Myocardial Ischemia-reperfusion Model Research Articles

Comprehensive analysis of the cardiac proteome in a rat model of myocardial ischemia-reperfusion using a TMT-based quantitative proteomic strategy

BackgroundTraditional studies of the cardiac proteome have mainly investigated in an animal model by two-dimensional gel electrophoresis (2-DE). However, the results have not been of satisfactory quality for an understanding of the underlying mechanism. Recent quantitative proteomic methods have been improved to overcome these limitations. To comprehensively study the cardiac proteome in a rat model of ischemia-reperfusion (IR), we developed a tandem mass tag (TMT)-based quantitative proteomic strategy. Furthermore, using this strategy, we examined the molecular mechanisms underlying the prevention of myocardial infarction by the intake of Triticum aestivum L. extract (TALE), a representative dietary fiber grain.MethodsCardiac proteomes were analyzed by 2-DE as a gel-based approach, and TMT labeling coupled with two-dimensional liquid chromatography (2D-LC) and tandem mass spectrometry (MS/MS) as a non-gel-based quantitative approach. Additionally, gene ontology annotation was conducted by PANTHER database. Several proteins of interest were verified by a Western blot analysis.ResultsTotal 641 proteins were identified commonly from two independent MS datasets using 2D-LC MS/MS. Among these, we identified 151 IR-related proteins that were differentially expressed between the sham-operation group and IR group, comprising 62 up-regulated proteins and 89 down-regulated proteins. Most of the reduced proteins were involved in metabolic processes. In addition, 57 of the IR-related proteins were affected by TALE intake, representing 25 up-regulated proteins and 32 down-regulated proteins. In particular, TALE intake leads to a switch in metabolism to reduce the loss of high-energy phosphates and the accumulation of harmful catabolites (especially reactive oxygen species (ROS)) and to maintain cytoskeleton balance, leading to a reduction in cardiac IR injury.ConclusionsOur study provides a comprehensive proteome map of IR-related proteins and potential target proteins and identifies mechanisms implicated in the prevention of myocardial infarction by TALE intake in a rat IR model.

MiR-19a suppress apoptosis of myocardial cells in rats with myocardial ischemia/reperfusion through PTEN/Akt/P-Akt signaling pathway.

To detect differentially expressed micro ribonucleic acids (miRNAs) in rats with myocardial ischemia/reperfusion (MIR), and to explore the influence of miR-19a on MIR rats and its mechanism. Firstly, the Sprague-Dawley (SD) rats were used to prepare MIR models, RNAs were extracted, and miRNA sequencing analysis was carried out to determine differentially expressed miRNAs related to MIR. Secondly, the predicted target genes of miR-19a were collected, and WebGestalt was applied to analyze gene ontology (GO) and pathway enrichment. Thirdly, the expression of the related proteins and the apoptosis of myocardial cells in MIR rats were detected via Western blotting. Fourthly, the interaction between miR-19a and the target gene phosphatase and tensin homolog (PTEN) was examined through Luciferase reporter assay. Compared with that in the Sham operation (Sham) group, the miR-19a expression in rat myocardial tissues in the MIR group was significantly increased (p<0.05). Compared with those in the miR-negative control (miR-NC) group, the messenger RNA (mRNA) and protein expressions of PTEN in the miR-19a group were notably decreased (p<0.05). In comparison with the miR-NC group, miR-19a group had elevated expression of phosphorylated protein kinase B (p-Akt) (p<0.05). The Luciferase reporter gene assay manifested the direct binding of miR-19a to PTEN mRNA. MiR-19a inhibits the PTEN expression by directly binding to the 3'-UTR of PTEN mRNA, thus activating the Akt/p-Akt signaling pathway to suppress the apoptosis of myocardial cells in MIR injury.

A miR-1275 mimic protects myocardiocyte apoptosis by regulating the Wnt/NF-κB pathway in a rat model of myocardial ischemia-reperfusion-induced myocardial injury.

This study evaluated the cardioprotective effects of a miR-1275 mimic in a rat model of myocardial ischemia-reperfusion (I/R)-induced myocardial injury (MI). Three groups of rats were established: a sham-operated group, a MI group and a MI+miR-1275 group pretreated for 1week i.p. with a miR-1275 mimic at a concentration of 30pmol/mL. MI was induced by I/R. The levels of myocardial enzymes in serum were estimated in all rats, together with haemodynamic functions. The effects of the miR-1275 mimic were determined based on the serum concentrations of inflammatory mediators in the treated vs. sham and MI rats. In addition, western blot assay and immunohistochemical analyses were performed to examine the effect of the miR-1275 mimic on the Wnt/NF-kB signalling pathway in MI rats. Treatment with the miR-1275 mimic attenuated the altered levels of myocardial enzymes and haemodynamic functions seen in MI rats. The myocardial infarct was smaller in rats treated with the miR-1275 mimic than in MI rats. The miR-1275 mimic also reduced myocardiocyte apoptosis and ameliorated the altered Wnt/KF-kB pathway. These results demonstrate the efficacy of the miR-1275 mimic in preventing myocardial I/R-induced MI in rats, by regulating the Wnt/NF-κB pathway.

Protective effect of FBXL10 in myocardial ischemia reperfusion injury via inhibiting endoplasmic reticulum stress.

The aim of the study was to investigate the mechanism and effect of FBXL10 in myocardial ischemia reperfusion injury in vivo and in vitro. The myocardial ischemia reperfusion (I/R) model was established by 30min of coronary occlusion followed by 2h of reperfusion in rats. Western blot and TUNEL assay were used to measure the apoptosis during I/R. The expression levels of endoplasmic reticulum related proteins in myocardial tissues and H9c2 cells were detected by immunohistochemistry staining and immunofluorescence staining. Flow cytometry and CCK-8 were used to detect the apoptosis and viability of H9c2 cells. The results revealed that FBXL10 significantly reduced myocardial infarction, improved the pathological morphology of myocardium, markedly reduced inflammatory response in the myocardial ischemia reperfusion rats. Moreover the expressions of endoplasmic reticulum stress key proteins were caused by I/R were suppressed significantly by FBXL10 treatment, including CHOP, GRP78, ATF4 and p-PERK. Additionally FBXL10 inhibited the expression of endoplasmic reticulum stress key proteins in H/R H9c2 cells. Furthermore, FBXL10 reduced the levels of apoptotic cells and inflammatory response compared with I/R and H/R group. Taken together, we found that FBXL10 could attenuate I/R injury through inhibiting endoplasmic reticulum stress (ERs).

Pharmaceutical Development and Safety Evaluation of a GMP-Grade Fucoidan for Molecular Diagnosis of Cardiovascular Diseases.

The adhesion molecule P-selectin is present on the cell surface of both activated endothelium and activated platelets. The present study describes the pharmaceutical development, safety evaluation, and preclinical efficacy of a micro-dosed radiotracer. The macromolecular nanoscale assembly consisted of a natural compound made of a sulfated fucose-rich polysaccharides (fucoidan) and a radionuclide (technetium-99m) for the detection of P-selectin expression in cardiovascular diseases. After extraction and fractionation from brown seaweeds, the good manufacturing practice (GMP) production of a low molecular weight (LMW) fucoidan of 7 kDa was achieved and full physicochemical characterization was performed. The regulatory toxicology study in rats of the GMP batch of LMW fucoidan revealed no adverse effects up to 400 μg/kg (×500 higher than the expected human dose) and pseudoallergy was not seen as well. In a myocardial ischemia-reperfusion model in rats, the GMP-grade LMW fucoidan labeled with technetium-99m detected P-selectin upregulation in vivo. The present study supports the potential of using 99mTc-fucoidan as an imaging agent to detect activated endothelium in humans.

Paeoniflorin on Rat Myocardial Ischemia Reperfusion Injury of Protection and Mechanism Research

Objective: To study the myocardial benefit effect and mechanism of paeoniflorin on myocardial ischemia reperfusion injury (MIRI) in rats. Methods: Hundred SD rats were randomly divided into 5 groups: sham group, model group, Paeoniflorin (15 mg/kg) group, Paeoniflorin (30 mg/kg) group, and Paeoniflorin (60 mg/kg) group. Myocardial ischemia reperfusion model was established in each group except the sham group. The myocardial infarction and morphological changes were measured by the TTC staining and HE staining respectively. Myocardial caspase-3 was detected by immunohistochemistry. In addition, the protein levels of Bcl-2 and Bax and the expression ratio of p-erk, p-jnk, and p-p38 were detected by Western blot. Myocardial superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were measured by the assay kit. Results: Paeoniflorin (30 mg/kg) and Paeoniflorin (60 mg/kg) can obviously alleviate myocardial infarction caused by MIRI (p < 0.05). HE staining showed that the myocardial morphology in the treatment group was obviously better than that in the model group. WB and immunohistochemistry showed that Paeoniflorin (30 mg/kg) and Paeoniflorin (60 mg/kg) can significantly increase the reduced protein level of bcl-2 (p < 0.05) and reduce the increased protein level of caspase3, bax ­p-erk, p-jnk, and p-p38 caused by MIRI (p < 0.05). The activity of SOD was increased and the level of MDA was decreased after Paeoniflorin treatment. Conclusion: Paeoniflorin preconditioning has a protective effect on MIRI in rats. Its mechanism is related to reducing oxidative stress and apoptosis by inhibiting the expression of apoptosis-related signaling pathway.

Effect of vagus nerve stimulation on tissue damage and function loss in a mouse myocardial ischemia-reperfusion model.

In cardiac ischemia, acute inflammatory responses further increase the detrimental effect on myocardial tissue. Since vagus nerve stimulation (VS) attenuates inflammatory responsiveness this study examines the effect of VS on myocardial damage development in a cardiac ischemia-reperfusion (IR) mouse model. 54 male C57Bl/6j mice were subjected to an IR procedure with or without prior VS. The effects on inflammatory responsiveness, infarct size, cardiac function, neutrophils, lymphocytes and vascular endothelial growth factor (VEGF) in the infarcted myocardium were measured at 48 h after intervention. Group results were compared with unpaired Mann-Whitney or Kruskall-Wallis test. A significant decrease in inflammatory responsiveness was not verified by decreased TNFα levels in blood from VS and IR treated mice. The percentage infarct size over area at risk was smaller in the group with VS + IR compared with IR (22.4 ± 10.2% vs 37.6 ± 9.0%, p = 0.003). The degree of the reduction in cardiac function was not different between the IR groups with or without VS and no group differences were found in amounts of neutrophils, CD3+ lymphocytes and VEGF in the reperfused mouse heart. The present study does not provide clear evidence of a reducing role for VS on cardiac function loss. This could mean that VS has a less inhibiting effect on myocardial inflammation than may be expected from the literature.

Bone marrow mesenchymal stem cell-derived exosomes protect against myocardial infarction by promoting autophagy.

Exosomes have been demonstrated to be effective in the treatment of a variety of cardiac disorders. However, the effects of mesenchymal stem cell (MSC) exosomes on myocardial infarction is yet to be determined. The current study aimed to investigate the potential therapeutic effects of MSC exosomes on myocardial injuries that are caused by myocardial infarction. MSCs were isolated from rat bone marrow and were used for exosome enrichment using culture medium. Confirmation that MSCs and exosomes had been successfully extracted was performed using flow cytometry, electron microscopy and western blot analysis. A rat myocardial ischemia reperfusion (I/R) model was established by ligation of the left anterior descending coronary artery. Rat myocardial injuries were determined using 2,3,5-triphenyltetrazolium chloride, Masson and TUNEL staining. H9c2 cell proliferation, apoptosis and migration were analyzed using 5-ethynyl-2′-deoxyuridine, Hoechst staining, flow cytometry and Transwell assays. Marker gene expression was evaluated using reverse transcription-quantitative PCR, western blot analysis and immunofluorescence. Rat MSC exosomes were revealed to suppress myocardial injury and the myocardiocyte functions that were induced by I/R. The results also demonstrated decreased apoptotic protease activating factor-1 and increased autophagy-related protein 13 expression. The H9c2 cell proliferation and migration inhibition, as well as cell apoptosis during hypoxia-reoxygenation (H/R), were suppressed by rat MSC exosomes, with an alteration of the expression of apoptotic and autophagic genes also being demonstrated. The application of autophagy inhibitor 3-methyladenine significantly mitigated the effect of exosomes on H9c2 cell proliferation and apoptosis, which were induced by H/R. Rat MSC exosomes inhibited myocardial infarction pathogenesis, possibly by regulating autophagy.

MiR-181a affects myocardial ischemia-reperfusion injury in rats via regulating akt signaling pathway.

The aim of this study was to explore the influence of the micro ribonucleic acid (miR)-181a on myocardial ischemia-reperfusion injury (MIRI) in rats by regulating the protein kinase B (Akt) signaling pathway. A total of 30 male Sprague-Dawley rats were randomly divided into three groups, including: sham operation group (Sham group), ischemia-reperfusion group (I/R group), and miR group (MiR-181a group). The model of myocardial ischemia-reperfusion was successfully established in rats. The concentration of blood nitric oxide (NO) was detected by the relative kits. Myocardial apoptosis in rats of the three groups was detected using terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Furthermore, the expressions of myocardial cell apoptosis-related proteins and tumor necrosis factor-α (TNF-α), and the degree of Akt phosphorylation were determined by Western blotting. Compared with Sham group and miR-181a group, I/R group exhibited significantly elevated left ventricular end-diastolic pressure (LVEDP) (p<0.05). However, the left ventricular end-systolic pressure (LVESP), stroke work (SW), differential pressure (DP), end-systolic pressure-volume relationship (ESPVR), and end-diastolic pressure-volume relationship (EDPVR) significantly decreased in the I/R group (p<0.05). In comparison with miR-181a group, the apoptosis index of myocardial cells was remarkably elevated in the I/R group, showing statistically significant differences (p<0.05). The protein bands were analyzed using the Quantity One detection software. The results demonstrated that, compared with the Sham group, I/R group showed significantly elevated expressions of cysteine-aspartic protease (Caspase)-3 and TNF-α in rat myocardial tissues (p<0.05). However, the protein levels of Akt and endothelial NO synthase (eNOS) phosphorylation and NO in rat myocardial cells were significantly down-regulated (p<0.05). MiR-181a activates Akt to promote the phosphorylation of its downstream protein eNOS, inhibit the apoptosis of myocardial cells, and alleviate MIRI.

A novel therapeutic peptide targeting myocardial reperfusion injury.

Regulated cell death is a main contributor of myocardial ischaemia-reperfusion (IR) injury during acute myocardial infarction. In this context, targeting apoptosis could be a potent therapeutical strategy. In a previous study, we showed that DAXX (death-associated protein) was essential for transducing the FAS-dependent apoptotic signal during IR injury. The present study aims at evaluating the cardioprotective effects of a synthetic peptide inhibiting FAS:DAXX interaction. An interfering peptide was engineered and then coupled to the Tat cell penetrating peptide (Tat-DAXXp). Its internalization and anti-apoptotic properties were demonstrated in primary cardiomyocytes. Importantly, an intravenous bolus injection of Tat-DAXXp (1 mg/kg) 5 min before reperfusion in a murine myocardial IR model decreased infarct size by 48% after 24 h of reperfusion. In addition, Tat-DAXXp was still efficient after a 30-min delayed administration, and was completely degraded and eliminated within 24 h thereby reducing risks of potential side effects. Importantly, Tat-DAXXp reduced mouse early post-infarction mortality by 67%. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting IR injury validating Tat-DAXXp as a promising candidate for therapeutic application.

Resina draconis inhibits the endoplasmic-reticulum-induced apoptosis of myocardial cells via regulating miR-423-3p/ERK signaling pathway in a tree shrew myocardial ischemia–reperfusion model

Ischemia-reperfusion (IR) is one of the significant medical problems in China. Triphenyltetrazolium chloride (TTC) staining is used to detect the status of the infarct size, and real-time PCR and western blotting are used to detect expressions of genes. TUNEL assay has been used to detect apoptosis. Using a tree shrew myocardial IR model, we found that in the reperfusion period, resina draconis (RD) treatment reduced the infarct size by TTC staining, and significantly enhanced the superoxide dismutase expression and down-regulated the malondialdehyde concentration in a dose-dependent manner. In hearts showing IR, Bax was increased and Bcl-2 was reduced, and RD treatment inhibited the IR-induced Bax expression and up-regulated the IR suppressed level of Bcl-2. TUNEL assay showed that IR induced the apoptosis of myocardial cells, and RD treatment suppressed the IR-induced apoptosis. CHOP and GRP78 were also upregulated in IR hearts, and RD treatment could significantly attenuate the CHOP and GRP78 levels compared with IR group. We further found that IR decreased the miR-423-3p expression and upregulated its target gene ERK both in mRNA and protein levels, and RD treatment upregulated miR-423-3p expression and downregulated ERK expression compared with the IR group. Importantly, miR-423-3p mimics inhibited IR increased ERK, CHOP and GRP78 expressions, and enhanced IR decreased Bcl-2 expression, and inhibited the IR-induced apoptosis of myocardial cells. The findings of this study suggest that RD treatment inhibited the endoplasmic reticulum induced apoptosis of myocardial cells via regulating miR-423-3p/ERK signaling pathway in a tree shrew myocardial IR model.

Protective effect of gastrodin on myocardial ischemia-reperfusion injury and the expression of Bax and Bcl-2.

The protective effects of gastrodin on myocardial ischemia-reperfusion injury in rats and the underlying mechanism were investigated. Sprague Dawley (SD) rats were randomly divided into three groups, of which the gastrodin group was treated with gastrodin, and the other two groups were treated with normal saline. In the myocardial ischemia-reperfusion model group, myocardial ischemia was induced by ligation of the left anterior descending coronary artery, and myocardial reperfusion was performed by ligature removal. Only thread without ligation for the sham operation group was conducted. The rats were euthanized 8 days after surgery. Heart tissues were harvested and used for measurement of apoptotic rate and expression levels of apoptosis-related proteins. Serum levels of cytokines were measured also using blood samples. Compared with the myocardial ischemia-reperfusion model group, significant reduction of cardiomyocyte apoptosis was observed in the gastrodin group (P<0.05). In the gastrodin group, the protein and mRNA expression levels for Bax and activated caspase-3 decreased, while for Bcl-2 increased (P<0.05). Gastrodin can downregulate inflammatory cytokines (P<0.05) and upregulate anti-inflammatory cytokines such as IL-10 (P<0.05) in serum of SD rats. Therefore, gastrodin played a protective role in myocardial ischemia-reperfusion injury by regulating the expression levels of apoptosis-related signaling proteins and inflammatory cytokines.

Cardioprotective effects of a synthetic peptide targeting the extrinsic apoptotic pathway in a mouse model of ischemia-reperfusion

Apoptosis is a main contributor of myocardial reperfusion injury during acute infarction. In a previous study, we showed that reperfusion injury was mediated by the FAS-dependent apoptotic signal mobilizing the DAXX (death-associated protein) adaptor protein. To evaluate the cardioprotective effects of a synthetic peptide that uncouples FAS to the DAXX downstream pathway. The SPOT technology was used to design a synthetic peptide interfering with FAS:DAXX interaction. This peptide was coupled to the Tat cell penetrating peptide (Tat-DAXXp). Infarct size (TTC staining) and apoptosis (DNA fragmentation) were evaluated in the left ventricle of mice subjected to a surgical protocol of reversible coronary artery ligation and treated by the peptide at the onset of reperfusion. Cellular internalization of the peptide was measured using a fluorescent peptide both in primary cultures of cardiomyocytes or in left ventricles after surgery. Anti-apoptotic properties of the synthetic peptide were demonstrated in primary cardiomyocytes. In vivo, one bolus of Tat-DAXXp (1 mg/kg), injected intravenously 5 min before reperfusion in a murine myocardial ischemia-reperfusion model decreased infarct size by 48% after 24 hours of reperfusion. After a 30-min delayed administration, Tat-DAXXp was still able to protect against reperfusion-induced apoptosis and was completely degraded and eliminated within 24 hours thereby reducing risks of potential side effects. Importantly, post-infarction mortality was reduced by 67% by Tat-DAXXp treatment. Mechanistically, cardioprotection was supported by both anti-apoptotic and pro-survival effects, and an improvement of myocardial functional recovery as evidenced in ex vivo experiments. Our study demonstrates that a single dose of Tat-DAXXp injected intravenously at the onset of reperfusion leads to a strong cardioprotection in vivo by inhibiting ischemia-reperfusion injury.

Modification with CREKA Improves Cell Retention in a Rat Model of Myocardial Ischemia Reperfusion.

Poor cell homing limits the efficacy of cardiac cellular therapy. The homing peptide, cysteine-arginine-glutamic acid-lysine-alanine (CREKA), targets fibrin effectively which is involved in the repair process of tissue injury. Here, we assessed if CREKA-modified stem cells had enhanced fibrin-mediated homing ability resulting in better functional recovery and structural preservation in a rat myocardial injury model. CREKA-modified mesenchymal stem cells (CREKA-MSCs) were obtained via membrane fusion with CREKA-modified liposomes. The fibrin targeting ability of CREKA-MSCs was examined both in vitro and in vivo. Under both static and flow conditions in vitro, CREKA significantly enhanced MSCs binding ability to fibrin clots (2.6- and 2.3-fold, respectively). CREKA-MSCs showed 6.5-fold higher accumulation than unmodified MSCs in injured rat myocardium one day after administration, resulting in better structural preservation and functional recovery. Fibrin is, therefore, a novel target for enhancing homing of transplanted cells to injured myocardium, and the delivery system of fibrin-targeting is on behalf of a universalizable platform technology for regenerative medicine. Stem Cells 2019;37:663-676.

In vivo gum arabic-coated tetrahydrobiopterin protects against myocardial ischemia reperfusion injury by preserving eNOS coupling

AimsExogenous tetrahydrobiopterin (BH4), an indispensable cofactor of endothelial nitric oxide synthase (eNOS), supplementation has been proved to be of advantage to improve cardiovascular function. Nevertheless, due to its highly redox-sensitive and easy to be oxidized, there is an urgent need to develop an appropriate BH4 formulation for clinical therapy. Gum Arabic (GA) has been considered as an alternative biopolymer for the stabilization and coating of drugs. The effects of GA on protecting BH4 from being oxidized were investigated in a rat model of myocardial ischemia-reperfusion (I/R). Main methodsRats were subjected to 60-min of in vivo left coronary artery occlusion and varying periods of reperfusion with or without pre-ischemic GA-coated BH4 supplementation (10 mg/kg, oral). Myocardial infarction, fibrotic area and left ventricle ejection fraction were correlated with cardiac BH4 content, eNOS protein, NOS enzyme activity, and ROS/NO generation. Key findingsPretreatment of rats with GA-coated 6R-BH4, 24 h before myocardial ischemia, resulted in smaller myocardial infarction, improved left ventricular function and inhibited fibrosis, correlated with maintained high levels of cardiac BH4 content, preserved eNOS activation and dimerization, and decreased ROS generation. However in uncoated group, 6R-BH4 treatment did not reduce acute and chronic myocardial I/R injury compared with control I/R rats, which was closely related with the marked loss of myocardial BH4 levels during I/R. SignificanceThese findings provide evidence that in vivo pre-ischemic oral GA-coated BH4 administration preserves eNOS function secondary to maintaining cardiac BH4 content, and confers cardioprotection after I/R.

2-Arachidonoylglycerol mobilizes myeloid cells and worsens heart function after acute myocardial infarction.

Myocardial infarction (MI) leads to an enhanced release of endocannabinoids and a massive accumulation of neutrophils and monocytes within the ischaemic myocardium. These myeloid cells originate from haematopoietic precursors in the bone marrow and are rapidly mobilized in response to MI. We aimed to determine whether endocannabinoid signalling is involved in myeloid cell mobilization and cardiac recruitment after ischaemia onset. Intravenous administration of endocannabinoid 2-arachidonoylglycerol (2-AG) into wild type (WT) C57BL6 mice induced a rapid increase of blood neutrophil and monocyte counts as measured by flow cytometry. This effect was blunted when using cannabinoid receptor 2 knockout mice. In response to MI induced in WT mice, the lipidomic analysis revealed significantly elevated plasma and cardiac levels of the endocannabinoid 2-AG 24 h after infarction, but no changes in anandamide, palmitoylethanolamide, and oleoylethanolamide. This was a consequence of an increased expression of 2-AG synthesizing enzyme diacylglycerol lipase and a decrease of metabolizing enzyme monoacylglycerol lipase (MAGL) in infarcted hearts, as determined by quantitative RT-PCR analysis. The opposite mRNA expression pattern was observed in bone marrow. Pharmacological blockade of MAGL with JZL184 and thus increased systemic 2-AG levels in WT mice subjected to MI resulted in elevated cardiac CXCL1, CXCL2, and MMP9 protein levels as well as higher cardiac neutrophil and monocyte counts 24 h after infarction compared with vehicle-treated mice. Increased post-MI inflammation in these mice led to an increased infarct size, an impaired ventricular scar formation assessed by histology and a worsened cardiac function in echocardiography evaluations up to 21 days. Likewise, JZL184-administration in a myocardial ischaemia-reperfusion model increased cardiac myeloid cell recruitment and resulted in a larger fibrotic scar size. These findings suggest that changes in endocannabinoid gradients due to altered tissue levels contribute to myeloid cell recruitment from the bone marrow to the infarcted heart, with crucial consequences on cardiac healing and function.

Integrative analysis of differentially expressed genes and miRNAs predicts complex T3-mediated protective circuits in a rat model of cardiac ischemia reperfusion

Thyroid hormone (T3) dyshomeostasis in the cardiac ischemia-reperfusion (IR) setting negatively impacts on mitochondria function and extracellular matrix remodeling. The modulation of cardiac miRNAs may represent the underlying molecular mechanisms, but a systems biology perspective investigating this critical issue in depth is still lacking. A rat model of myocardial IR, with or without an early short-term T3-replacement, was used to predict putative T3-dependent miRNA-gene interactions targeted to mitochondria quality control and wound healing repair. As evidenced by mRNA and miRNA expression profiling, the T3 supplementation reverted the expression of 87 genes and 11 miRNAs that were dysregulated in the untreated group. In silico crossing and functional analysis of the T3-associated differentially expressed transcripts, identified a signature of interconnected miRNA-gene regulatory circuits that confer resistance to noxious cascades of acute stress. In this network the T3-down-regulated Tp53, Jun and Sp1 transcription factors emerge as critical nodes linking intrinsic cell death and oxidative stress pathways to adverse remodeling cascades. The data presented here provide a novel insight into the molecular basis of T3 cardioprotection in the early post-IR phase and highlight the contribution of a previously unappreciated complex T3-regulatory network that may be helpful in translating T3 replacement into clinical practice.

Tanshinone modulates the expression of Bcl-2 and Bax in cardiomyocytes and has a protective effect in a rat model of myocardial ischemia-reperfusion

ObjectiveTo investigate the protective activity of tanshinone in a rat model of myocardial ischemia-reperfusion and determine its effect on the expression of Bcl-2 and Bax in cardiomyocytes. MethodsWe established a rat model of myocardial ischemia-reperfusion. Rats were randomly divided into blank (no surgery); saline; and low-dose (2 mg/ml), medium-dose (15 mg/ml), and high-dose (50 mg/ml) tanshinone groups. We measured heart rate and troponin (cTnI) levels, performed TUNEL to detect cardiomyocyte apoptosis, and detected LDH and CK-MB activities in serum by ELISA. We performed RT-qPCR and western blot to detect the expression of Bcl-2 and Bax mRNA and protein in cardiomyocytes. ResultsRats treated with tanshinone experienced more stable heart rate after ischemia-reperfusion compared with those in the saline control group. cTnI decreased after ischemia-reperfusion in mice injected with tanshinone, while cTnI in saline-treated mice increased significantly compared with that in the blank control group. TUNEL staining showed that there were greater apoptotic cardiomyocytes in the saline group, but the tanshinone groups showed fewer apoptotic cardiomyocytes. LDH and CK-MB activities were significantly increased after reperfusion in the saline group (p<0.01) and also in the low- and medium-dose tanshinone groups (p<0.05). However, no significant differences were found in the high-dose tanshinone group (p>0.05). The expression levels of Bcl-2 mRNA and protein in cardiomyocytes of rats were higher in the three tanshinone groups in a dose-sensitive manner than those in the blank and saline groups (p<0.05). By contrast, the expression levels of Bax mRNA and protein were reduced in the three tanshinone groups in a dose-sensitive manner compared to those in the blank and saline groups (p<0.05). ConclusionTanshinone shows a protective effect in a dose-dependent manner in a rat model of myocardial ischemia-reperfusion, suggesting its potential therapeutic use.

Autonomic imbalance in cardiac surgery: A potential determinant of the failure in remote ischemic preconditioning

Remote ischemic preconditioning (RIPC) is a cardioprotective strategy against myocardial damage by ischemia-reperfusion. Many in-vivo and ex-vivo animal researches have demonstrated that RIPC decreases significantly the ischemia-reperfusion myocardial damage, by up to 58% in isolated rat heart. Cardiac artery bypass graft surgery (CABG) is a clinical model of myocardial ischemia-reperfusion and a clinical potential application to RIPC. However, although RIPC has shown successful results in experimental studies, clinical trials on CABG have failed to demonstrate a benefit of RIPC in humans. Strikingly, the main proposed factors associated with this translational failure also impair the balance of the autonomic nervous system (ANS), which has shown to play a key role in RIPC cardioprotection in animal models. Comorbidities, chronic pharmacological treatment and anesthesic drugs - common conditions in CABG patients - cause an ANS imbalance through parasympathetic activity decrement. On the other hand, ANS and specially the parasympathetic branch are essentials to get cardioprotection by RIPC in animal models. Consequently, we propose that ANS imbalance in CABG patients would explain the failure of RIPC clinical trials. Whether our hypothesis is true, many patients could be benefited by RIPC: a cheap, simple and virtually broad-available cardioprotective maneuver. In this paper we discuss the evidence that support this hypothesis and its clinical implications.

IL-37 protects myocardial ischemia reperfusion injury in mice through mediating inflammation response

Objective: To investigate the mechanism of IL-37 in alleviating myocardial ischemia reperfusion injury by regulating inflammatory response balance. Method: 30 Wistar male mice were randomly and equally assigned into Sham group (Sham operation), IL-37 intervention group (I/R plus IL-37 intervention, short for experimental group) and control intervention group (I/R+normal saline, short for control group), respectively. Sham operation or myocardial Ischemia Reperfusion (I/R) model were performed in Wistar male mice. Mice in experimental group received human recombined IL-37 intervention. The infarction area and cardiac function were examined. The expression of TNF-α, IL-1β, IL-6 and TGF-β in serum and myocardial infarction border zone were measured. MPO activity was examined as an indicator of neutrophil recruitment. Result: Compared with control group, I/R infarction area of mice in experimental group were significantly decreased (P<0.01), while EF and FS was increased remarkably (P<0.05). Compared with Sham group, the level of TNF-α, IL-1β, IL-6 and TGF-β in serum and myocardial infarction border zone was increased both in control and experimental group (P<0.01), with significantly increased MPO activity (P<0.01). Moreover, the level of TNF-α, IL-1β, IL-6 and MPO in serum and myocardial infarction border zone in experimental group was significantly lower than that in control group (P<0.01). However, the TGF-β level was higher in experimental group than that in control group (P<0.01). Conclusions: IL-37 could decrease I/R infarction area and improve cardiac function after myocardial ischemia reperfusion injury. The protective effect of IL-37 against I/R injury might be associated with down-regulating the secretion of proinflammatory factors, inhibiting MPO expression and up-regulating the secretion of anti-inflammatory cytokines.