Ischemic-reperfused Hearts Research Articles

Role of δ-opioid receptors in hydromorphone postconditioning-induced maintenance of electrophysiological stability during ischemia-reperfusion in isolated rat hearts

Objective To evaluate the role of δ-opioid receptors in hydromorphone postconditioning-induced maintenance of electrophysiological stability during ischemia-reperfusion (I/R) in isolated rat hearts. Methods Healthy male Sprague-Dawley rats, aged 2-3 months, weighing 280-360 g, were used in this study.The animals were anesthetized with intraperitoneal pentobarbital 60 mg/kg.Their hearts were immediately removed and perfused in a Langendorff apparatus.Thirty-two isolated hearts were divided into 4 groups after successful preparation of Langendorff perfusion model (n=8 each) using a random number table: control group (group C), group I/R, hydromorphone postconditioning group (group HP) and hydromorphone plus δ-opioid receptor antagonist naltridole postconditioning group (group HNP). In HP and HNP groups, the hearts were perfused for 10 min with K-H solution containing 4.1 ng/ml hydromorphone and 4.1 ng/ml hydromorphone plus 5 μmol/L naltridole, respectively, and then with K-H solution for 50 min.At 20 min of stabilization (T0) and 10, 25 and 60 min of reperfusion (T1-2), heart rate (HR), monophasic action potential (MAP) duration at 90% repolarization (MAPD90) of the two layers (endocardium, epicardium) of the anterior left ventricular wall were recorded.Transmural dispersion of repolarization (TDR) was calculated.The development of arrhythmia, time for restoration of spontaneous heart beat and duration of arrhythmia were recorded during the period of reperfusion. Results Compared with group C, MAPD90 of endocardium at T1-2 and MAPD90 of epicardium at T1 were significantly prolonged in I/R and HP groups, HR was significantly decreased at T2-3, MAPD90 of endocardium and epicardium was prolonged at T1-3 in group HNP, TDR was significantly enlarged at T1 in group I/R and at T2 in group HNP, and TDR was decreased at T3 in group HP (P 0.05), the time for restoration of spontaneous heart beat was significantly shortened, and TDR was decreased at T1 in HP and HNP groups, duration of arrhythmia was significantly shortened, and MAPD90 of endocardium was shortened at T1 in group HP, and HR was significantly decreased at T2-3, MAPD90 of endocardium and epicardium was prolonged at T1-3, and TDR was decreased at T2-3 in group HNP (P 0.05), HR was significantly decreased at T2-3, MAPD90 of endocardium and epicardium was prolonged at T1-3, and TDR was increased at T3 in group HNP (P<0.05). Conclusion The mechanism underlying hydromorphone postconditioning-induced maintenance of electrophysiological stability during I/R is related to activating δ-opioid receptors in isolated rat hearts. Key words: Receptors, opioid, delta; Hydromorphone; Postconditioning; Myocardial reperfusion injury; Cardiac electrophysiology

Effect of hydromorphone postconditioning on electrophysiological stability during ischemia-reperfusion in isolated rat hearts

Objective To evaluate the effect of hydromorphone postconditioning on the electrophysiological stability during ischemia-reperfusion (I/R) in isolated rat hearts. Methods Healthy adult male Sprague-Dawley rats, aged 2-3 months, weighing 280-360 g, were heparinized and anesthetized with pentobarbital sodium.Their hearts were excised and perfused in a Langendorff apparatus with K-H solution saturated with 95% O2-5% CO2 at 37 ℃.Twenty-four Langendorff-perfused hearts were divided into 3 groups (n=8 each) using a random number table: control group (group C), I/R group and hydromorphone postconditioning group (group HM). The isolated hearts were subjected to 60 min ischemia followed by 60 min reperfusion to establish the model of isolated heart I/R injury.The isolated hearts were perfused with K-H solution containing 4.1 ng/ml hydromorphone for 10 min starting from onset of reperfusion in group HM.Heart rate, electrocardiogram, coronary flow, and monophasic action potential amplitude, in the left ventricular endocardium, mid-myocardium and epicardium the maximal increase rate (Vmax) at the 0 phase, and monophasic action potential duration at 50% and 90% repolarization (MAPD50 and MAPD90, respectively) were recorded at 20 min of stabilization (T0) and 10, 25, 40 and 60 min of reperfusion (T1-4). The transmural dispersion of repolarization (TDR) was calculated, and the time for restoration of spontaneous heart beat was recorded. Results There was no significant difference in the heart rate, coronary flow or monophasic action potential amplitude between the three groups (P>0.05). Compared with group C, Vmax in the epicardium was significantly decreased, MAPD50 and MAPD90 in the three transmural layers were prolonged, and TDR was prolonged in group I/R (P 0.05). Conclusion Hydromorphone postconditioning is helpful in maintaining the electrophysiological stability during I/R in isolated rat hearts. Key words: Hydromorphone; Myocardial reperfusion injury; Cardiac electrophysiology; Ischemic postconditioning

Restoring diabetes-induced autophagic flux arrest in ischemic/reperfused heart by ADIPOR (adiponectin receptor) activation involves both AMPK-dependent and AMPK-independent signaling

ABSTRACTMacroautophagy/autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of ADIPOQ, a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout (adipoq−/−) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq−/− largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq−/− mice with AdipoRon, a novel ADIPOR (adiponectin receptor) agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P < 0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-class III PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacological AMPK inhibition or genetic Prkaa2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. ADIPOR activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.

Abstract 362: Restoring Diabetes-induced Autophagic Flux Arrestment in Ischemic/Reperfused Heart by Adiponectin Receptor Activation Involves Both AMPK-Dependent and AMPK-Independent Signaling

Autophagy is increasingly recognized as an important regulator of myocardial ischemia-reperfusion (MI-R) injury. However, whether and how diabetes may alter autophagy in response to MI-R remains unknown. Deficiency of adiponectin (AdipoQ), a cardioprotective molecule, markedly increases MI-R injury. However, the role of diabetic hypoadiponectinemia in cardiac autophagy alteration after MI-R is unclear. Utilizing normal control (NC), high-fat-diet-induced diabetes, and Adipoq knockout ( adipoq -/- ) mice, we demonstrated that autophagosome formation was modestly inhibited and autophagosome clearance was markedly impaired in the diabetic heart subjected to MI-R. adipoq -/- largely reproduced the phenotypic alterations observed in the ischemic-reperfused diabetic heart. Treatment of diabetic and adipoq -/- mice with AdipoRon, a novel adiponectin receptor agonist, stimulated autophagosome formation, markedly increased autophagosome clearance, reduced infarct size, and improved cardiac function (P&lt;0.01 vs vehicle). Mechanistically, AdipoRon caused significant phosphorylation of AMPK-BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) and enhanced lysosome protein LAMP2 expression both in vivo and in isolated adult cardiomyocytes. Pharmacologic AMPK inhibition or genetic AMPK alpha2 mutation abolished AdipoRon-induced BECN1 (Ser93/Thr119)-PtdIns3K (Ser164) phosphorylation and AdipoRon-stimulated autophagosome formation. However, AdipoRon-induced LAMP2 expression, AdipoRon-stimulated autophagosome clearance, and AdipoRon-suppressed superoxide generation were not affected by AMPK inhibition. Treatment with MnTMPyP (a superoxide scavenger) increased LAMP2 expression and stimulated autophagosome clearance in simulated ischemic-reperfused cardiomyocytes. However, no additive effect between AdipoRon and MnTMPyP was observed. Collectively, these results demonstrate that hypoadiponectinemia impairs autophagic flux, contributing to enhanced MI-R injury in the diabetic state. Adiponectin receptor activation restores AMPK-mediated autophagosome formation and antioxidant-mediated autophagosome clearance, representing a novel intervention effective against MI-R injury in diabetic conditions.

S1P receptor 1-Mediated Anti-Renin-Angiotensin System Cardioprotection: Pivotal Role of Mast Cell Aldehyde Dehydrogenase Type 2.

In the ischemic-reperfused (I/R) heart, renin-containing mast cells (MC) release enzymatically active renin, activating a local renin-angiotensin system (RAS), causing excessive norepinephrine release and arrhythmic dysfunction. Activation of Gi-receptors on MC and/or ischemic preconditioning (IPC) prevent renin release, thus providing anti-RAS cardioprotection. We questioned whether sphingosine-1-phosphate (S1P), a sphingolipid produced in the I/R heart, might afford anti-RAS cardioprotection by activating Gi-coupled S1P1 receptors (S1P1R) on MC. We report that activation of Gi-coupled S1P1R in cardiac MC confers IPC-like anti-RAS cardioprotection due to S1P1R-mediated inhibition of I/R-induced cardiac MC degranulation and renin release. This results from an initial translocation of protein kinase C subtype-ε and subsequent activation of aldehyde dehydrogenase type 2 (ALDH2), culminating in the elimination of the MC-degranulating effects of acetaldehyde and other toxic species produced during I/R. Inhibition of toxic aldehydes-induced MC-renin release prevents local RAS activation, reduces infarct size, and alleviates arrhythmias. Notably, these cardioprotective effects are lacking in hearts and MC from gene-targeted knock-in mice (ALDH2*2) in which ALDH2 enzymatic activity is maximally reduced. Thus, ALDH2 appears to play a pivotal role in this protective process. Our findings suggest that MC S1P1R may represent a new pharmacologic and therapeutic target for the direct alleviation of RAS-induced cardiac dysfunctions, including ischemic heart disease and congestive heart failure.

Cavin-1 deficiency modifies myocardial and coronary function, stretch responses and ischaemic tolerance: roles of NOS over-activity.

Caveolae and associated cavin and caveolins may govern myocardial function, together with responses to mechanical and ischaemic stresses. Abnormalities in these proteins are also implicated in different cardiovascular disorders. However, specific roles of the cavin-1 protein in cardiac and coronary responses to mechanical/metabolic perturbation remain unclear. We characterised cardiovascular impacts of cavin-1 deficiency, comparing myocardial and coronary phenotypes and responses to stretch and ischaemia-reperfusion in hearts from cavin-1 +/+ and cavin-1 -/- mice. Caveolae and caveolins 1 and 3 were depleted in cavin-1 -/- hearts. Cardiac ejection properties in situ were modestly reduced in cavin-1 -/- mice. While peak contractile performance in ex vivo myocardium from cavin-1 -/- and cavin-1 +/+ mice was comparable, intrinsic beating rate, diastolic stiffness and Frank-Starling behaviour (stretch-dependent diastolic and systolic forces) were exaggerated in cavin-1 -/- hearts. Increases in stretch-dependent forces were countered by NOS inhibition (100µM L-NAME), which exposed negative inotropy in cavin-1 -/- hearts, and were mimicked by 100µM nitroprusside. In contrast, chronotropic differences appeared largely NOS-independent. Cavin-1 deletion also induced NOS-dependent coronary dilatation, ≥3-fold prolongation of reactive hyperaemic responses, and exaggerated pressure-dependence of coronary flow. Stretch-dependent efflux of lactate dehydrogenase and cardiac troponin I was increased and induction of brain natriuretic peptide and c-Fos inhibited in cavin-1 -/- hearts, while ERK1/2 phospho-activation was preserved. Post-ischaemic dysfunction and damage was also exaggerated in cavin-1 -/- hearts. Diverse effects of cavin-1 deletion reveal important roles in both NOS-dependent and -independent control of cardiac and coronary functions, together with governing sarcolemmal fragility and myocardial responses to stretch and ischaemia.

Role of miRNA-1 in regulating connexin 43 in ischemia-reperfusion heart injury: a rat model.

MiRNA-1 may participate in regulating ischemia-reperfusion injury (IRI) by affecting the expression and distribution of connexin 43 (Cx43). The aim of this study is to investigate miR-1 expression and its potential role in regulating Cx43 during ischemic postconditioning (IPOST) in a rat model. Fifty-five Wistar male rats were randomly divided into five groups: N, IR, IPOST, agomir-1, and antagomir-1 group. The hearts were perfused with the Langendorff system. The reperfusion arrhythmia (RA) and myocardial infarct size were observed and recorded. The miRNA-1 expression and the Cx43 expression and distribution were assessed by RT-PCR, immunoblotting, and immunohistochemistry. First, the RA score in the IR group was higher than that in the control group, whereas there was no difference between the IPOST and antagomir-1 groups. Second, the myocardial infarct size was larger in the agomir-1 than in the IPOST group; there was no difference between the antagomir-1 and the IPOST group. Third, the miRNA-1 expression increased by 78% in the agomir-1 group but decreased by 32% in the antagomir-1 group compared with the IPOST group. Fourth, compared with the Control group, the Cx43 expression in the IR group decreased, the Cx43 expression decreased in the agomir-1 group compared with the IPOST group. Fifth, the distribution of Cx43 was irregular and disorganized in the IR and agomir-1 groups. In the IPOST and antagomir-1 groups, Cx43 was neatly distributed in the intercalated disk area. Our findings suggest that IPOST can inhibit the up-regulation of miRNA-1 induced by ischemia-reperfusion and that the down-regulation of miRNA-1 can prevent the decrease and redistribution of Cx43, which will protect the heart from IRI.

Effects of Chronic and Acute Zinc Supplementation on Myocardial Ischemia-Reperfusion Injury in Rats.

The present study aims to explore the effects of chronic and acute zinc sulfate supplementation on myocardial ischemia-reperfusion injury in rats. The study registered 50 adult male rats which were divided into five groups in equal numbers as follows: group 1, normal control; group 2, sham; group 3, myocardial ischemia reperfusion (My/IR): the group which was fed on a normal diet and in which myocardial I/R was induced; group 4, myocardial ischemia reperfusion + chronic zinc: (5mg/kgi.p. zinc sulfate for 15days); and group 5, myocardial ischemia reperfusion + acute zinc: the group which was administered 15mg/kgi.p. zinc sulfate an hour before the operation and in which myocardial I/R was induced. The collected blood and cardiac tissue samples were analyzed using spectrophotometric method to determine levels of MDA, as an indicator of tissue injury, and GSH, as an indicator of antioxidant activity. The highest plasma and heart tissue MDA levels were measured in group 3 (p<0.05). Group 5 had lower MDA values than group 3, while group 4 had significantly lower MDA values than groups 3 and 5 (p<0.05). The highest erythrocyte GSH values were found in group 4 (p<0.05). Erythrocyte GSH values in group 5 were higher than those in group 3 (p<0.05). The highest GSH values in heart tissue were measured in group 4 (p<0.05). The results of the study reveal that the antioxidant activity inhibited by elevated oxidative stress in heart ischemia reperfusion in rats is restored partially by acute zinc administration and markedly by chronic zinc supplementation.

Fluorescent Nanocomposite for Visualizing Cross-Talk between MicroRNA-21 and Hydrogen Peroxide in Ischemia-Reperfusion Injury in Live Cells and In Vivo.

MicroRNAs (miRNAs) and reactive oxygen species (ROS) are concurrently implicated in heart ischemia-reperfusion (IR) injury. There may exist mutual cross-talk between miRNAs and ROS in cardiac IR injury process. In this study, we developed a novel crown-like silica@polydopamine-DNA-CeO2 nanocomposite by assembly of silica@polydopamine-DNA1 nanoparticles decorated with satellite CeO2-DNA2 nanoparticles for detecting and imaging of microRNA-21 (miR-21) and hydrogen peroxide (H2O2) in simulated IR injury in living cells and in vivo. The miRNA-21 was found to be regulated by H2O2 via PI3K/AKT signaling pathway for the first time in H9C2 cells in simulated ischemia-reperfusion injury. H2O2 and miRNA-21 are overproduced during mimicked heart ischemia-reperfusion injury, suggesting that they are closely related to reperfusion injury. All these results reveal that there is definite cross-talk between miR-21 and H2O2 in IR injury. The current method can provide a promising strategy to further explore the interplaying roles between ROS and miRNAs in other pathological processes.

Therapeutic Potential and Mechanisms of Berberine in Cardiovascular Disease

Berberine is originally isolated from extracts of the Chinese herb Coptis chinensis. It was initially used to treat diarrhea. Over the past two decades, berberine has been shown to be effective for improving glycemic and lipid profiles. Interest in its protective effects against diabetes and cardiovascular risk is beginning to emerge, and research investigating the pharmacological activity of berberine is developing rapidly and being reported internationally. Some clinical evidence has demonstrated the ability of berberine to prevent endothelial dysfunction, myocardial infarction, and arrhythmia in patients with cardiovascular risks. Numerous molecular targets of berberine have been explored, including adenosine monophosphate-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), and NADPH oxidase (NOX). The aim of this review is to focus on the therapeutic potential of berberine in cardiovascular disease and to describe its underlying mechanisms in the pathogenesis of artherosclerosis, hypertension, heart failure, ischemia reperfusion heart injury, stroke, and arrhythmias.

Nanoparticles-Mediated Targeting of Pioglitazone Reduces Myocardial Ischemia-Reperfusion Injury and Cardiac Remodeling by Antagonizing Monocyte/Macrophage-mediated Inflammation in Preclinical Animal Models

Background: Monocyte-mediated inflammation is a major mechanism of myocardial ischemia-reperfusion (IR) injury and cardiac remodeling. However, no decisive anti-inflammatory therapy has been developed for clinical setting. Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, has unique anti-inflammatory effects on monocyte/macrophage. Here we tested the hypothesis that nanoparticle-mediated targeting of pioglitazone into cardiomyocytes and inflammatory monocytes ameliorates IR injury and cardiac remodeling in preclinical animal models. Methods and Results: We formulated poly(lactic acid/glycolic acid) nanoparticle containing pioglitazone (Pio-NPs). In mouse IR model, nanoparticles were delivered predominantly to circulating monocytes and to cardiomyocytes and macrophages in the IR heart. Intravenous treatment with Pio-NPs at the time of reperfusion reduced IR injury, which was canceled by the pretreatment with PPARγ antagonist GW9662. In contrast, pioglitazone solution showed no therapeutic effects. Pio-NPs reduced inflammatory gene expression and inhibited the recruitment of Ly6Chigh inflammatory monocytes into IR heart. In mouse MI model, intravenous treatment with Pio-NPs for 3 days after LAD ligation reduced recruitment of macrophage and polarized macrophages toward an anti-inflammatory M2 phenotype, which, leads to improvement of cardiac remodeling and mortality. Finally, in a conscious mini-pig model of myocardial IR injury, Pio-NPs induced cardioprotection from IR injury, indicating the pre-clinical proof of concept. Conclusion: Nanoparticle-mediated targeting of pioglitazone can be developed as a novel modality by antagonizing monocyte-mediated inflammation in myocardial infarction. Background: Monocyte-mediated inflammation is a major mechanism of myocardial ischemia-reperfusion (IR) injury and cardiac remodeling. However, no decisive anti-inflammatory therapy has been developed for clinical setting. Pioglitazone, a peroxisome proliferator-activated receptor (PPAR)γ agonist, has unique anti-inflammatory effects on monocyte/macrophage. Here we tested the hypothesis that nanoparticle-mediated targeting of pioglitazone into cardiomyocytes and inflammatory monocytes ameliorates IR injury and cardiac remodeling in preclinical animal models. Methods and Results: We formulated poly(lactic acid/glycolic acid) nanoparticle containing pioglitazone (Pio-NPs). In mouse IR model, nanoparticles were delivered predominantly to circulating monocytes and to cardiomyocytes and macrophages in the IR heart. Intravenous treatment with Pio-NPs at the time of reperfusion reduced IR injury, which was canceled by the pretreatment with PPARγ antagonist GW9662. In contrast, pioglitazone solution showed no therapeutic effects. Pio-NPs reduced inflammatory gene expression and inhibited the recruitment of Ly6Chigh inflammatory monocytes into IR heart. In mouse MI model, intravenous treatment with Pio-NPs for 3 days after LAD ligation reduced recruitment of macrophage and polarized macrophages toward an anti-inflammatory M2 phenotype, which, leads to improvement of cardiac remodeling and mortality. Finally, in a conscious mini-pig model of myocardial IR injury, Pio-NPs induced cardioprotection from IR injury, indicating the pre-clinical proof of concept. Conclusion: Nanoparticle-mediated targeting of pioglitazone can be developed as a novel modality by antagonizing monocyte-mediated inflammation in myocardial infarction.

Saffron (Crocus sativus) pretreatment confers cardioprotection against ischemia-reperfusion injuries in isolated rabbit heart.

Restoration of blood flow to the ischemic myocardium is imperative to avoid demise of cardiomyocytes, but is paradoxically associated with irreversible damage to cardiac tissues due to the excessive generation of reactive oxygen species (ROS). We have previously reported that saffron, a natural antioxidant, attenuated ischemia-reperfusion (IR) injuries in vitro; however, its role in a meaningful cardiac recovery remains unknown. Here, we show that saffron supplement (oral administration for 6weeks) reduced myocardial damage and restored cardiac function in an IR model of rabbit hearts. This was evidenced by improved left ventricle pressure, heart rate and coronary flow, and left ventricle end diastolic pressure (LVEDP) in IR hearts (isolated from rabbits pre-exposed to saffron (S/IR)). Electrophysiological recordings revealed a significant decline in both premature ventricle contraction and ventricle tachycardia/fibrillation in S/IR compared to IR hearts. This was paralleled by increased expression of the contractile proteins α-actinin and Troponin C in the myocardium of S/IR hearts. Histological examination combined to biochemical analysis indicated that hearts pre-exposed to saffron exhibited reduced infarct size, lower lipid peroxidation, with increased glutathione peroxidase activity, and oxidation of nitro blue tetrazolium (by reactive oxygen species). Furthermore, in contrast with IR hearts, saffron pretreatment induced restoration of the phosphorylation level of the survival proteins Akt and 4EBP1 and reduced activity of p38. Collectively, our data demonstrate that the natural antioxidant saffron plays a pivotal role in halting IR-associated cardiac injuries and emerges as a novel preventive tool for ischemic heart disease.

Atrial natriuretic peptide induces peroxisome proliferator activated receptor γ during cardiac ischemia-reperfusion in swine heart.

Atrial natriuretic peptide is a cardiac atrium-derived hormone and its cardioprotective effects have recently been confirmed, but the actual mechanism underlying these effects has not been well elucidated. In this study, we proposed that atrial natriuretic peptide achieves its effects in part via peroxisome proliferator activated receptor γ, a nuclear receptor. Hemodynamic data in swine heart ischemia-reperfusion model were measured under the conditions of no medication for control (Group N, n=8) or that of carperitide (synthetic human atrial natriuretic peptide) systemic administration (Group A, n=8). After 30min of left anterior descending artery total occlusion and 4h of reperfusion, peroxisome proliferator activated receptor γ mRNA and protein expressions in cardiac muscle were examined. The mRNA expression of Liver X receptor α, the downstream agent of peroxisome proliferator activated receptor γ, was also evaluated. Creatine kinase-myocardial band and Troponin T elevations after reperfusion were evaluated as markers of cardiac damage. The dP/dT decrease during reperfusion was ameliorated in Group A. Peroxisome proliferator activated receptor γ mRNA expression in Group A was significantly higher in ischemic area than that in Group N, although the difference was not significant in the marginal and non-ischemic areas. The peroxisome proliferator activated receptor γ protein expression in ischemic area was also significantly dominant in Group A. Atrial natriuretic peptide may achieve its cardioprotective effects in part via the activation of the peroxisome proliferator activated receptor γ pathway, particularly in central areas of ischemic lesions.

Nanoparticle-Mediated Delivery of Irbesartan Induces Cardioprotection from Myocardial Ischemia-Reperfusion Injury by Antagonizing Monocyte-Mediated Inflammation.

Myocardial ischemia-reperfusion (IR) injury limits the therapeutic effect of early reperfusion therapy for acute myocardial infarction (AMI), in which the recruitment of inflammatory monocytes plays a causative role. Here we develop bioabsorbable poly-lactic/glycolic acid (PLGA) nanoparticles incorporating irbesartan, an angiotensin II type 1 receptor blocker with a peroxisome proliferator-activated receptor (PPAR)γ agonistic effect (irbesartan-NP). In a mouse model of IR injury, intravenous PLGA nanoparticles distribute to the IR myocardium and monocytes in the blood and in the IR heart. Single intravenous treatment at the time of reperfusion with irbesartan-NP (3.0 mg kg−1 irbesartan), but not with control nanoparticles or irbesartan solution (3.0 mg kg−1), inhibits the recruitment of inflammatory monocytes to the IR heart, and reduces the infarct size via PPARγ-dependent anti-inflammatory mechanisms, and ameliorates left ventricular remodeling 21 days after IR. Irbesartan-NP is a novel approach to treat myocardial IR injury in patients with AMI.

MiR-28 inhibits cardiomyocyte survival through suppressing PDK1/Akt/mTOR signaling

MicroRNAs play critical roles in regulating cell survival under multiple pathological conditions of heart diseases. Oxidative stress-induced apoptosis contributes greatly to heart ischemia-reperfusion injury. Herein, we describe a novel regulatory role of miR-28 on the survival of cardiomyocytes. We show that miR-28 was upregulated in cardiomyocytes treated with hydrogen peroxide (H2O2). MiR-28 gain of function sensitized cell apoptosis, whereas miR-28 loss of function partially rescued cell apoptosis induced by H2O2. Importantly, we observed a significant reduction in Akt/mammalian target of rapamycin (mTOR) signaling activity after miR-28 treatment. Luciferase activity assay and western blot analysis both revealed that, phosphoinositide-dependent kinase-1 (PDK1), which is critical for Akt activation, was directly and negatively modulated by miR-28. Our results therefore indicate that miR-28 regulates oxidative stress-induced cell apoptosis in heart muscle cells, which possibly involves a PDK1/Akt/mTOR-dependent mechanism. MIR-28 could serve as a critical therapeutic target to diminish oxidative stress-induced cell death in the heart.

Dual effects of VEGF-B on activating cardiomyocytes and cardiac stem cells to protect the heart against short- and long-term ischemia-reperfusion injury.

AimsTo investigate whether vascular endothelial growth factor B (VEGF-B) improves myocardial survival and cardiac stem cell (CSC) function in the ischemia–reperfusion (I/R) heart and promotes CSC mobilization and angiogenesis.Methods and resultsOne hour after myocardial ischemia and infarction, rats were treated with recombinant human VEGF-B protein following 24 h or 7 days of myocardial reperfusion. Twenty-four hours after myocardial I/R, VEGF-B increased pAkt and Bcl-2 levels, reduced p-p38MAPK, LC3-II/I, beclin-1, CK, CK-MB and cTnt levels, triggered cardiomyocyte protection against I/R-induced autophagy and apoptosis, and contributed to the decrease of infarction size and the improvement of heart function during I/R. Simultaneously, an in vitro hypoxia-reoxygenation (H/R)-induced H9c2 cardiomyocyte injury model was used to mimic I/R injury model in vivo; in this model, VEGF-B decreased LDH release, blocked H/R-induced apoptosis by inhibiting cell autophagy, and these special effects could be abolished by the autophagy inducer, rapamycin. Mechanistically, VEGF-B markedly activated the Akt signaling pathway while slightly inhibiting p38MAPK, leading to the blockade of cell autophagy and thus protecting cardiomyocyte from H/R-induced activation of the intrinsic apoptotic pathway. Seven days after I/R, VEGF-B induced the expression of SDF-1α and HGF, resulting in the massive mobilization and homing of c-Kit positive cells, triggering further angiogenesis and vasculogenesis in the infracted heart and contributing to the improvement of I/R heart function.ConclusionVEGF-B could contribute to a favorable short- and long-term prognosis for I/R via the dual manipulation of cardiomyocytes and CSCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-0847-3) contains supplementary material, which is available to authorized users.

Abstract 581: Divergent Roles for Mertk and Axl in Resident Vs Recruited Phagocytes After Reperfusion of Ischemic Hearts

Objective: Following myocardial infarction (MI), recruited and resident phagocytes are mobilized at the heart to promote clearance of dying myocardial cells by efferocytosis. This occurs in part through the actions of the macrophage receptor tyrosine kinases, Tyro 3, Axl, and Mertk (TAM), although potential differential contributions of TAMs on recruited vs resident phagocytes, as well as the contribution to clinically-relevant reperfusion, are unknown. We set out to test the significance of recruited vs resident Mertk + phagocytes during ischemia/reperfusion (I/R) and in comparison to Axl + phagocytes. Methods and Results: In contrast to MI, I/R resulted in elevated inflammation and early loss of Mertk on phagocytes in the infarcted tissue with a concomitant increase in serum levels of soluble Mertk in both mice and humans. In mice, Mertk-deficiency during I/R led to decreased cardiac efferocytosis, increased infarct size, and depressed cardiac contractility, newly revealing the importance of efferocytosis during clinically-relevant reperfusion. These endpoints were similarly affected after blocking blood-borne recruited monocytes with CCR2 antagonists, thereby specifically implicating resident Mertk + phagocytes to cardiac repair. We observed the highest expression of Mertk on Ly6C lo MHCII lo cardiac resident macrophages, which resulted in increased efferocytosis-efficiency by these cells compared to other cardiac phagocytes. Shedding of Mertk from Ly6C lo MHCII lo cardiac resident macrophages was observed after I/R and specific inhibition of Mertk cleavage using mice that express a cleavage-resistant Mertk reduced infarct size after I/R, identifying Mertk cleavage as a new therapeutic target after reperfusion injury. Interestingly, Axl was selectively expressed on Ly6C lo MHCII hi cardiac resident macrophages and surprisingly, Axl-deficiency during I/R led to reduced infarct size and improved cardiac contractility, suggesting differential roles between TAMs during cardiac repair. Conclusions: These data newly support the importance of efferocytosis pathways post-I/R and reveal both Axl and specifically Mertk on resident macrophages as key molecular mediators of inflammation resolution during atherothrombosis.

Lipopolysaccharide pretreatment promotes cardiac stem cell migration through heat shock protein 90-dependent β-catenin activation

AimsTo investigate the effect of lipopolysaccharide (LPS) on the migration of cardiac stem cells (CSCs) and whether β-catenin is involved in its underlying mechanism. Main methodsCSCs were isolated from neonatal Sprague–Dawley rat hearts. Migration was detected using 24-well transwell system in vitro cultured CSCs and using carboxyfluorescein diacetate (CFDA)-labelled method in myocardial infarction rat model. The expression of toll like receptor 4 (TLR4), β-catenin, heat shock protein 90 (HSP90) was analyzed using western blotting. Key findingsExposure of CSCs to higher LPS (1μg/mL) for 24h inhibited the cell migration. However, LPS (0.01, 0.1μg/mL) significantly increased the number of migrated CSCs, which reached a peak at 0.01μg/mL. LPS (0.01μg/mL) pretreatment promoted the migration of CFDA-labeled CSCs into the risk area in ischemia–reperfusion rat heart. And injection of LPS-pretreated CSCs also caused a significant decrease in infarct size when compared with LPS-untreated CSCs group. Lower dose of LPS did not influence the expression of TLR4 and total β-catenin protein. However, it enhanced the levels of active β-catenin, nuclear β-catenin, and HSP90 protein. Compared with LPS group, after preincubated with HSP90 inhibitor 17-AAG, the LPS-induced enhancement of active β-catenin protein and nuclear β-catenin protein was abolished. In addition, 17-AAG also prevented the lower dose of LPS-induced cell migration. SignificanceThe results suggested that low dose of LPS pretreatmemt induced increased CSC migration, reduced the infarct size of ischemia–reperfusion heart. The mechanism might be due to the activation and translocation of β-catenin via HSP90-dependent manner.

Nuclear matrix metalloproteinase-2 in the cardiomyocyte and the ischemic-reperfused heart

Matrix metalloproteinases (MMPs) are zinc-dependent proteases involved in intra- and extra-cellular matrix remodeling resulting from oxidative stress injury to the heart. MMP-2 was the first MMP to be localized to the nucleus; however, its biological functions there are unclear. We hypothesized that MMP-2 is present in the nucleus under normal physiological conditions but increases during myocardial ischemia-reperfusion (I/R) injury-induced oxidative stress, proteolyzing nuclear structural proteins. Lamins are intermediate filament proteins that provide structural support to the nucleus and are putative targets of MMP-2. To identify lamin susceptibility to MMP-2 proteolysis, purified lamin A or B was incubated with MMP-2 in vitro. Lamin A, but not lamin B, was proteolysed by MMP-2 into an approximately 50kDa fragment, which was also predicted by in silico cleavage site analysis. Immunofluorescent confocal microscopy and subcellular fractionation showed MMP-2 both in the cytosol and nuclei of neonatal rat ventricular myocytes. Rat hearts were isolated and perfused by the Langendorff method aerobically, or subjected to I/R injury in the presence or absence of o-phenanthroline, an MMP inhibitor. Nuclear fractions extracted from I/R hearts showed increased MMP-2 activity, but not protein level. The level of troponin I, a known sarcomeric target of MMP-2, was rescued in I/R hearts treated with o-phenanthroline, demonstrating the efficacy of MMP inhibition. However, lamin A or B levels remained unchanged in I/R hearts. MMP-2 has a widespread subcellular distribution in cardiomyocytes, including a significant presence in the nucleus. The increase in nuclear MMP-2 activity seen during stunning injury here, indicates yet unknown biological actions, other than lamin proteolysis, which may require more severe ischemia to effect.

0513 : Key role of endothelium in eNOS-dependent cardioprotection with exercise training

Exercise training is a preconditioning strategy to reduce heart ischemiareperfusion (IR) injuries. The activation of endothelial isoform of NOS (eNOS) by exercise is now recognized as playing a key part. However, eNOS is expressed in both coronary endothelial cells and cardiomyocytes, and which eNOS cellular compartment mediates the protective effects of exercise remains unknown. In this study we investigated the role of cardiomyocytes vs. endothelial eNOS in exercise cardioprotection. Rats (n=90) were assigned to sedentary (Sed) or exercised (Ex) group, and experiments were performed in isolated heart, cardiomyocytes or isolated left coronary artery (LCA). First, we observed that exercise increases eNOS activation (phosphorylation on Ser1177) and S-nitrosylation in whole heart but not at cardiomyocyte level. To investigate functional involvement of eNOS at myocyte level during IR cardioprotection, we exposed isolated cardiomyocytes to an anoxia-reoxygenation (AR) procedure in presence or not of L-NAME, an eNOS inhibitor. Exercise reduced cells death and improved cells contractility after AR, but no effect of L-NAME was observed, suggesting an eNOS-independent effect of exercise. Next, to evaluate the contribution of endothelial cells in exercise cardioprotection, an IR procedure was performed on Langendorff hearts, and LCA were isolated after reperfusion. The alteration of endothelium-dependent coronary relaxation induced by IR was mainly reduced in Ex group. To further explore the contribution of endothelium in this cardioprotection, we perfused hearts with Triton X-100 before IR to abolish coronary endothelial cells activity. This inactivation totally suppressed cardioprotective effects of exercise on both heart functional recovery after IR and infarct size, whereas no effect was observed in Sed group. Altogether, the results suggest that coronary endothelial cells rather than cardiomyocytes play a key role in the eNOSdependent cardioprotection of exercise. The author hereby declares no conflict of interest