score (AS) under basal (spontaneous arrhythmias: sAS) and pacing (pAS) conditions. At the cellular level (HL1 cells), HBP overactivation (GlcN) was assessed on conduction velocity characterized by micro-electrode arrays recordings, and properties of voltage gated ionic channels involved in cellular excitability and conduction by patch clamp experiments. Results In vivo GlcN did not modify PCr/ATP of the left ventricle under basal conditions. This ratio was significantly decreased during beta-adrenergic stress, while a similar increased heart rate was reported. When hearts were isolated and perfused ex vivo , GlcN induced a decrease of the heart rate and MVO 2 temporarily, and an increase of the sAS and pAS. Physiological activation of HBP with gln did not impact the ex vivo cardiac function and the sAS, but further increased the probability of atrial arrhythmias during pacing. In HL1 cells, GlcN reduced the rate of spontaneous depolarization, modified the direction and the velocity of the impulse propagation, and altered activation and inactivation properties of the sodium channel. Conclusion Acute HBP activation is associated with an increased vulnerability for arrhythmias and alteration of impulse conduction, suggesting its potential role in the atrial and ventricular arrhythmias. The author hereby declares no conflict of interest [ABSTRACT FROM AUTHOR]

Background Papillary muscles are an important source of ventricular tachycardia (VT). Yet little is known about the role of the right ventricular (RV) endocavity structure, the moderator band (MB). The aim of this study was to determine the characteristics of the MB that may predispose to arrhythmia substrates. Methods Ventricular wedge preparations with intact MBs were studied from humans (n=2) and sheep (n=15; 40-50 kg). RV endocardium was optically mapped, and electrical recordings were measured along the MB and septum. S1S2 pacing of the RV free wall, MB, or combined S1-RV S2-MB sites were assessed. Human (n=2) and sheep (n=4) MB tissue constituents were assessed histologically. Results The MB structure was remarkably organized as 2 excitable, yet uncoupled compartments of myocardium and Purkinje. In humans, action potential duration heterogeneity between MB and RV myocardium was found (324.6±12.0 versus 364.0±8.4 ms; P<0.0001). S1S2-MB pacing induced unidirectional propagation via MB myocardium, permitting sustained macroreentrant VT. In sheep, the incidence of VT for RV, MB, and S1-RV S2-MB pacing was 1.3%, 5.1%, and 10.3%. Severing the MB led to VT termination, confirming a primary arrhythmic role. Inducible preparations had shorter action potential duration in the MB than RV (259.3±45.2 versus 300.7±38.5 ms; P<0.05), whereas noninducible preparations showed no difference (312.0±30.3 versus 310.0±24.6 ms, respectively). Conclusions The MB presents anatomic and electrical compartmentalization between myocardium and Purkinje fibers, providing a substrate for macroreentry. The vulnerability to sustain VT via this mechanism is dependent on MB structure and action potential duration gradients between the RV free wall and MB. [ABSTRACT FROM AUTHOR]

Introduction We recently found the presence of a pro-arrhythmic electrophysiological substrate with increased collagen content and slow conduction in the healthy right ventricular outflow tract (RVOT). An epicardial layer of fat is commonly found in the RVOT but whether fat penetrates in the myocardium and regulates RVOT structure and electrophysiology remains unknown. Objective To assess the presence and localization of fat infiltrations (FI) in the healthy RVOT. Methods The presence of FI was assessed histologically in Masson's trichrome and Oil-Red-O stained sections from young healthy pigs ( n = 4) and human donor hearts with no cardiac disease history ( n = 5). High resolution MRI (9.4 T Bruker Biospin) was used to localize fat in formaldehyde-fixed human RVOTs. The expression of selected targets was assessed by qPCR and Western Blot in the subepicardial layer (EPI) of the RV free wall (RVFW) and RVOT of healthy pigs. Results Sporadic FIs could be observed in the pig RVOT but were absent in the RVFW. In human hearts, FIs were present in the RVFW, LV free wall but were larger in the RVOT. High resolution MRI showed the presence of 3 layers in the human RVOT wall with a thick epicardial fat layer, a transition layer composed of myocardial fibers and fat (up to 50% of wall thickness) and a muscular endocardial layer. Intramyocardial fat content increased with age. The mRNA expression of perilipin (PLIN1) and the adipogenic hormone adiponectin (ADIPOQ) was significantly higher in the RVOT EPI compared to the RVFW ( P < 0.05). Interestingly, Activin-A protein expression was significantly increased ( P < 0.05) in the RVOT compared to the RVFW EPI ( P < 0.05). Conclusion FIs are in direct contact with the adjacent myocardium in the healthy porcine and human RVOT. Activin-A is likely to contribute to the higher collagen content found in the healthy RVOT as observed in the atria. Thus, these infiltrations are likely to participate to RVOT slower conduction and arrhythmogenesis. [ABSTRACT FROM AUTHOR]

Background The investigation of single cardiac myocytes from healthy and diseased hearts of various species is a valuable tool to explore cardiac physio/ pathophysiology. The application of cell isolation to human donor tissue has been proofed to be difficult due to the limited amount of human tissue (mainly human right atrial appendages during cardiac surgery). Another limitation is the low viability of cardiomyocytes after isolation. In this study, we present a method to obtain single cardiac myocytes from different regions of human heart. Methods and results Human hearts rejected for transplantation were obtained from Bordeaux hospital. This protocol was approved by the Agence de la Biomédecine. Left atrial (LA) and ventricular (LV) myocytes were obtained by enzymatic dissociation. The ventricles and right atrium were removed and used for other studies (e.g. high resolution optical mapping). LA was cannulated by the circumflex artery and mounted into a Langendorff perfusion system after suture of the leaky atrial branches. LA was perfused with a Ca2+-free solution (~10 min), then collagenase and protease solution (0.08 mM Ca2+) and recirculated for ~25 min. Enzymes were washed out with a 0.2 mM Ca2+ solution. LA was separated into 4 regions: Endocardium, Epicardium, roof and pulmonary vein; LV myocytes were also obtained. Cells were re-suspended into a 1.8 mM Ca2+ solution by steps. Ca2+ transients were recorded (Fura-2, field stimulation) using an IonOptix system and cell membrane was stained with di-8 ANEPPS and visualized under confocal microscopy. Ca2+ tolerant myocytes were obtained from the 4 LA regions and LV. Human cardiac myocytes respond to electrical stimulation and Ca2+ transient can be recorded. Analysis of functional and structural data will be presented. Conclusion Isolation of single cardiac myocytes from human samples is a tedious task, but we present data showing reliable method to obtain functional and structural insights. The author hereby declares no conflict of interest [ABSTRACT FROM AUTHOR]

Background Calcium leak from the sarcoplasmic reticulum (SR) favors cardiac sudden death through a calcium overload mechanism. Here we investigate the crosstalk between the calcium overload SR-mediated and the mitochondrial permeability transition pore (mPTP) opening during a ventricular arrhythmic episode. Methods and results a burst pacing protocol was developed to induce ventricular arrhythmia in the perfused rat heart. Surface electrocardiogram, left ventricular pressure, and oxygen consumption were monitored. Epicardial fluorescence was recorded from either a mitochondrial membrane potential dye or an intracellular calcium dye. ECG signals were used to assess both arrhythmia complexity and duration. Hearts were perfused with tacrolimus (FK506) alone or in combination with cyclosporin A (CsA) to promote SR calcium leak or inhibit mPTP, respectively. Prior to arrhythmia induction, FK506 promoted mitochondrial depolarization and intracellular calcium overload in a CsA-sensitive manner. More than 90% of hearts developed complex ventricular arrhythmias in the FK506 group and this was accompanied by a strong mitochondrial membrane depolarization. In contrast, in the FK506 + CsA group, 100% of the hearts spontaneously defibrillate after a few seconds of arrhythmias. In this group, following arrhythmia induction, mitochondrial depolarization amplitude, intracellular calcium overload and heart efficiency depression was decreased in comparison to the FK506 group. Conclusions Here we suggest that the FK506-induced calcium overload promotes mitochondrial calcium accumulation and leads to mPTP opening. We propose that mPTP opening should be considered as an arrhythmogenic substrate. The author hereby declares no conflict of interest [ABSTRACT FROM AUTHOR]

Background: Changes in mitochondria have been implicated in atrial fibrillation (AF), but their manifestations and significance are poorly understood. Here, we studied changes in mitochondrial morphology and function during AF and assessed the effect of a mitochondrial-targeted intervention in a large animal model., Methods and Results: Atrial cardiomyocytes (ACMs) were isolated from dogs in electrically-driven AF for periods of 24 hours to 3 weeks and from humans with/without longstanding persistent AF. Mitochondrial Ca 2+ -concentration ([Ca 2+ ] Mito ), reactive oxygen species (mtROS) production, membrane potential (ΔΨ m ), permeability transition-pore (mPTP) opening and flavin adenine dinucleotide (FAD) were measured via confocal microscopy; nicotine adenine dinucleotide (NADH) under ultraviolet light. mtROS-production increased within 24 hours and superoxide-dismutase type-2 was significantly reduced from 3-day AF. [Ca 2+ ] Mito and mPTP-opening frequency/duration increased progressively during AF. Mitochondrial depolarization was detectable 24 hours after AF-onset. NADH increased by 15% at 24-hour AF, concomitant with increased pyruvate-dehydrogenase expression, then gradually decreased. Mitochondria enlarged and elongated at 24-hour and 3-day AF, followed by progressive fragmentation, rupture and shrinkage. Mitochondrial fusion protein-1 (MFN1) was reduced from 3-day to 3-week AF and phosphorylated dynamin-related protein-1 (p-DRP1ser-616) increased after 1 week of canine AF and in human AF. Addition of the mitochondrial antioxidant MitoTempo attenuated action-potential shortening and L-type Ca 2+ -current (I CaL )-downregulation in canine and human AF ACMs in vitro . Administration of the orally-active mitochondrial-targeted ubiquinone mitoquinone to dogs during 3-week AF prevented mitochondrial Ca 2+ -overload, mtROS-overproduction, structural damage and abnormalities in ΔΨ m and respiration. Functionally, mitoquinone reduced AF-induced Ca 2+ -current downregulation, action-potential abbreviation, contractile dysfunction and fibrosis, preventing AF-substrate development and AF-sustainability., Conclusions: Mitochondria show a series of changes during AF, with early hyperfunction and enhanced ROS-generation, followed by progressive damage and dysfunction. Mitochondrial-targeted therapy prevents mitochondrial dysfunction and attenuates adverse AF-related remodeling, positioning mitochondrial protection as a potential novel therapeutic target in AF.

Background and Purpose: Pulmonary hypertension (PH) is a cardiovascular disease characterised by an increase in pulmonary arterial (PA) resistance leading to right ventricular (RV) failure. Reactive oxygen species (ROS) play a major role in PH. OP2113 is a drug with beneficial effects on cardiac injuries that targets mitochondrial ROS. The aim of the study was to address the in vivo therapeutic effect of OP2113 in PH., Experimental Approach: PH was induced by 3 weeks of chronic hypoxia (CH-PH) in rats treated with OP2113 or its vehicle via subcutaneous osmotic mini-pumps. Haemodynamic parameters and both PA and heart remodelling were assessed. Reactivity was quantified in PA rings and in RV or left ventricular (LV) cardiomyocytes. Oxidative stress was detected by electron paramagnetic resonance and western blotting. Mitochondrial mass and respiration were measured by western blotting and oxygraphy, respectively., Key Results: In CH-PH rats, OP2113 reduced the mean PA pressure, PA remodelling, PA hyperreactivity in response to 5-HT, the contraction slowdown in RV and LV and increased the mitochondrial mass in RV. Interestingly, OP2113 had no effect on haemodynamic parameters, both PA and RV wall thickness and PA reactivity, in control rats. Whereas oxidative stress was evidenced by an increase in protein carbonylation in CH-PH, this was not affected by OP2113., Conclusion and Implications: Our study provides evidence for a selective protective effect of OP2113 in vivo on alterations in both PA and RV from CH-PH rats without side effects in control rats., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Cardiac alternans has been associated with an increased propensity to lethal tachyarrhythmias such as ventricular tachycardia and fibrillation (VT/VF). Myocardial infarction (MI), resulting from restricted oxygen supply to the heart, is a known substrate for VT/VF. Here, we investigate the utility of cardiac alternans as a predictor of tachyarrhythmias in a chronic MI ovine model. In-vivo electrophysiological studies were performed to assess the change in microvolt T-wave alternans (TWA) with induction of acute ischemia following coronary artery occlusion. 24-hour telemetry was performed in an ambulatory animal for 6 weeks to monitor the progression of TWA with chronic MI. At 6 weeks, ex-vivo optical mapping experiments were performed to assess the spatiotemporal evolution of alternans in sham (n=5) and chronic MI hearts (n=8). Our results demonstrate that chronic MI leads to significant electrophysiological changes in the cardiac substrate. Significant increase in TWA is observed post occlusion and a steady rise in alternans is seen with progression of chronic MI. Compared to sham, chronic MI hearts show significant presence of localized action potential amplitude alternans, which spatially evolve with an increase in pacing frequency. Clinical Relevance - Our results demonstrate that localized alternans underlie arrhythmogenesis in chronic MI hearts and microvolt TWA can serve as a biomarker of disease progression during chronic MI.

Structural remodeling is a common consequence of chronic pathological stresses imposed on the heart. Understanding the architectural and compositional properties of diseased tissue is critical to determine their interactions with arrhythmic behavior. Microscale tissue remodeling, below the clinical resolution, is emerging as an important source of lethal arrhythmia, with high prevalence in young adults. Challenges remain in obtaining high imaging contrast at sufficient microscale resolution for preclinical models, such as large mammalian whole hearts. Moreover, tissue composition-selective contrast enhancement for three-dimensional high-resolution imaging is still lacking. Non-destructive imaging using micro-computed tomography shows promise for high-resolution imaging. The objective was to alleviate sufferance from X-ray over attenuation in large biological samples. Hearts were extracted from healthy pigs (N = 2), and sheep (N = 2) with either induced chronic myocardial infarction and fibrotic scar formation or induced chronic atrial fibrillation. Excised hearts were perfused with: a saline solution supplemented with a calcium ion quenching agent and a vasodilator, ethanol in serial dehydration, and hexamethyldisilizane under vacuum. The latter reinforced the heart structure during air-drying for 1 week. Collagen-dominant tissue was selectively bound by an X-ray contrast-enhancing agent, phosphomolybdic acid. Tissue conformation was stable in air, permitting long-duration microcomputed tomography acquisitions to obtain high-resolution (isotropic 20.7 µm) images. Optimal contrast agent loading by diffusion showed selective contrast enhancement of the epithelial layer and sub-endocardial Purkinje fibers in healthy pig ventricles. Atrial fibrillation (AF) hearts showed enhanced contrast accumulation in the posterior walls and appendages of the atria, attributed to greater collagen content. Myocardial infarction hearts showed increased contrast selectively in regions of cardiac fibrosis, which enabled the identification of interweaving surviving myocardial muscle fibers. Contrast-enhanced air-dried tissue preparations enabled microscale imaging of the intact large mammalian heart and selective contrast enhancement of underlying disease constituents.

The current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of cellular respiration, undergo profound changes in post mortem tissues. The objective of this study was to demonstrate that mitochondrial activity constitutes a putative early fish freshness marker. The structure of gilthead sea bream (Sparus aurata) muscle tissue was evaluated over time by transmission electron microscopy. Respiration was assessed in mitochondria isolated from sea bream fillets using oxygraphy. Membrane potential (ΔΨ m ) was determined by fluorescence (Rhodamine 123). Mitochondrial activity of fillets stored at +4 °C was studied for 6 days. Changes in mitochondrial cristae structure appeared from Day 3 highlighting the presence of dense granules. ΔΨ m and mitochondrial activity were significantly disrupted in sea bream fillets after 96 h of storage at +4 °C. Mitochondrial activity constituted a reliable and early indicator of fish freshness., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Findings: Here, we demonstrate that OP2113 (5-(4-Methoxyphenyl)-3H-1,2-dithiole-3-thione, CAS 532-11-6), synthesized and used as a drug since 1696, does not act as an unspecific antioxidant molecule (i.e., as a radical scavenger) but unexpectedly decreases mitochondrial reactive oxygen species (ROS/H2O2) production by acting as a specific inhibitor of ROS production at the IQ site of complex I of the mitochondrial respiratory chain. Studies performed on isolated rat heart mitochondria also showed that OP2113 does not affect oxidative phosphorylation driven by complex I or complex II substrates. We assessed the effect of OP2113 on an infarct model of ex vivo rat heart in which mitochondrial ROS production is highly involved and showed that OP2113 protects heart tissue as well as the recovery of heart contractile activity., Conclusion / Significance: This work represents the first demonstration of a drug authorized for use in humans that can prevent mitochondria from producing ROS/H2O2. OP2113 therefore appears to be a member of the new class of mitochondrial ROS blockers (S1QELs) and could protect mitochondrial function in numerous diseases in which ROS-induced mitochondrial dysfunction occurs. These applications include but are not limited to aging, Parkinson's and Alzheimer's diseases, cardiac atrial fibrillation, and ischemia-reperfusion injury., Competing Interests: We report here that D.D. and P.D. are listed as inventors on pending and issued patent applications related to the molecule OP2113 described in this work (USA US62/215,215; France 15 184 217.6; France EP 17 159 691.9). P.D. is one of the founders of the OP2 drugs company. The funding sources had no role in the study design or the analysis and interpretation of data or in the decision to submit the article for publication. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

We critically assess the proposal that succinate-fuelled reverse electron flow (REF) drives mitochondrial matrix superoxide production from Complex I early in reperfusion, thus acting as a key mediator of ischemia/reperfusion (IR) injury. Real-time surface fluorescence measurements of NAD(P)H and flavoprotein redox state suggest that conditions are unfavourable for REF during early reperfusion. Furthermore, rapid loss of succinate accumulated during ischemia can be explained by its efflux rather than oxidation. Moreover, succinate accumulation during ischemia is not attenuated by ischemic preconditioning (IP) despite powerful cardioprotection. In addition, measurement of intracellular reactive oxygen species (ROS) during reperfusion using surface fluorescence and mitochondrial aconitase activity detected major increases in ROS only after mitochondrial permeability transition pore (mPTP) opening was first detected. We conclude that mPTP opening is probably triggered initially by factors other than ROS, including increased mitochondrial [Ca 2+ ]. However, IP only attenuates [Ca 2+ ] increases later in reperfusion, again after initial mPTP opening, implying that IP regulates mPTP opening through additional mechanisms. One such is mitochondria-bound hexokinase 2 (HK2) which dissociates from mitochondria during ischemia in control hearts but not those subject to IP. Indeed, there is a strong correlation between the extent of HK2 loss from mitochondria during ischemia and infarct size on subsequent reperfusion. Mechanisms linking HK2 dissociation to mPTP sensitisation remain to be fully established but several related processes have been implicated including VDAC1 oligomerisation, the stability of contact sites between the inner and outer membranes, cristae morphology, Bcl-2 family members and mitochondrial fission proteins such as Drp1., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mitochondrial permeability transition pore (mPTP) opening is critical for ischemia / reperfusion (I/R) injury and is associated with increased [Ca2+] and reactive oxygen species (ROS). Here we employ surface fluorescence to establish the temporal sequence of these events in beating perfused hearts subject to global I/R. A bespoke fluorimeter was used to synchronously monitor surface fluorescence and reflectance of Langendorff-perfused rat hearts at multiple wavelengths, with simultaneous measurements of hemodynamic function. Potential interference by motion artefacts and internal filtering was assessed and minimised. Re-oxidation of NAD(P)H and flavoproteins on reperfusion (detected using autofluorescence) was rapid (t0.5 < 15 s) and significantly slower following ischemic preconditioning (IP). This argues against superoxide production from reduced Complex 1 being a critical mediator of initial mPTP opening during early reperfusion. Furthermore, MitoPY1 (a mitochondria-targeted H2O2-sensitive fluorescent probe) and aconitase activity measurements failed to detect matrix ROS increases during early reperfusion. However, two different fluorescent cytosolic ROS probes did detect ROS increases after 2-3 min of reperfusion, which was shown to be after initiation of mPTP opening. Cyclosporin A (CsA) and IP attenuated these responses and reduced infarct size. [Ca2+]i (monitored with Indo-1) increased progressively during ischemia, but dropped rapidly within 90 s of reperfusion when total mitochondrial [Ca2+] was shown to be increased. These early changes in [Ca2+] were not attenuated by IP, but substantial [Ca2+] increases were observed after 2-3 min reperfusion and these were prevented by both IP and CsA. Our data suggest that the major increases in ROS and [Ca2+] detected later in reperfusion are secondary to mPTP opening. If earlier IP-sensitive changes occur that might trigger initial mPTP opening they are below our limit of detection. Rather, we suggest that IP may inhibit initial mPTP opening by alternative mechanisms such as prevention of hexokinase 2 dissociation from mitochondria during ischemia., Competing Interests: The authors have declared that no competing interests exist.

Optical mapping of Ca(2+)-sensitive fluorescence probes has become an extremely useful approach and adopted by many cardiovascular research laboratories to study a spectrum of myocardial physiology and disease conditions. Optical mapping data are often displayed as detailed pseudocolor images, providing unique insight for interpreting mechanisms of ectopic activity, action potential and Ca(2+) transient alternans, tachycardia, and fibrillation. Ca(2+)-sensitive fluorescent probes and optical mapping systems continue to evolve in the ongoing effort to improve therapies that ease the growing worldwide burden of cardiovascular disease. In this technical review we provide an updated overview of conventional approaches for optical mapping of Cai (2+) within intact myocardium. In doing so, a brief history of Cai (2+) probes is provided, and nonratiometric and ratiometric Ca(2+) probes are discussed, including probes for imaging sarcoplasmic reticulum Ca(2+) and probes compatible with potentiometric dyes for dual optical mapping. Typical measurements derived from optical Cai (2+) signals are explained, and the analytics used to compute them are presented. Last, recent studies using Cai (2+) optical mapping to study arrhythmias, heart failure, and metabolic perturbations are summarized., (Copyright © 2016 the American Physiological Society.)

Mitochondrial membrane potential is the major regulator of mitochondrial functions, including coupling efficiency and production of reactive oxygen species (ROS). Both functions are crucial for cell bioenergetics. We previously presented evidences for a specific modulation of adenine nucleotide translocase (ANT) appearing during aging that results in a decrease in membrane potential - and therefore ROS production-but surprisingly increases coupling efficiency under conditions of low ATP turnover. Careful study of the bioenergetic parameters (oxidation and phosphorylation rates, membrane potential) of isolated mitochondria from skeletal muscles (gastrocnemius) of aged and young rats revealed a remodeling at the level of the phosphorylation system, in the absence of alteration of the inner mitochondrial membrane (uncoupling) or respiratory chain complexes regulation. We further observed a decrease in mitochondrial affinity for ADP in aged isolated mitochondria, and higher sensitivity of ANT to its specific inhibitor atractyloside. This age-induced modification of ANT results in an increase in the ADP concentration required to sustain the same ATP turnover as compared to young muscle, and therefore in a lower membrane potential under phosphorylating-in vivo-conditions. Thus, for equivalent ATP turnover (cellular ATP demand), coupling efficiency is even higher in aged muscle mitochondria, due to the down-regulation of inner membrane proton leak caused by the decrease in membrane potential. In the framework of the radical theory of aging, these modifications in ANT function may be the result of oxidative damage caused by intra mitochondrial ROS and may appear like a virtuous circle where ROS induce a mechanism that reduces their production, without causing uncoupling, and even leading in improved efficiency. Because of the importance of ROS as therapeutic targets, this new mechanism deserves further studies.

Mitochondrial permeability transition pore (mPTP) opening plays a critical role in cardiac reperfusion injury and its prevention is cardioprotective. Tumour cell mitochondria usually have high levels of hexokinase isoform 2 (HK2) bound to their outer mitochondrial membranes (OMM) and HK2 binding to heart mitochondria has also been implicated in resistance to reperfusion injury. HK2 dissociates from heart mitochondria during ischaemia, and the extent of this correlates with the infarct size on reperfusion. Here we review the mechanisms and regulations of HK2 binding to mitochondria and how this inhibits mPTP opening and consequent reperfusion injury. Major determinants of HK2 dissociation are the elevated glucose-6-phosphate concentrations and decreased pH in ischaemia. These are modulated by the myriad of signalling pathways implicated in preconditioning protocols as a result of a decrease in pre-ischaemic glycogen content. Loss of mitochondrial HK2 during ischaemia is associated with permeabilization of the OMM to cytochrome c, which leads to greater reactive oxygen species production and mPTP opening during reperfusion. Potential interactions between HK2 and OMM proteins associated with mitochondrial fission (e.g. Drp1) and apoptosis (B-cell lymphoma 2 family members) in these processes are examined. Also considered is the role of HK2 binding in stabilizing contact sites between the OMM and the inner membrane. Breakage of these during ischaemia is proposed to facilitate cytochrome c loss during ischaemia while increasing mPTP opening and compromising cellular bioenergetics during reperfusion. We end by highlighting the many unanswered questions and discussing the potential of modulating mitochondrial HK2 binding as a pharmacological target., (© 2014 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of The British Pharmacological Society.)

Rationale: High-fat diet with obesity-associated co-morbidities triggers cardiac remodeling and renders the heart more vulnerable to ischemia/reperfusion injury. However, the effect of high-fat diet without obesity and associated co-morbidities is presently unknown., Objectives: To characterize a non-obese mouse model of high-fat diet, assess the vulnerability of hearts to reperfusion injury and to investigate cardiac cellular remodeling in relation to the mechanism(s) underlying reperfusion injury., Methods and Results: Feeding C57BL/6J male mice high-fat diet for 20 weeks did not induce obesity, diabetes, cardiac hypertrophy, cardiac dysfunction, atherosclerosis or cardiac apoptosis. However, isolated perfused hearts from mice fed high-fat diet were more vulnerable to reperfusion injury than those from mice fed normal diet. In isolated cardiomyocytes, high-fat diet was associated with higher diastolic intracellular Ca2+ concentration and greater damage to isolated cardiomyocytes following simulated ischemia/reperfusion. High-fat diet was also associated with changes in mitochondrial morphology and expression of some related proteins but not mitochondrial respiration or reactive oxygen species turnover rates. Proteomics, western blot and high-performance liquid chromatography techniques revealed that high-fat diet led to less cardiac oxidative stress, higher catalase expression and significant changes in expression of putative components of the mitochondrial permeability transition pore (mPTP). Inhibition of the mPTP conferred relatively more cardio-protection in the high-fat fed mice compared to normal diet., Conclusions: This study shows for the first time that high-fat diet, independent of obesity-induced co-morbidities, triggers changes in cardiac oxidative state, calcium handling and mitochondria which are likely to be responsible for increased vulnerability to cardiac insults.

With aging, most skeletal muscles undergo a progressive loss of mass and strength, a process termed sarcopenia. Aging-related defects in mitochondrial energetics have been proposed to be causally involved in sarcopenia. However, changes in muscle mitochondrial oxidative phosphorylation with aging remain a highly controversial issue, creating a pressing need for integrative approaches to determine whether mitochondrial bioenergetics are impaired in aged skeletal muscle. To address this issue, mitochondrial bioenergetics was first investigated in vivo in the gastrocnemius muscle of adult (6 months) and aged (21 months) male Wistar rats by combining a modular control analysis approach with (31) P magnetic resonance spectroscopy measurements of energetic metabolites. Using this innovative approach, we revealed that the in vivo responsiveness ('elasticity') of mitochondrial oxidative phosphorylation to contraction-induced increase in ATP demand is significantly reduced in aged skeletal muscle, a reduction especially pronounced under low contractile activities. In line with this in vivo aging-related defect in mitochondrial energetics, we found that the mitochondrial affinity for ADP is significantly decreased in mitochondria isolated from aged skeletal muscle. Collectively, the results of this study demonstrate that mitochondrial bioenergetics are effectively altered in vivo in aged skeletal muscle and provide a novel cellular basis for this phenomenon., (© 2013 the Anatomical Society and John Wiley & Sons Ltd.)