Langendorff-perfused Rat Hearts Research Articles

MC225, a Novel Probe for P-glycoprotein PET Imaging at the Blood-brain Barrier: In Vitro Cardiovascular Safety Evaluation.

The P-glycoprotein (P-gp) substrate MC225, at concentrations ≤10 nM, is a valuable radiotracer for positron emission tomography imaging of P-gp function in rats and mice. The aim of this study was to evaluate its potential toxic hazard toward the cardiovascular system through an in-depth analysis of its effects on rat aorta rings, on CaV1.2 channel current (ICa1.2) of A7r5 cells and on Langendorff-perfused rat heart. In aortic rings, MC225 relaxed phenylephrine-induced contraction in a concentration-dependent and endothelium-independent manner, with an IC50 value of about 1 μM. At concentrations ≥3 μM, it antagonized the response to cumulative concentrations of K. MC225, 1 and 10 μM, inhibited ICa1.2 by 15% and 31%, respectively, without affecting either current activation or inactivation kinetics. In Langendorff-perfused rat hearts, only 10 μM MC225 significantly decreased left ventricular pressure and increased coronary perfusion pressure while reducing heart rate and prolonging the cardiac cycle length as well as the atrioventricular conduction time (PQ interval) on the electrocardiogram. Lower concentrations of the drug were ineffective. These findings demonstrate that MC225-induced cardiovascular effects took place at concentrations that are at least 2 orders of magnitude higher than those allowing in vivo measurement of P-gp function. Therefore, MC225 represents a promising positron emission tomography tool for in vivo straightforward P-gp quantification.

Phoenixin-14: detection and novel physiological implications in cardiac modulation and cardioprotection.

Phoenixin-14 (PNX) is a newly identified peptide co-expressed in the hypothalamus with the anorexic and cardioactive Nesfatin-1. Like Nesfatin-1, PNX is able to cross the blood-brain barrier and this suggests a role in peripheral modulation. Preliminary mass spectrography data indicate that, in addition to the hypothalamus, PNX is present in the mammalian heart. This study aimed to quantify PNX expression in the rat heart, and to evaluate whether the peptide influences the myocardial function under basal condition and in the presence of ischemia/reperfusion (I/R). By ELISA the presence of PNX was detected in both hypothalamus and heart. In plasma of normal, but not of obese rats, the peptide concentrations increased after meal. Exposure of the isolated and Langendorff perfused rat heart to exogenous PNX induces a reduction of contractility and relaxation, without effects on coronary pressure and heart rate. As revealed by immunoblotting, these effects were accompanied by an increase of Erk1/2, Akt and eNOS phosphorylation. PNX (EC50 dose), administered after ischemia, induced post-conditioning-like cardioprotection. This was revealed by a smaller infarct size and a better systolic recovery with respect to those detected on hearts exposed to I/R alone. The peptide also activates the cardioprotective RISK and SAFE cascades and inhibits apoptosis. These effects were also observed in the heart of obese rats. Our data provide a first evidence on the peripheral activity of PNX and on its direct cardiomodulatory and cardioprotective role under both normal conditions and in the presence of metabolic disorders.

Ursodeoxycholic acid prevents ventricular conduction slowing and arrhythmia by restoring T-type calcium current in fetuses during cholestasis.

BackgroundIncreased maternal serum bile acid concentrations in intrahepatic cholestasis of pregnancy (ICP) are associated with fetal cardiac arrhythmias. Ursodeoxycholic acid (UDCA) has been shown to demonstrate anti-arrhythmic properties via preventing ICP-associated cardiac conduction slowing and development of reentrant arrhythmias, although the cellular mechanism is still being elucidated.MethodsHigh-resolution fluorescent optical mapping of electrical activity and electrocardiogram measurements were used to characterize effects of UDCA on one-day-old neonatal and adult female Langendorff-perfused rat hearts. ICP was modelled by perfusion of taurocholic acid (TC, 400μM). Whole-cell calcium currents were recorded from neonatal rat and human fetal cardiomyocytes.ResultsTC significantly prolonged the PR interval by 11.0±3.5% (P<0.05) and slowed ventricular conduction velocity (CV) by 38.9±5.1% (P<0.05) exclusively in neonatal and not in maternal hearts. A similar CV decline was observed with the selective T-type calcium current (ICa,T) blocker mibefradil 1μM (23.0±6.2%, P<0.05), but not with the L-type calcium current (ICa,L) blocker nifedipine 1μM (6.9±6.6%, NS). The sodium channel blocker lidocaine (30μM) reduced CV by 60.4±4.5% (P<0.05). UDCA co-treatment was protective against CV slowing induced by TC and mibefradil, but not against lidocaine. UDCA prevented the TC-induced reduction in the ICa,T density in both isolated human fetal (−10.2±1.5 versus −5.5±0.9 pA/pF, P<0.05) and neonatal rat ventricular myocytes (−22.3±1.1 versus −9.6±0.8 pA/pF, P<0.0001), whereas UDCA had limited efficacy on the ICa,L.ConclusionOur findings demonstrate that ICa,T plays a significant role in ICP-associated fetal cardiac conduction slowing and arrhythmogenesis, and is an important component of the fetus-specific anti-arrhythmic activity of UDCA.

Angiotensin II-preconditioning is associated with increased PKCε/PKCδ ratio and prosurvival kinases in mitochondria.

Angiotensin II-preconditioning (APC) has been shown to reproduce the cardioprotective effects of ischaemic preconditioning (IPC), however, the molecular mechanisms mediating the effects of APC remain unknown. In this study, Langendorff-perfused rat hearts were subjected to IPC, APC or both (IPC/APC) followed by ischaemia-reperfusion (IR), to determine translocation of PKCε, PKCδ, Akt, Erk1/2, JNK, p38 MAPK and GSK-3β to mitochondria as an indicator of activation of the protein kinases. In agreement with previous observations, IPC, APC and IPC/APC increased the recovery of left ventricular developed pressure (LVDP), reduced infarct size (IS) and lactate dehydrogenase (LDH) release, compared to controls. These effects were associated with increased mitochondrial PKCε/PKCδ ratio, Akt, Erk1/2, JNK, and inhibition of permeability transition pore (mPTP) opening. Chelerythrine, a pan-PKC inhibitor, abolished the enhancements of PKCε but increased PKCδ expression, and inhibited Akt, Erk1/2, and JNK protein levels. The drug had no effect on the APC- and IPC/APC-induced cardioprotection as previously reported, but enhanced the post-ischaemic LVDP in controls. Losartan, an angiotensin II type 1 receptor (AT1-R) blocker, abolished the APC-stimulated increase of LVDP and reduced PKCε, Akt, Erk1/2, JNK, and p38. Both drugs reduced ischaemic contracture and LDH release, and abolished the inhibition of mPTP by the preconditioning. Chelerythrine also prevented the reduction of IS by APC and IPC/APC. These results suggest that the cardioprotection induced by APC and IPC/APC involves an AT1-R-dependent translocation of PKCε and survival kinases to the mitochondria leading to mPTP inhibition. In chelerythrine-treated hearts, however, alternate mechanisms appear to maintain cardiac function.

MiR-125a, miR-139 and miR-324 contribute to Urocortin protection against myocardial ischemia-reperfusion injury

Urocortin 1 and 2 (Ucn-1 and Ucn-2) have established protective actions against myocardial ischemia-reperfusion (I/R) injuries. However, little is known about their role in posttranscriptional regulation in the process of cardioprotection. Herein, we investigated whether microRNAs play a role in urocortin-induced cardioprotection. Administration of Ucn-1 and Ucn-2 at the beginning of reperfusion significantly restored cardiac function, as evidenced ex vivo in Langendorff-perfused rat hearts and in vivo in rat subjected to I/R. Experiments using microarray and qRT-PCR determined that the addition of Ucn-1 at reperfusion modulated the expression of several miRNAs with unknown role in cardiac protection. Ucn-1 enhanced the expression of miR-125a-3p, miR-324-3p; meanwhile it decreased miR-139-3p. Similarly, intravenous infusion of Ucn-2 in rat model of I/R mimicked the effect of Ucn-1 on miR-324-3p and miR-139-3p. The effect of Ucn-1 involves the activation of corticotropin-releasing factor receptor-2, Epac2 and ERK1/2. Moreover, the overexpression of miR-125a-3p, miR-324-3p and miR-139-3p promoted dysregulation of genes expression involved in cell death and apoptosis (BRCA1, BIM, STAT2), in cAMP and Ca2+ signaling (PDE4a, CASQ1), in cell stress (NFAT5, XBP1, MAP3K12) and in metabolism (CPT2, FoxO1, MTRF1, TAZ). Altogether, these data unveil a novel role of urocortin in myocardial protection, involving posttranscriptional regulation with miRNAs.

\u03b2 adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca2+ release by tentacle extract from the jellyfish Cyanea capillata

BackgroundIntracellular Ca2+ overload induced by extracellular Ca2+ entry has previously been confirmed to be an important mechanism for the cardiotoxicity as well as the acute heart dysfunction induced by jellyfish venom, while the underlying mechanism remains to be elucidated.MethodsUnder extracellular Ca2+-free or Ca2+-containing conditions, the Ca2+ fluorescence in isolated adult mouse cardiomyocytes pre-incubated with tentacle extract (TE) from the jellyfish Cyanea capillata and β blockers was scanned by laser scanning confocal microscope. Then, the cyclic adenosine monophosphate (cAMP) concentration and protein kinase A (PKA) activity in primary neonatal rat ventricular cardiomyocytes were determined by ELISA assay. Furthermore, the effect of propranolol against the cardiotoxicity of TE was evaluated in Langendorff-perfused rat hearts and intact rats.ResultsThe increase of intracellular Ca2+ fluorescence signal by TE was significantly attenuated and delayed when the extracellular Ca2+ was removed. The β adrenergic blockers, including propranolol, atenolol and esmolol, partially inhibited the increase of intracellular Ca2+ in the presence of 1.8 mM extracellular Ca2+ and completely abolished the Ca2+ increase under an extracellular Ca2+-free condition. Both cAMP concentration and PKA activity were stimulated by TE, and were inhibited by the β adrenergic blockers. Cardiomyocyte toxicity of TE was antagonized by β adrenergic blockers and the PKA inhibitor H89. Finally, the acute heart dysfuction by TE was antagonized by propranolol in Langendorff-perfused rat hearts and intact rats.ConclusionsOur findings indicate that β adrenergic receptor/cAMP/PKA signaling contributes to the intracellular Ca2+ overload through intracellular Ca2+ release by TE from the jellyfish C. capillata.

Abstract 431: Upregulation of Small Conductance Ca 2+ Activated K + Channels Promotes Formation of Mitochondrial Supercomplexes and Increases Respiration Reserve

Introduction: Small conductance calcium (Ca 2+ )-activated K + (SK) channels are present in the inner mitochondria membrane of ventricular cardiomyocytes. We recently reported that pharmacological enhancement of mito-SKs protects from Ca 2+ dependent arrhythmia by reducing production of reactive oxygen species (ROS) by mitochondria thereby normalizing oxidation state and activity of Ca 2+ release channels ryanodine receptors in hypertrophic rat hearts. Recent advances demonstrate that respiratory components of the mitochondrial electron transport chain can organize into supercomplexes (SCs) resulting in more efficient electron transfer and reduced generation of ROS. Hypothesis: Functional upregulation of mito-SK channels protects from Ca 2+ -dependent arrhythmia via promotion of mitochondrial SC formation and improved respiration. Methods: H9C2 myoblasts were infected with adenoviral vectors to overexpress SK2 and Cox7rp. After 48 hours in culture, cell respiration was measured using Seahorse XFe Analyzer; ROS was measured using general ROS indicator CFDA; and mitochondria were isolated to assess SC formation using Blue-Native electrophoresis (BN-PAGE). The effects of SK enhancer NS309 (2 μM, 10 min.) on SC formation were studied in Langendorff perfused rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Results: rSK2 overexpression in H9C2s reduced intracellular ROS ~ 30%, enhanced maximum respiration rate vs. controls (0.74±0.04 and 0.58±0.03 pM/min/μg respectively, p&lt;0.05); and caused a robust shift (&gt;2 fold) of complex IV towards large mitochondrial SCs (~1.2 mDa). As a control, similar results were obtained with overexpression of Cox7rp protein that promotes SC formation. In addition, BN-PAGE experiments in isolated rat hearts demonstrated that robust decrease in high MW Complexes I and IV in hypertrophy was fully restored to control levels upon treatment with SK enhancer NS309. Conclusion: Upregulation of SK channels reduces mito-ROS by an increase in respiration reserve which is linked to enhanced mitochondrial SC formation. Our data suggest that targeting mito-SK channels has therapeutic potential to treat cardiac arrhythmia in conditions accompanied by oxidative stress.

Cardiovascular Effects of High-Molecular-Weight Compounds of Humic Nature.

The active ingredient extracted from the peat humic substances was characterized by physicochemical parameters evaluated by UV- and IR-spectroscopy, titration, and elemental (C, H, N) analysis. The cardiovascular effects of this ingredient were examined on isolated Langendorff-perfused rat heart. It was found that the active substance in a concentration range of 0.01-0.1 mg/ml produced a vasodilating effect; in addition, it decreased the end-diastolic and left-ventricular developed pressures.

The IK1/Kir2.1 channel agonist zacopride prevents and cures acute ischemic arrhythmias in the rat.

Arrhythmogenesis in acute myocardial infarction (MI) is associated with depolarization of resting membraine potential (RMP) and decrease of inward rectifier potassium current (IK1) in cardiomyocytes. However, clinical anti-arrhythmic agents that primarily act on RMP by enhancing the IK1 channel are not currently available. We hypothesized that zacopride, a selective and moderate agonist of the IK1/Kir2.1 channels, prevents and cures acute ischemic arrhythmias. To test this viewpoint, adult Sprague-Dawley (SD) rats were subjected to MI by ligating the left main coronary artery. The antiarrhythmic effects of zacopride (i.v. infusion) were observed in the settings of pre-treatment (zacopride given 3 min prior to coronary occlusion), post-treatment (zacopride given 3 min after coronary occlusion) and therapeutic treatment (zacopride given 30 s after the onset of the first sustained ventricular tachycardia (VT)/ventricular fibrillation (VF) post MI). In all the three treatment modes, zacopride (15 μg/kg) inhibited MI-induced ventricular tachyarrhythmias, as shown by significant decreases in the premature ventricular contraction (PVC) and the duration and incidence of VT or VF. In Langendorff perfused rat hearts, the antiarrhythmic effect of 1 μmol/L zacopride were reversed by 1 μmol/L BaCl2, a blocker of IK1 channel. Patch clamp results in freshly isolated rat ventricular myocytes indicated that zacopride activated the IK1 channel and thereby reversed hypoxia-induced RMP depolarization and action potential duration (APD) prolongation. In addition, zacopride (1 μmol/L) suppressed hypoxia- or isoproterenol- induced delayed afterdepolarizations (DADs). In Kir2.x transfected Chinese hamster ovary (CHO) cells, zacopride activated the Kir2.1 homomeric channel but not the Kir2.2 or Kir2.3 channels. These results support our hypothesis that moderately enhancing IK1/Kir2.1 currents as by zacopride rescues ischemia- and hypoxia- induced RMP depolarization, and thereby prevents and cures acute ischemic arrhythmias. This study brings a new viewpoint to antiarrhythmic theories and provides a promising target for the treatment of acute ischemic arrhythmias.

Hydrogen Sulfide Improves Cardiomyocyte Function in a Cardiac Arrest Model.

BackgroundCardioplegic arrest is a common procedure for many types of cardiac surgery, and different formulations have been proposed to enhance its cardio-protective effect. Hydrogen sulfide is an important signaling molecule that has cardio-protective properties. We therefore studied the cardio-protective effect of hydrogen sulfide in cardiac cell culture and its potential therapeutic use in combination with cardioplegia formulations.Materials/MethodsWe added hydrogen sulfide donor GYY4137 to HL-1 cells to study its protective effect in nutrient starved conditions. In addition, we tested the potential use of GYY4137 when it is added into two different cardioplegia formulations: Cardi-Braun® solution and del Nido solution in an ex vivo Langendorff perfused rat hearts model.ResultsWe observed that eight-hour pre-treatment with GYY4137 significantly suppressed apoptosis in nutrient-starved HL-1 cells (28% less compared to untreated cells; p<0.05), maintained ATP content, and reduced protein synthesis. In ex vivo experiments, Cardi-Braun® and del Nido cardioplegia solutions supplemented with GYY4137 significantly reduced the pro-apoptotic protein caspase-3 content and preserved ATP content. Furthermore, GYY4137 supplemented cardioplegia solutions decreased the S-(5-adenosyl)-L-methionine/S-(adenosyl)-L-homocysteine ratio, reducing the oxidative stress in cardiac tissue. Finally, heart beating analysis revealed the preservation of the inter-beat interval and the heart rate in del Nido cardioplegia solution supplemented with GYY4137.ConclusionsGYY4137 preconditioning preserved energetic state during starved conditions, attenuating the cardiomyocytes apoptosis in vitro. The addition of GYY4137 to cardioplegia solutions prevented apoptosis, ATP consumption, and oxidative stress in perfused rat hearts, restoring its electrophysiological status after cardiac arrest. These findings suggested that GYY4137 sulfide donor may improve the cardioplegia solution performance during cardiac surgery.

A NOVEL NECROPTOSIS INHIBITORS (NECROSULFONAMIDE AND NECROSTATIN-1S) DEMONSTRATE THERAPEUTIC POTENTIAL FOR MYOCARDIAL ISCHEMIA-REPERFUSION INJURY

Background: We investigated the effect of novel highly active necroptosis inhibitors, necrosulfonamide (NSA) and necrostatin-1s (Nec-1s), on myocardial tolerance to ischemia-reperfusion injury (IRI). Methods: The isolated Langendorff-perfused rat hearts were subjected to 35-min global ischemia

Label-free detection of myocardial ischaemia in the perfused rat heart by spontaneous Raman spectroscopy

Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the “stopped-flow,” global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm−1, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm−1 enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.

Properties of New Voltage Sensitive Dyes in Cardiac Field

These last years, combination of high spatiotemporal resolution techniques with the development of fluorescent voltage sensitive dyes (VSDs) have significantly improved our understandings of electrophysiological functions of electrically excitable organs (heart, brain). These dyes allow to measure simultaneously membrane potential changes from multiple sites of biological preparations in a non-invasive way. However, conventional dyes have excitation spectra in the blue/green range, which limits the depth of penetration due to high absorbance and photon scattering properties. Therefore, the development of longer wavelength VSDs could have many advantages namely: action potentials recording from tissue's deeper layers, enhanced voltage sensitivity, and improved spectral properties such that simultaneous monitoring of multiple parameters (i.e. ion transient, pH).The goal of our study is to develop and characterize novel VSDs with large Stoke shifts and near-infrared spectral properties. Three families of VSDs were thus synthesized from two heterocyclic fluorophores and their properties (spectral properties, membrane staining, and dye phototoxicity) assessed in isolated ventricular cells and in Langendorff perfused rat hearts. In addition, special attention is paid to Signal:background ratios and signal kinetics of these new dyes by epifluorescence imaging.Optimal peak emission wavelengths for all dyes was found between 700 nm and 750 nm. The largest observed Stoke shift was found with fluorophore 1 family (about 250 nm). Signal:background ratios ranged from 3% (fluorophore 2) to 7% (fluorophore 3), compared to 5.5% for the usual conventional dye, Di-4-ANEPPS.Rapid signal decay indicative of internalization was observed for one VSD family. However, in other two, signal stability was improved compared to Di-4-ANEPPS. Overall, dyes made with fluorophore 3 show improved voltage sensitivity and potential for applications.

HEMO2life as a protective additive to Celsior solution for static storage of donor hearts prior to transplantation

Background: Prior to heart transplantation, static storage of donor hearts is currently limited to 4–5 h, despite profound hypothermia (4–8 °C). Because heart transplantation is an emergency procedure, improved protection to extend safe storage duration would be advantageous. We investigated whether the naturally respiratory pigment HEMO2life®, which is effective at hypothermia for the passive release of oxygen via oxygen gradient, could improve long-term preservation.Methods: Isolated Langendorff-perfused rat hearts (n = 12/group) were equilibrated (20 min) and function (left ventricular developed pressure: LVDP) measured by intraventricular balloon before arrest with cold (7.5 °C) Celsior® solution, either alone (control) or with the addition of HEMO2life® (Hemarina SA, Morlaix, France) at 1 g/L. Cold storage lasted 8 h prior to reperfusion (60 min) and recovery (as % of pre-ischemic function) was assessed. Hearts (minced and homogenized) were also assessed by TTC staining as a measure of viability and two hearts from each group were sliced and assessed by TTC staining for infarct size. Values are expressed as mean ± standard error of mean and analyzed by Student’s t-test.Results: Hearts recovered rapidly in both groups to a plateau by 20 min of reperfusion; control and HEMO2life® final recovery (60 min) was 45 ± 2% and 57 ± 1% (P < 0.05) respectively. Left ventricular end-diastolic pressure recovered to a similar extent in both groups (between 31 to 35 mmHg), as did heart rate (final recovery between 84 to 89% pre-ischemic value); however, coronary flow was significantly (P < 0.05) higher in HEMO2life® group (7.5 ± 0.7 ml/min) compared to control (5.4 ± 0.4 ml/min). Viability and infarct size measurements were similar between groups.Conclusion: The addition of the natural oxygen releasing pigment HEMO2life® to Celsior® preservation solution significantly improved post-ischemic recovery of heart function. This additive may have major therapeutic potential for clinical heart transplantation.

Acute Intravenous Infusion of Immunoglobulins Protects Against Myocardial Ischemia-Reperfusion Injury Through Inhibition of Caspase-3

Background/Aims: To investigate the cardioprotective effects of intravenous immunoglobulins (IVIG) in rats subjected to regional myocardial ischemia reperfusion (I/R). Methods: Langendorff-perfused rat hearts were used in this study. Hearts subjected to regional ischemia served as a negative untreated control. The effects of IVIG pre- and post-ischemic treatment on left ventricular function, coronary vascular dynamics and contractility were assessed. IVIG were administered in either a low or high dose. The infarct size was determined using triphenyltetrazolium chloride and through biochemical assays using the measured creatine kinase and lactate dehydrogenase levels. Apoptosis was evaluated by the TUNEL assay, and the caspase-3 expression level was assessed by immunoblotting. The cytokine levels were measured by ELISA. Results: Low and high doses of immunoglobulins administered 2 hours before sacrifice, before the ischemic insult or at reperfusion resulted in a significant improvement in cardiac hemodynamics, coronary vascular dynamics and heart contractility. A significant decrease in the infarct size and cardiac enzymes was also evident compared to those in the control. IVIG administered as an infusion at reperfusion or pre-treatment resulted in a marked decrease in myocyte apoptosis, which was associated with decreased levels of caspase-3 expression in the supernatants of homogenized left ventricles. Infusion of IVIG both pre-ischemia and at reperfusion did not show the same protective effects. Conclusions: This study demonstrates a novel protection to the heart by low and high doses of IVIG given either pre- or post-ischemia.

Real-Time Fluorescence Measurements of ROS and [Ca2+] in Ischemic / Reperfused Rat Hearts: Detectable Increases Occur only after Mitochondrial Pore Opening and Are Attenuated by Ischemic Preconditioning.

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.

Elucidating Mitochondrial Electron Transport Chain Supercomplexes in the Heart During Ischemia–Reperfusion

Mitochondrial supercomplexes (SCs) are the large supramolecular assembly of individual electron transport chain (ETC) complexes that apparently provide highly efficient ATP synthesis and reduce electron leakage and reactive oxygen species (ROS) production. Oxidative stress during cardiac ischemia-reperfusion (IR) can result in degradation of SCs through oxidation of cardiolipin (CL). Also, IR induces calcium overload and enhances reactive oxygen species (mitROS) in mitochondria that result in the opening of the nonselective permeability transition pores (PTP). The opening of the PTP further compromises cellular energetics and increases mitROS ultimately leading to cell death. Here, we examined the role of PTP-induced mitROS in disintegration of SCs during cardiac IR. The relationship between mitochondrial PTP, ROS, and SCs was investigated using Langendorff-perfused rat hearts subjected to global ischemia (25 min) followed by short-time (5 min) or long-time (60 min) reperfusion in the presence or absence of the PTP inhibitor, sanglifehrin A (SfA), and the mitochondrial targeted ROS and electron scavenger, XJB-5-131. Also, the effects of CL deficiency on SC degradation, PTP, and mitROS were investigated in tafazzin knockdown (TazKD) mice. Cardiac IR induced PTP opening and mitROS generation, inhibited by SfA. Percent distributions of SCs were significantly affected by IR, and the effects were dependent on the reperfusion time and reversed by SfA and XJB-5-131. TazKD mice demonstrated a 40% lower SC I + III+IV with reduced basal mitochondrial PTP, ROS, and ETC complex activity. Innovation and Conclusion: Sustained reperfusion after cardiac ischemia induces disintegration of mitochondrial SCs, and PTP-induced ROS presumably play a causal role in SC disassembly. Antioxid. Redox Signal. 27, 57-69.

T1 High glucose and myocardial ischaemia/reperfusion injury: the role of the sodium/glucose transporter, SGLT1

Background Hyperglycaemia, frequently found at the time of acute myocardial infarction, is associated with excess cardiovascular mortality. The sodium-glucose transporter SGLT1 is present in mammalian myocardium, but its role in acute ischaemia/reperfusion injury is currently unknown. We hypothesise high glucose at reperfusion increases infarct size following injurious ischaemia and this excess injury can be ameliorated by inhibition of SGLT1. Methods and results Langendorff-perfused Sprague-Dawley rat (SDR) heart were subjected to 35 min regional ischaemia and 60min variable glucose (3.5–22 mmol/L) reperfusion, with osmolality controlled by reciprocal dose mannitol. Reperfusion High Glucose (RHG) significantly dose-dependently increased infarct size (45 to 65%, p Discussion and conclusion We demonstrate for the first time that high glucose at reperfusion exacerbates myocardial injury following lethal myocardial ischaemia that can be ameliorated by inhibition of SGLT, which appears not directly targeted at cardiomyocytes, but rather non-myocyte myocardial cell populations. These data therefore provide the first data for a novel repurposing of SGLT inhibitors in non-diabetic patients presenting with acute coronary syndromes complicated by hyperglycaemia, and provides a tantalising insight into the mechanisms of hyperglycaemic-driven excess cardiovascular mortality.

Lebetin 2, a Snake Venom-Derived Natriuretic Peptide, Attenuates Acute Myocardial Ischemic Injury through the Modulation of Mitochondrial Permeability Transition Pore at the Time of Reperfusion.

Cardiac ischemia is one of the leading causes of death worldwide. It is now well established that natriuretic peptides can attenuate the development of irreversible ischemic injury during myocardial infarction. Lebetin 2 (L2) is a new discovered peptide isolated from Macrovipera lebetina venom with structural similarity to B-type natriuretic peptide (BNP). Our objectives were to define the acute cardioprotective actions of L2 in isolated Langendorff-perfused rat hearts after regional or global ischemia-reperfusion (IR). We studied infarct size, left ventricular contractile recovery, survival protein kinases and mitochondrial permeability transition pore (mPTP) opening in injured myocardium. L2 dosage was determined by preliminary experiments at its ability to induce cyclic guanosine monophosphate (cGMP) release without changing hemodynamic effects in normoxic hearts. L2 was found to be as effective as BNP in reducing infarct size after the induction of either regional or global IR. Both peptides equally improved contractile recovery after regional IR, but only L2 increased coronary flow and reduced severe contractile dysfunction after global ischemia. Cardioprotection afforded by L2 was abolished after isatin or 5-hydroxydecanote pretreatment suggesting the involvement of natriuretic peptide receptors and mitochondrial KATP (mitoKATP) channels in the L2-induced effects. L2 also increased survival protein expression in the reperfused myocardium as evidenced by phosphorylation of signaling pathways PKCε/ERK/GSK3β and PI3K/Akt/eNOS. IR induced mitochondrial pore opening, but this effect was markedly prevented by L2 treatment. These data show that L2 has strong cardioprotective effect in acute ischemia through stimulation of natriuretic peptide receptors. These beneficial effects are mediated, at least in part, by mitoKATP channel opening and downstream activated survival kinases, thus delaying mPTP opening and improving IR-induced mitochondrial dysfunction.

The novel H2S-donor 4-carboxyphenyl isothiocyanate promotes cardioprotective effects against ischemia/reperfusion injury through activation of mitoKATP channels and reduction of oxidative stress

The endogenous gasotransmitter hydrogen sulphide (H2S) is an important regulator of the cardiovascular system, particularly of myocardial function. Moreover, H2S exhibits cardioprotective activity against ischemia/reperfusion (I/R) or hypoxic injury, and is considered an important mediator of “ischemic preconditioning”, through activation of mitochondrial potassium channels, reduction of oxidative stress, activation of the endogenous “anti-oxidant machinery” and limitation of inflammatory responses. Accordingly, H2S-donors, i.e. pro-drugs able to generate exogenous H2S, are viewed as promising therapeutic agents for a number of cardiovascular diseases. The novel H2S-donor 4-carboxy phenyl-isothiocyanate (4CPI), whose vasorelaxing effects were recently reported, was tested here in different experimental models of myocardial I/R.In Langendorff-perfused rat hearts subjected to I/R, 4CPI significantly improved the post-ischemic recovery of myocardial functional parameters and limited tissue injury. These effects were antagonized by 5-hydroxydecanoic acid (a blocker of mitoKATP channels). Moreover, 4CPI inhibited the formation of reactive oxygen species. We found the whole battery of H2S-producing enzymes to be present in myocardial tissue: cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST). Notably, 4CPI down-regulated the post-ischemic expression of CSE.In Langendorff-perfused mouse hearts, 4CPI reduced the post-ischemic release of norepinephrine and the incidence of ventricular arrhythmias. In both rat and mouse hearts, 4CPI did not affect the degranulation of resident mast cells.In isolated rat cardiac mitochondria, 4CPI partially depolarized the mitochondrial membrane potential; this effect was antagonized by ATP (i.e., the physiological inhibitor of KATP channels). Moreover, 4CPI abrogated calcium uptake in the mitochondrial matrix.Finally, in an in vivo model of acute myocardial infarction in rats, 4CPI significantly decreased I/R-induced tissue injury.In conclusion, H2S-donors, and in particular isothiocyanate-based H2S-releasing drugs like 4CPI, can actually be considered a suitable pharmacological option in anti-ischemic therapy.