Myocardial Ischemia And Reperfusion Research Articles

Matrix metalloproteinase-2 proteolyzes mitofusin-2 and impairs mitochondrial function during myocardial ischemia-reperfusion injury.

During myocardial ischemia and reperfusion (IR) injury matrix metalloproteinase-2 (MMP-2) is rapidly activated in response to oxidative stress. MMP-2 is a multifunctional protease that cleaves both extracellular and intracellular proteins. Oxidative stress also impairs mitochondrial function which is regulated by different proteins, including mitofusin-2 (Mfn-2), which is lost in IR injury. Oxidative stress and mitochondrial dysfunction trigger the NLRP3 inflammasome and the innate immune response which invokes the de novo expression of an N-terminal truncated isoform of MMP-2 (NTT-MMP-2) at or near mitochondria. We hypothesized that MMP-2 proteolyzes Mfn-2 during myocardial IR injury, impairing mitochondrial function and enhancing the inflammasome response. Isolated hearts from mice subjected to IR injury (30min ischemia/40min reperfusion) showed a significant reduction in left ventricular developed pressure (LVDP) compared to aerobically perfused hearts. IR injury increased MMP-2 activity as observed by gelatin zymography and increased degradation of troponin I, an intracellular MMP-2 target. MMP-2 preferring inhibitors, ARP-100 or ONO-4817, improved post-ischemic recovery of LVDP compared to vehicle perfused IR hearts. In muscle fibers isolated from IR hearts the rates of mitochondrial oxygen consumption and ATP production were impaired compared to those from aerobic hearts, whereas ARP-100 or ONO-4817 attenuated these reductions. IR hearts showed higher levels of NLRP3, cleaved caspase-1 and interleukin-1β in the cytosolic fraction, while the mitochondria-enriched fraction showed reduced levels of Mfn-2, compared to aerobic hearts. ARP-100 or ONO-4817 attenuated these changes. Co-immunoprecipitation showed that MMP-2 is associated with Mfn-2 in aerobic and IR hearts. ARP-100 or ONO-4817 also reduced infarct size and cell death in hearts subjected to 45min ischemia/120min reperfusion. Following myocardial IR injury, impaired contractile function and mitochondrial respiration and elevated inflammasome response could be attributed, at least in part, to MMP-2 activation, which targets and cleaves mitochondrial Mfn-2. Inhibition of MMP-2 activity protects against cardiac contractile dysfunction in IR injury in part by preserving Mfn-2 and suppressing inflammation.

Cardiac-specific overexpression of Claudin-5 exerts protection against myocardial ischemia and reperfusion injury

Claudin-5 has recently attracted increasing attention by its potential as a novel treatment target in the early stage of heart failure. However, whether Claudin-5 produces beneficial effects on myocardial ischemia and reperfusion (IR) injury has not been elucidated yet. In this study, we identified reduced levels of Claudin-5 in the hearts of mice subjected to acute myocardial IR injury and murine HL-1 cardiomyocytes subjected to hypoxia and reoxygenation (HR). We then constructed cardiac-specific Cldn5-overexpressing mice using an adeno-associated virus (AAV9) vector and demonstrated that Cldn5 overexpression ameliorated cardiac dysfunction and myocardial damage in mice subjected to myocardial IR injury. Moreover, Cldn5 overexpression attenuated myocardial oxidative stress (DHE and protein levels of Nrf2, HO-1, and NQO1), inflammatory response (levels of MPO, F4/80, Ly6C, and circulating inflammatory cells), mitochondrial dysfunction (protein levels of PGC-1α, NRF1, and TFAM), endoplasmic reticulum stress (protein levels of GRP78, ATF6, and CHOP and p-PERK), energy metabolism disorder (p-AMPK and ACC), and apoptosis (TUNEL assay and protein levels of Bax and Bcl2) in mice subjected to myocardial IR. Next, we generated Cldn5 knockdown cells by lentiviral shRNA and observed that Cldn5 knockdown inhibited cell viability and affected the expression or activation of these IR-related signalings in HL-1 cardiomyocytes subjected to HR. Mechanistically, SIRT1 was proved to be involved in regulating the expression of Claudin-5 by co-immunoprecipitation analysis and Sirt1 knockdown experiments. Our data demonstrated that targeting Claudin-5 may represent a promising approach for preventing and treating acute myocardial IR injury.

A1 and A2b adenosine receptors regulate GPX4 against ferroptosis of cardiomyocytes in myocardial infarction rat model and in vitro

AimThe regulation of GPX4 by A1AR and A2bAR was investigated, and whether the inhibition of A1AR and A2bAR on ferroptosis of myocardial cell is related to GPX4 was also discussed. Methodswe constructed a rat model of myocardial ischemia and reperfusion (MIR) model and hypoxia/reoxygenation (H/R) model of H9C2 cells, and MIR rats were intraperitoneally injected with A1AR and A2bAR agonists and antagonists. TTC staining, DHE, TUNEL, western blot experiments, immunohistochemistry assay were implemented to analyze the influence of A1AR and A2bAR on ferroptosis and potential role of GPX4. To further authenticate the result of non-specific agonists and antagonists, we transfected siRNA interference or overexpression vectors into cells. CCK8, flow cytometry and western blot were performed to evaluate cell proliferation and apoptosis, and the expression of GPX4 and ferroptosis-related proteins. ResultsThe experimental results showed that reduced expression of A1AR, A2bAR and GPX4 was found after MIR. A1AR and A2bAR activation by agonists increased GPX4 expression and decreased production of lipid ROS, further inhibiting apoptosis of cardiomyocytes. In addition, we also analyzed the effect of A1AR and A2bAR on ferroptosis-related proteins. We found that expression of FIH1 protein increased and expression of ACSL4 and NOX1 proteins decreased. Consistent with results in vivo, cellular data also indicated that A1AR and A2bAR overexpression could increase proliferation ability of H9C2, and inhibit apoptosis and ROS production, upregulate GPX4 and FIH1, and downregulate ACSL4 and NOX1. ConclusionA1AR and A2bAR could regulate GPX4, thereby affecting ferroptosis of cardiomyocytes in a rat model of MIR.

Activation of Nrf2 by Lithospermic Acid Ameliorates Myocardial Ischemia and Reperfusion Injury by Promoting Phosphorylation of AMP-Activated Protein Kinase α (AMPKα).

Background: As a plant-derived polycyclic phenolic carboxylic acid isolated from Salvia miltiorrhiza, lithospermic acid (LA) has been identified as the pharmacological management for neuroprotection and hepatoprotection. However, the role and mechanism of lithospermic acid in the pathological process of myocardial ischemia-reperfusion injury are not fully revealed. Methods: C57BL/6 mice were subjected to myocardial ischemia and reperfusion (MI/R) surgery and pretreated by LA (50 mg/kg, oral gavage) for six consecutive days before operation. The in vitro model of hypoxia reoxygenation (HR) was induced by hypoxia for 24 h and reoxygenation for 6 h in H9C2 cells, which were subsequently administrated with lithospermic acid (100 μM). Nrf2 siRNA and dorsomorphin (DM), an inhibitor of AMPKα, were used to explore the function of AMPKα/Nrf2 in LA-mediated effects. Results: LA pretreatment attenuates infarct area and decreases levels of TnT and CK-MB in plasm following MI/R surgery in mice. Echocardiography and hemodynamics indicate that LA suppresses MI/R-induced cardiac dysfunction. Moreover, LA ameliorates oxidative stress and cardiomyocytes apoptosis following MI/R operation or HR in vivo and in vitro. In terms of mechanism, LA selectively activates eNOS, simultaneously increases nuclear translocation and phosphorylation of Nrf2 and promotes Nrf2/HO-1 pathway in vivo and in vitro, while cardioprotection of LA is abolished by pharmacological inhibitor of AMPK or Nrf2 siRNA in H9C2 cells. Conclusion: LA protects against MI/R-induced cardiac injury by promoting eNOS and Nrf2/HO-1 signaling via phosphorylation of AMPKα.

Abstract 9633: Caveolin-3 Nitrative Modification Exacerbates Ischemic Heart Failure in Pre-Diabetic Mice by Disassembling Cardioprotective Signalosomes

Rationale: Diabatic heart is “universally” resistant to protective interventions, including insulin, adiponectin exacerbating ischemic heart failure (HF). Caveolin (Cav) serves as scaffolding regulator, coordinating signal transduction in cell survival. However, whether and how diabetes alter Cav3-centered signalosome integrity, contributing to diabetic ischemic HF remains unclear.We hypothesized that preserving Cav3 signalosome integrity restore cardioprotective signaling, protecting against diabetic exacerbation of ischemia HF. Methods and Results: Mice with normal diet (ND) or high-fat-diet (HFD) were subjected to myocardial ischemia and reperfusion (MI/R). Administration of insulin or adiponectin markedly attenuated MI injury (60 minutes MI/24 hours reperfusion) in ND mice, an effect lost in diabetic animals (12 weeks HFD, n=18-20/group). Interestingly, cardioprotective effect of insulin and adiponectin was blunted as early as 4 weeks of HFD (pre-diabetes). In ND heart, Cav3 interacts with AdipoR1 and insulin receptor-β (IRβ), forming signalosomes. Those complexes were significantly reduced in pre-diabetic heart. However, the level of Cav3, AdipoR1, AdipoR2, and IRβ remain unchanged. Among multiple post-translational modifications, Cav3 nitration was the dominant in pre-diabetic heart. SIN-1 (a nitration donor) reduced Cav3/AdipoR1 and Cav3/IRβ complex, and blocked their transmembrane signaling. Mass spectrometry identified Tyr 73 is the site modified in pre-diabetic cardiomyocytes. Phenylalanine substitution of Tyr 73 (Cav3 Y73F ) blocked SIN-1 induced Cav3 nitration, restored signalosome assembling, and rescued insulin and adiponectin transmembrane signaling in diabetic cardiomyocytes. Finally, AAV9-mediated cardiomyocyte specific Cav3 Y73F re-expression blocked HFD-induced Cav3 nitration, restored transmembrane signaling, rescued the protective action against ischemic HF. Conclusion: Nitration of Cav3 at Tyr 73 and complex dissociation is responsible for cardiac insulin/adiponectin resistance in pre-diabetic heart, contributing to ischemic HF progression. Early intervention preserving Cav3-centered signalosome integrity is an effective novel strategy against diabetic ischemic HF.

Role of NLRP3 Inflammasome in Myocardial Ischemia-Reperfusion Injury and Ventricular Remodeling

Reperfusion therapy is the optimal therapy for acute myocardial infarction (AMI), but acute inflammatory injury and chronic heart failure (HF) after myocardial ischemia and reperfusion (MI/R) remain the leading cause of death after AMI. Pyroptosis, a newly discovered form of cell death, has been proven to play a significant role in the acute reperfusion process and the subsequent chronic process of ventricular remodeling. Current research shows that multiple stimuli activate the pyroptotic signaling pathway and contribute to cell death and nonbacterial inflammation after MI/R. These stimuli promote the assembly of the nucleotide-binding and oligomerization-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by activating NLRP3. The mature NLRP3 inflammasome cleaves procaspase-1 to active caspase-1, which leads to mature processing of interleukin (IL)-18, IL-1β, and gasdermin D (GSDMD) protein. That eventually results in cell lysis and generation of nonbacterial inflammation. The present review summarizes the mechanism of NLRP3 inflammasome activation after MI/R and discusses the role that NLRP3-mediated pyroptosis plays in the pathophysiology of MI/R injury and ventricular remodeling. We also discuss potential mechanisms and targeted therapy for which there is evidence supporting treatment of ischemic heart disease.

Remote Ischemic Perconditioning Ameliorates Myocardial Ischemia and Reperfusion-Induced Coronary Endothelial Dysfunction and Aortic Stiffness in Rats.

Background:Vascular stiffness and endothelial dysfunction are accelerated by acute myocardial infarction (AMI) and subsequently increase the risk for recurrent coronary events.Aim:To explore whether remote ischemic perconditioning (RIPerc) protects against coronary and aorta endothelial dysfunction as well as aortic stiffness following AMI.Methods:Male OFA-1 rats were subjected to 30 min of occlusion of the left anterior descending artery (LAD) followed by reperfusion either 3 or 28 days with or without RIPerc. Three groups: (1) sham operated (Sham, without LAD occlusion); (2) myocardial ischemia and reperfusion (MIR) and (3) MIR + RIPerc group with 3 cycles of 5 minutes of IR on hindlimb performed during myocardial ischemia were used. Assessment of vascular reactivity in isolated septal coronary arteries (non-occluded) and aortic rings as well as aortic stiffness was assessed by wire myography either 3 or 28 days after AMI, respectively. Markers of pro-inflammatory cytokines, adhesion molecules were assessed by RT-qPCR and ELISA.Results:MIR promotes impaired endothelial-dependent relaxation in septal coronary artery segments, increased aortic stiffness and adverse left ventricular remodeling. These changes were markedly attenuated in rats treated with RIPerc and associated with a significant decline in P-selectin, IL-6 and TNF-α expression either in infarcted or non-infarcted myocardial tissue samples.Conclusions:Our study for the first time demonstrated that RIPerc alleviates MIR-induced coronary artery endothelial dysfunction in non-occluded artery segments and attenuates aortic stiffness in rats. The vascular protective effects of RIPerc are associated with ameliorated inflammation and might therefore be caused by reduced inflammatory signaling.

Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes.

Dysregulation of long non-coding RNA (IncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is associated with the risk of myocardial infarction (MI). Therefore, the present study aimed to determine the mechanisms underlying this association, which is currently poorly understood, to the best of our knowledge. The current study used an in vitro myocardial ischemia and reperfusion (MI/R) model, in which H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R), which demonstrated that ANRIL expression was downregulated and that ANRIL positively regulated sirtuin 1 (SIRT1) expression following H/R injury. Subsequently, it was demonstrated that ANRIL upregulated SIRT1 expression by sponging microRNA-181a (miR-181a). In addition, ANRIL overexpression reduced lactate dehydrogenase release and apoptosis of H9c2 cardiomyocytes exposed to H/R, indicating that ANRIL prevented H/R-induced cardiomyocyte injury. Moreover, both miR-181a overexpression and SIRT1 knockdown significantly decreased the protective effects of ANRIL on H/R-induced cardiomyocyte injury, thus demonstrating that SIRT1 upregulation via sponging miR-181a is a critical mechanism that mediates the function of ANRIL. These results provided a novel mechanistic insight into the role of ANRIL in H/R-injured cardiomyocytes and suggested that the ANRIL/miR-181a/SIRT1 axis may be a therapeutic target for reducing MI/R injury.

Cardioprotective effects of resveratrol following myocardial ischemia and reperfusion.

Resveratrol (RSV), a plant origin polyphenol, has shown beneficial cardiovascular effects. In this study, isolated hearts from male Wistar rats were studied using the Langendorff technique. Following 30min stabilization, the hearts underwent 30min global ischemia and 120min reperfusion. The perfusion solution in the test group contained RSV (10μM). Hemodynamics of the hearts, the markers of myocardial damage including creatine kinase-MB (CK-MB), lactate dehydrogenase (LDH), and troponin I were studied during the study. Furthermore, the infarct size and the markers of oxidative stress including catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GPX) were assayed in the homogenates of the hearts. The release of nitrite from the hearts and the occurrence of ventricular arrhythmias were also monitored throughout the experiment. Resveratrol caused a significant improvement in the restoration of the mechanical performance of the hearts following myocardial ischemia and reperfusion (MIR). Besides, the infarct size, CK-MB, LDH, and troponin I declined in the test group. Besides, the cardiac release of nitrite increased, and the redox status of the heart was improved as indicated by the levels of CAT, SOD, GPX, and MDA. Finally, the treatment caused significant decreases in the occurrences of single and salvo arrhythmias, ventricular tachycardia, and ventricular fibrillation. The current study suggests strong cardioprotective and antiarrhythmic effects for RSV following MIR.

Mitochondrial and mitochondrial-independent pathways of myocardial cell death during ischaemia and reperfusion injury.

Acute myocardial infarction causes lethal injury to cardiomyocytes during both ischaemia and reperfusion (IR). It is important to define the precise mechanisms by which they die in order to develop strategies to protect the heart from IR injury. Necrosis is known to play a major role in myocardial IR injury. There is also evidence for significant myocardial death by other pathways such as apoptosis, although this has been challenged. Mitochondria play a central role in both of these pathways of cell death, as either a causal mechanism is the case of mitochondrial permeability transition leading to necrosis, or as part of the signalling pathway in mitochondrial cytochrome c release and apoptosis. Autophagy may impact this process by removing dysfunctional proteins or even entire mitochondria through a process called mitophagy. More recently, roles for other programmed mechanisms of cell death such as necroptosis and pyroptosis have been described, and inhibitors of these pathways have been shown to be cardioprotective. In this review, we discuss both mitochondrial and mitochondrial‐independent pathways of the major modes of cell death, their role in IR injury and their potential to be targeted as part of a cardioprotective strategy. This article is part of a special Issue entitled ‘Mitochondria as targets of acute cardioprotection’ and emerged as part of the discussions of the European Union (EU)‐CARDIOPROTECTION Cooperation in Science and Technology (COST) Action, CA16225.

κ-opioid receptor activation promotes mitochondrial fusion and enhances myocardial resistance to ischemia and reperfusion injury via STAT3-OPA1 pathway

Mitochondrial dynamics, determining mitochondrial morphology, quality and abundance, have recently been implicated in myocardial ischemia and reperfusion (MI/R) injury. The roles of κ-opioid receptor activation in cardioprotection have been confirmed in our previous studies, while the underlying mechanism associated with mitochondrial dynamics remains unclear. This study aims to investigate the effect of κ-opioid receptor activation on the pathogenesis of MI/R and its underlying mechanisms. MI/R mouse model and hypoxia-reoxygenation cardiomyocyte model were established in this study. Mitochondrial dynamics were analyzed with transmission electron microscopy in vivo and confocal microscopy in vitro. STAT3 phosphorylation and OPA1 expression were detected by Western blotting. We show here that κ-opioid receptor activation with its selective receptor agonist U50,488H promoted mitochondrial fusion and enhanced myocardial resistance to MI/R injury, while these protective effects were blockaded by nor-BNI, a selective κ-opioid receptor antagonist. In addition, κ-opioid receptor activation increased STAT3 phosphorylation and OPA1 expression, which were blockaded by nor-BNI. Furthermore, inhibition of STAT3 phosphorylation by stattic, a specific STAT3 inhibitor, repressed the effects of κ-opioid receptor activation on promoting OPA1 expression and mitochondrial fusion, as well as inhibiting cell apoptosis and oxidative stress both in vivo and in vitro during MI/R injury. Overall, our data for the first time provide evidence that κ-opioid receptor activation promotes mitochondrial fusion and enhances myocardial resistance to MI/R injury via STAT3-OPA1 pathway. Targeting the pathway regulated by κ-opioid receptor activation may be a potential therapeutic strategy for MI/R injury.

Preliminary evaluation of 18F‑AlF‑NOTA‑MAL‑Cys40‑Exendin‑4 in rodent heart after myocardial ischemia and reperfusion.

Glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1R) exert cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in animal models and human clinical trials. Receptor imaging with positron emission tomography (PET) provides a non-invasive method for monitoring GLP-1R expression. In the present study, a fluorine-18-labeled aluminum fluoride exendin-4 analog [18F-AlF conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA)-maleimide (MAL)-Cys40-exendin-4] was synthesized and evaluated in a rat MI/R model for GLP-1R imaging. NOTA-MAL-Cys40-exendin-4 was synthesized by coupling Cys40-exendin-4 with NOTA-MAL. NOTA-MAL-Cys40-exendin-4 was then conjugated with 18F-AlF to obtain 18F-AlF-NOTA-MAL-Cys40-exendin-4. The yield of 18F-AlF-NOTA-MAL-Cys40-exendin-4 was 18.5±3.4% (not decay corrected). The process was completed within ~30 min. In rat MI/R models, the tracer exhibited specific binding to GLP-1R and an appropriate signal-to-noise ratio. At 8 h post-MI/R, tracer uptake reached its peak [0.35±0.053% of injected dose (%ID)/g; n=6] in ischemic myocardium. Localized tracer uptake decreased 1 day (0.20±0.032 %ID/g; n=6) and 3 days (0.16±0.017 %ID/g; n=6) post-MI/R compared with 8 h post-MI/R, but still remained higher compared with sham-operated groups (0.06±0.012 %ID/g; n=6). Pre-injected unlabeled exendin-4 effectively blocked tracer accumulation (0.09±0.041 %ID/g; n=6). In conclusion, 18F-AlF-NOTA-MAL-Cys40-exendin-4 demonstrated favorable characteristics for GLP-1R imaging following MI/R. PET imaging using 18F-AlF-NOTA-MAL-Cys40-exendin-4 in rodent hearts after MI/R revealed a dynamic pattern of GLP-1R upregulation.

Mitochondrial Dysfunction and Apoptosis Are Attenuated on κ-Opioid Receptor Activation Through AMPK/GSK-3β Pathway After Myocardial Ischemia and Reperfusion.

Previous studies have shown that κ-opioid receptor activation possesses cardioprotection against myocardial ischemia and reperfusion (MI/R) injury. The current study was designed to investigate whether mitochondrial dysfunction after MI/R is regulated by the κ-opioid receptor and to further explore the underlying mechanisms involved. MI/R rat model was established in vivo, and a hypoxia and reoxygenation cardiomyocytes model was used in vitro. Mitochondrial morphology and function as well as myocardial apoptosis were determined. Our data indicated that treatment with U50,488H (a selective κ-opioid receptor agonist) not only reduced apoptosis but also significantly improved mitochondrial morphology and function. These effects were blocked by nor-binaltorphimine (nor-BNI, a selective κ-opioid receptor antagonist), Compound C (an AMPK inhibitor), and AR-A014418 (a GSK3β inhibitor). Moreover, in cardiomyocytes, treatment with U50,488H significantly increased the expression in phosphorylation of AMPK and the phosphorylation of GSK3β. Treatment of cardiomyocytes with AMPKα siRNA decreased the phosphorylation of AMPK and GSK3β. Moreover, AMPK activation resulted in the phosphorylation of GSK3β. Our findings suggested that U50,488H exerted cardioprotective effects by improving mitochondrial morphology and function against MI/R injury through activation of the κ-opioid receptor-mediated AMPK/GSK3β pathway.

Selective ablation of the ligament of Marshall reduces ischemia and reperfusion-induced ventricular arrhythmias.

Cardiac sympathetic tone overdrive is a key mechanism of arrhythmia. Cardiac sympathetic nerves denervation, such as LSG ablation or renal sympathetic denervation, suppressed both the prevalence of VAs and the incidence of SCD. Accumulating evidence demonstrates the ligament of Marshall (LOM) is a key component of the sympathetic conduit between the left stellate ganglion (LSG) and the ventricles. The present study aimed to investigate the roles of the distal segment of LOM (LOMLSPV) denervation in ischemia and reperfusion (IR)-induced VAs, and compared that LSG denervation. Thirty-three canines were randomly divided into group 1 (IR group, n = 11), group 2 (LOMLSPV Denervation + IR, n = 9), and group 3 (LSG Denervation + IR, n = 13). Hematoxylin-Eosin (HE) and Immunohistochemistry staining revealed that LOMLSPV contained bundles of sympathetic but not parasympathetic nerves. IR increased the cardiac sympathetic tone [serum concentrations of noradrenaline (NE) and epinephrine (E)] and induced the prevalence of VAs [ventricular premature beat (VPB), salvo of VPB, ventricular tachycardia (VT), VT duration (VTD) and ventricular fibrillation (VF)]. Both LOMLSPV denervation and LSG denervation could reduce the cardiac sympathetic tone in Baseline (BS) [heart rate variability (HRV)]. Compared with group 1, LOMLSPV denervation and LSG denervation similarly reduced sympathetic tone [NE (1.39±0.068 ng/ml in group 2, 1.29±0.081 ng/ml in group 3 vs 2.32±0.17 ng/ml in group 1, P<0.05) and E (114.64±9.22 pg/ml in group 2, 112.60±9.69 pg/ml in group 3 vs 166.18±15.78 pg/ml in group 1, P<0.05),] and VAs [VT (0±3.00 in group 2, 0±1.75 in group 3 vs 8.00±11.00 in group 1, P<0.05) and VTD (0 ± 4 s in group 2, 0±0.88s in group 3 vs 10.0 ± 22.00s in group 1, P<0.05)] after 2h reperfusion. These findings indicated LOMLSPV denervation reduced the prevalence of VT by suppressing SNS activity. These effects are comparable to those of LSG denervation. In myocardial IR, the anti-arrhythmic effects of LOMLSPV Denervation may be related to the inhibition of the expression of NE and E.

3-Bromo-4,5-Dihydroxybenzaldehyde Protects Against Myocardial Ischemia and Reperfusion Injury Through the Akt-PGC1α-Sirt3 Pathway.

Natural marine products are useful candidates for the treatment of oxidative and inflammatory diseases, including myocardial ischemia. 3-bromo-4,5 - dihydroxybenzaldehyde (BDB), a natural bromophenol isolated from marine red algae, has been shown to display anti-microbial, anti-oxidative, anti-cancer, anti-inflammatory, and free radical scavenging activities. In this study, the potential protective effects of BDB against myocardial ischemia and reperfusion (IR) injury was investigated in an in vitro model mimicked by oxygen and glucose deprivation (OGD) in cardiomyocytes and in an in vivo model induced by coronary artery ligation in rats. The results showed that BDB attenuated the OGD-induced cytotoxicity in a dose-dependent manner, with no toxic effect when treated alone. BDB significantly decreased apoptosis and the cleavage of caspase-3 after OGD. We found that OGD-induced oxidative stress, as evidenced by increases of reactive oxygen species (ROS) and lipid peroxidation, as well as mitochondrial dysfunction, as measured by mitochondrial reporter gene, cytochrome c release and ATP synthesis, were markedly attenuated by BDB treatment. In addition, BDB increased the enzymatic activities of mitochondrial antioxidant enzymes, including IDH2, GSH-Px and SOD2. Western blot analysis showed that BDB increased Akt phosphorylation and upregulated the expression of Sirt3 and PGC1α after OGD. Furthermore, BDB-induced protection in cardiomyocytes was partially reversed by the Akt inhibitor and downregulation of PGC1α. BDB also attenuated myocardial contractile dysfunction and activated the Akt-PGC1α-Sirt3 pathway in vivo. All these data suggest that BDB protects against myocardial IR injury through activating the Akt-PGC1α-Sirt3 pathway.

Postconditioning Effect of PDE5 inhibitor, Sildenafil in Normal and Diabetic Rabbits following Myocardial Ischemia/Reperfusion injury.

BackgroundSeveral studies have demonstrated cardioprotective effects of the phosphodiesterase 5 inhibitor, sildenafil (SILD), in the setting of myocardial ischemia and reperfusion (MI/R) in mice and rabbits. However, it is unclear whether sildenafil exerts postconditioning effect in normal and diabetic rabbits.Methods and ResultsTo induce diabetes, alloxan monohydrate (125 mg/kg, i.v.) was administered in New Zealand white male rabbits (n=10; ~3 kg, 4 months old). Blood glucose level increased to 342±21 mg/dL following 2 weeks of alloxan administration. Balloon occluders were implanted on the top of the coronary artery and 7 days later, conscious rabbits were subjected to 45‐min ischemia (I) and 3 days of reperfusion (R) by inflating/deflating the balloon. Saline (as control) or Sildenafil (0.71 or 1.4 mg/kg; i.v.) was infused for 60 min starting 5 min before reperfusion. Myocardial infarct size was determined by triphenyltetrazolium chloride staining and troponin I (cTnI) was measured using ELISA method in the plasma. The result showed that sildenafil (both concentrations) significantly reduced myocardial infarct size (Figure A) and peak plasma levels of cardiac troponin I. Similarly, sildenafil reduced infarct size in diabetic rabbits (DM) following MI/R (Figure C). There were no differences in hemodynamics during MI/R or myocardial area‐at‐risk (AAR) (Figure B and D).ConclusionSildenafil exerts significant postconditioning effect in normal and diabetic rabbits of conscious I/R model that is potentially is free of confounding factors associated with open‐chest preparations.Support or Funding InformationNIHRO1HL134366 (AD &amp; RCK), RO1HL118808 (RCK), RO1HL133167 (RCK)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adiponectin has a pivotal role in the cardioprotective effect of CP-3(iv), a selective CD36 azapeptide ligand, after transient coronary artery occlusion in mice.

CD36 is a multiligand receptor involved in lipid metabolism. We investigated the mechanisms underlying the cardioprotective effect of CP-3(iv), an azapeptide belonging to a new class of selective CD36 ligands. The role of CP-3(iv) in mediating cardioprotection was investigated because CD36 signaling leads to activation of peroxisome proliferator-activated receptor-γ, a transcriptional regulator of adiponectin. CP-3(iv) pretreatment reduced infarct size by 54% and preserved hemodynamics in C57BL/6 mice subjected to 30 min coronary ligation and reperfusion but had no effect in CD36-deficient mice. The effects of CP-3(iv) were associated with an increase in circulating adiponectin levels, epididymal fat adiponectin gene expression, and adiponectin transcriptional regulators ( Pparg, Cebpb, Sirt1) after 6 h of reperfusion. Reduced myocardial oxidative stress and apoptosis were observed along with an increase in expression of myocardial adiponectin target proteins, including cyclooxygenase-2, phospho-AMPK, and phospho-Akt. Moreover, CP-3(iv) increased myocardial performance in isolated hearts, whereas blockade of adiponectin with an anti-adiponectin antibody abrogated it. CP-3(iv) exerts cardioprotection against myocardial ischemia and reperfusion (MI/R) injury and dysfunction, at least in part, by increasing circulating and myocardial adiponectin levels. Hence, both paracrine and endocrine effects of adiponectin may contribute to reduced reactive oxygen species generation and apoptosis after MI/R, in a CD36-dependent manner.-Huynh, D. N., Bessi, V. L., Ménard, L., Piquereau, J., Proulx, C., Febbraio, M., Lubell, W. D., Carpentier, A. C., Burelle, Y., Ong, H., Marleau, S. Adiponectin has a pivotal role in the cardioprotective effect of CP-3(iv), a selective CD36 azapeptide ligand, after transient coronary artery occlusion in mice.

Effects of hypoglycemia on myocardial susceptibility to ischemia\u2013reperfusion injury and preconditioning in hearts from rats with and without type 2 diabetes

BackgroundHypoglycemia is associated with increased mortality rate in patients with diabetes. The underlying mechanisms may involve reduced myocardial tolerance to ischemia and reperfusion (IR) or reduced capacity for ischemic preconditioning (IPC). As IPC is associated with increased myocardial glucose uptake (MGU) during reperfusion, cardioprotection is linked to glucose metabolism possibly by O-linked β-N-acetylglucosamine (O-GlcNAc). We aimed to investigate the impact of hypoglycemia in hearts from animals with diabetes on myocardial IR tolerance, on the efficacy of IPC and whether modulations of MGU and O-GlcNAc levels are involved in the underlying mechanisms.MethodsIn a Langendorff model using diabetic ZDF (fa/fa) and non-diabetic (fa/+) rats (n = 6–7 in each group) infarct size (IS) was evaluated after 40 min of global ischemia and 120 min reperfusion during hypoglycemia [(glucose) = 3 mmol/l] and normoglycemia [(glucose) = 11 mmol/l]. Myocardial glucose uptake and O-GlcNAc levels were evaluated during reperfusion. IPC was induced by 2 × 5 min of global ischemia prior to index ischemia.ResultsIS increased in hearts from animals with (p < 0.01) and without (p < 0.01) diabetes during hypoglycemia compared to normoglycemia. IPC reduced IS during normoglycemia in both animals with (p < 0.01) and without (p < 0.01) diabetes. During hypoglycemia, however, IPC only reduced IS in hearts from animals with diabetes (p < 0.05). IPC increased MGU during reperfusion and O-GlcNAc levels in animals with diabetes during hypo- (MGU: p < 0.05, O-GlcNAc: p < 0.05) and normoglycemia (MGU: p < 0.01, O-GlcNAc: p < 0.05) and in animals without diabetes only during normoglycemia (MGU: p < 0.05, O-GlcNAc: p < 0.01).ConclusionsHypoglycemia increases myocardial susceptibility to IR injury in hearts from animals with and without diabetes. In contrast to hearts from animals without diabetes, the hearts from animals with diabetes are amenable to cardioprotection during hypoglycemia. In parallel with IPC induced cardioprotection, MGU and O-GlcNAc levels increase suggesting that increased MGU and O-GlcNAc levels are involved in the mechanisms of IPC.

Curcumin Protects Against Myocardial Ischemia-Reperfusion Injury by Activating Endothelial Nitric Oxide Synthase in Rats

BackgroundThis study aims to investigate whether or not curcumin (Cur) protects against myocardial ischemia and reperfusion (MI/R) injury via endothelial nitric oxide synthase (eNOS) activation. Materials and MethodsThe rats were randomly assigned to a sham group, an MI/R group, or a MI/R + Cur group. Infarct size, cell apoptosis, oxidative stress, eNOS phosphorylation expression and eNOS expression were measured. ResultsCur reduced infarct size, cell apoptosis and oxidative stress compared with the MI/R group. Additionally, Cur significantly decreased eNOS phosphorylation. ConclusionsCur reduced MI/R injury via activation of eNOS. These findings suggest that Cur may be a promising therapeutic drug for MI/R injury.

Vagal nerve stimulation reduces infarct size via a mechanism involving the alpha-7 nicotinic acetylcholine receptor and downregulation of cardiac and vascular arginase.

Vagal nerve stimulation (VNS) protects from myocardial and vascular injury following myocardial ischaemia and reperfusion (IR) via a mechanism involving activation of alpha-7 nicotinic acetylcholine receptor (α7 nAChR) and reduced inflammation. Arginase is involved in development of myocardial IR injury driven by inflammatory mediators. The aim of the study was to clarify whether VNS downregulates myocardial and vascular arginase via a mechanism involving activation of α7 nAChR following myocardial IR. Anaesthetized rats were randomized to (i) sham-operated, (ii) control IR (30-min ischaemia and 2-h reperfusion, (iii) VNS throughout IR, (iv) the arginase inhibitor nor-NOHA+IR, (v) nor-NOHA+VNS+IR, (vi) selective α7 nAChR blockade by methyllycaconitine (MLA) followed by VNS throughout IR and (vii) MLA+IR. Infarct size was reduced by VNS compared to control IR (41 ± 3% vs. 67 ± 2% of the myocardium at risk, P < 0.001). Myocardial IR increased myocardial and aortic arginase activity 1.7- and 3.1-fold respectively (P < 0.05). VNS attenuated the increase in arginase activity compared to control IR both in the myocardium and aorta (P < 0.05). MLA partially abolished the cardioprotective effect of VNS and completely abrogated the effect of VNS on arginase activity. Arginase inhibition combined with VNS did not further reduce infarct size. Vagal nerve stimulation reduced infarct size and reversed the upregulation of arginase induced by IR both in the myocardium and aorta via a mechanism depending on α7 nAChR activation. The data suggest that the cardioprotective effect of VNS is mediated via reduction in arginase activity.