Mouse Model Of Acute Myocardial Infarction Research Articles

Pyroptosis inhibitors MCC950 and VX-765 mitigate myocardial injury by alleviating oxidative stress, inflammation, and apoptosis in acute myocardial hypoxia

Acute myocardial infarction (AMI) is a prevalent cardiovascular disease with high morbidity and mortality rates worldwide. Pyroptosis is an inflammatory form of programmed cell death that has been linked to various pathological conditions. However, its exact contribution to the onset and progression of heart injury in AMI has not yet fully elucidated. Herein, we established mouse AMI model by ligating the left anterior descending artery and performed transcriptome analysis during the early phase of AMI. Mouse HL-1 and human AC-16 cardiomyocytes were subjected to hypoxia to simulate ischemic injury in vitro. Our results revealed a significant activation of the inflammatory response at 3 h post-ligation, as confirmed by RNA sequencing. We identified the occurrence of NLRP3 inflammasome-mediated pyroptosis in the cardiac tissues of human cases with AMI, as well as in mouse models of AMI and hypoxia-induced cardiomyocytes, using immunohistochemistry staining and Western blotting assays. Concurrently, pharmacological inhibition of NLRP3 inflammasome-mediated pyroptosis with MCC950 and VX-765 effectively decreased hypoxia-induced cardiomyocytes injury, while mitigating myocardial oxidative stress, apoptosis and inflammation caused by hypoxia. Moreover, the circulating levels of gasdermin D (GSDMD), the pyroptosis executor, were remarkably elevated in the plasma of mice with early AMI and in the supernatant of hypoxia-exposed cardiomyocytes in a time-dependent manner using ELISA and Western blotting. Furthermore, the change in circulating GSDMD positively correlated with Creatine Kinase-MB (CK-MB) in the plasma of early-stage AMI mouse. In summary, these findings indicated a critical role for NLRP3 inflammasome-mediated pyroptosis in the progression of AMI, the administration of MCC950 and VX-765 may be attractive candidate therapeutic approaches for cardiac injury caused by acute hypoxia or even AMI. Additionally, the circulating GSDMD exhibits potential as a newly diagnostic biomarker for AMI.

CircDiaph3 aggravates H/R-induced cardiomyocyte apoptosis and inflammation through miR-338-3p/SRSF1 axis

Acute myocardial infarction (AMI) is one of the most prevalent cardiovascular diseases, accounting for a high incidence rate and high mortality worldwide. Hypoxia/reoxygenation (H/R)-induced myocardial cell injury is the main cause of AMI. Several studies have shown that circular RNA contributes significantly to the pathogenesis of AMI. Here, we established an AMI mouse model to investigate the effect of circDiaph3 in cardiac function and explore the functional role of circDiaph3 in H/R-induced cardiomyocyte injury and its molecular mechanism. Bioinformatics tool and RT-qPCR techniques were applied to detect circDiaph3 expression in human patient samples, heart tissues of AMI mice, and H/R-induced H9C2 cells. CCK-8 was used to examine cell viability, while annexin-V/PI staining was used to assess cell apoptosis. Myocardial reactive oxygen species (ROS) levels were detected by immunofluorescence. Western blot was used to detect the protein expression of anti-apoptotic Bcl-2 while pro-apoptotic Bax and cleaved-Caspase-3. Furthermore, ELISA was used to detect inflammatory cytokines production. While bioinformatics tool and RNA pull-down assay were used to verify the interaction between circDiaph3 and miR-338-3p. We found that circDiaph3 expression was high in AMI patients and mice, as well as in H/R-treated H9C2 cells. CircDiaph3 silencing ameliorated apoptosis and inflammatory response of cardiomyocytes in vivo. Moreover, the knockdown of cirDiaph3 mitigated H/R-induced apoptosis and the release of inflammatory mediators like IL-1β, IL-6, and TNF-α in H9C2 cells. Mechanistically, circDiaph3 induced cell apoptosis and inflammatory responses in H/R-treated H9C2 cells by sponging miR-338-3p. Overexpressing miR-338-3p in H/R-treated cells prominently reversed circDiaph3-induced effects. Notably, miR-338-3p inhibited SRSF1 expression in H/R-treated H9C2 cells. While overexpressing SRSF1 abrogated miR-338-3p-mediated alleviation of apoptosis and inflammation after H/R treatment. To summarize, circDiaph3 aggravates H/R-induced cardiomyocyte apoptosis and inflammation through the miR-338-3p/SRSF1 axis. These findings suggest that the circDiaph3/miR-338-3pp/SRSF1 axis could be a potential therapeutic target for treating H/R-induced myocardial injury.

KNOCKDOWN OF CIRC_0001379 ATTENUATES HYPOXIA/REOXYGENATION-INDUCED CARDIOMYOCYTE APOPTOSIS AND INFLAMMATORY RESPONSE BY MIR-98-5P/SOX6 AXIS.

Background: Aberrant expression of circular RNAs (circRNAs) has been revealed to have crucial roles in the pathological processes of cardiovascular disease. Here, this study aimed to investigate the role and mechanism of circ_0001379 in hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury to explore the potential action of circ_0001379 in acute myocardial infarction (AMI). Methods: Levels of genes and proteins were examined by quantitative real-time polymerase chain reaction and western blot. Cell counting kit-8 assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry were used to detect cardiomyocyte proliferation and apoptosis, respectively. The activity of IL-1β, IL-6, and TNF-α was determined by ELISA analysis. The target relationship between miR-98-5p and circ_0001379 or SOX6 (SRY-Box Transcription Factor 6) was verified by dual-luciferase reporter and RNA immunoprecipitation assays. Results: Circ_0001379 was highly expressed in AMI mouse model and H/R-induced cardiomyocytes. Functionally, circ_0001379 silencing attenuated H/R-evoked cardiomyocyte apoptosis and inflammatory response. Mechanistically, circ_0001379 functioned as a sponge for miR-98-5p, which directly targeted SOX6. Moreover, circ_0001379 could regulate SOX6 expression via sponging miR-98-5p. Further rescue experiments showed that inhibition of miR-98-5p reversed the protective effects of circ_0001379 silencing on H/R-induced cardiomyocytes. Besides that, miR-98-5p overexpression abolished H/R-evoked cardiomyocyte apoptosis and inflammatory response, while this condition was abated by SOX6. Conclusion: Circ_0001379 silencing protects cardiomyocytes from H/R-induced apoptosis and inflammatory response by miR-98-5p/SOX6 axis, suggesting a novel therapeutic strategy for AMI prevention.

Deficiency of miR-409-3p improves myocardial neovascularization and function through modulation of DNAJB9/p38 MAPK signaling

Angiogenesis is critical for tissue repair following myocardial infarction (MI), which is exacerbated under insulin resistance or diabetes. MicroRNAs are regulators of angiogenesis. We examined the metabolic regulation of miR-409-3p in post-infarct angiogenesis. miR-409-3p was increased in patients with acute coronary syndrome (ACS) and in a mouse model of acute MI. In endothelial cells (ECs), miR-409-3p was induced by palmitate, while vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGF) decreased its expression. Overexpression of miR-409-3p decreased EC proliferation and migration in the presence of palmitate, whereas inhibition had the opposite effects. RNA sequencing (RNA-seq) profiling in ECs identified DNAJ homolog subfamily B member 9 (DNAJB9) as a target of miR-409-3p. Overexpression of miR-409-3p decreased DNAJB9 mRNA and protein expression by 47% and 31% respectively, while enriching DNAJB9 mRNA by 1.9-fold after Argonaute2 microribonucleoprotein immunoprecipitation. These effects were mediated through p38 mitogen-activated protein kinase (MAPK). Ischemia-reperfusion (I/R) injury in EC-specific miR-409-3p knockout (KO) mice (miR-409ECKO) fed a high-fat, high-sucrose diet increased isolectin B4 (53.3%), CD31 (56%), and DNAJB9 (41.5%). The left ventricular ejection fraction (EF) was improved by 28%, and the infarct area was decreased by 33.8% in miR-409ECKO compared with control mice. These findings support an important role of miR-409-3p in the angiogenic EC response to myocardial ischemia.

Identification of Hypoxia-related Genes in Acute Myocardial Infarction using Bioinformatics Analysis.

Acute myocardial infarction (AMI) remains one of the most fatal diseases worldwide. Persistent ischemia and hypoxia are implicated as significant mechanisms in the development of AMI. However, no hypoxia-related gene targets of AMI have been identified to date. This study aimed to identify potential genes and drugs for AMI using bioinformatics analysis. Two datasets both related to AMI (GSE76387 and GSE161427) were downloaded from the Gene Expression Omnibus to identify differentially expressed genes (DEGs) between AMI and sham mice. Gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. A protein-protein interaction (PPI) network was constructed to identify hub genes using Cytoscape. Candidate genes were identified by the intersection of hub genes and hypoxia-related genes. Western blotting was used to validate the candidate genes in the AMI mouse model. Furthermore, the Drug-Gene Interaction Database was used to predict potential therapeutic drugs targeting all hub genes. Fifty-three upregulated and 16 downregulated genes closely related to AMI were identified. The DEGs were primarily enriched in protein, heparin, and integrin binding. KEGG analysis suggested that focal adhesion, PI3K-Akt signaling pathway, and extracellular matrix-receptor interaction are crucial pathways for AMI. The PPI network analysis identified 14 hub genes, two of which were hypoxia-related. Several agents were found to have therapeutic potential for AMI. This study suggests that connective tissue growth factors and the collagen family members may be candidate targets in treating AMI. Agents targeting these candidates may be potential treatments.

CircSMARCC1 and CircLRBA are potential biomarkers in forensic postmortem diagnosis of acute myocardial infarction

Postmortem diagnosis of acute myocardial infarction (AMI), especially early AMI, is a challenge for forensic scientists. Circular RNAs (circRNA) are a unique type of RNA with a closed loop structure and more stability, compared with linear RNA. We aimed at evaluating whether circRNAs are ideal postmortem diagnostic markers for AMI. We employed bioinformatics methods to screen for target circRNAs. Divergent and convergent primers were used to confirm the loop structure. Ribonuclease R (RNaseR) digestion and artificial simulated room temperature test were performed to evaluate the stability of circRNAs. Furthermore, RT-PCR analysis was performed to assess the expressions of target circRNAs in a mouse model of AMI and in autopsy cases, while the diagnostic significance of circRNAs was evaluated by the receiver-operator characteristic (ROC) curve. The bioinformatics analysis screened out circSMARCC1 and circLRBA as target circRNAs. Agarose gel electrophoresis revealed the loop structure of target circRNAs. RNaseR digestion and the artificial simulated room temperature test showed that the stability of circRNAs was good. In mouse AMI model, circSMARCC1 levels were elevated while circLRBA levels were suppressed. Finally, in forensic autopsy cases, circSMARCC1 levels were significantly elevated, while circLRBA levels were significantly suppressed in the MI and early-MI group, relative to the normal control group. The ROC curve analysis showed that both circSMARCC1 and circLRBA can distinguish between AMI and normal control cases. Futher, a combination of the two circRNAs can increase the diagnostic efficacy of AMI. Thus, circSMARCC1 and circLRBA are potential biomarkers for postmortem diagnosis of AMI.

Abstract 10618: Modified Rna Ccnd2 Promotes Myocardial Remuscularizationin Lv Infarct by Regulating Cardiomyocyte Cell Cycle

The adult mammalian heart has limited capacity to regenerate, due to postnatal cardiomyocyte cell cycle arrest. This study examined whether the cardiomyocyte cell-cycle reactivation can enhance myocardial remuscularization in hearts with acute myocardial infarction (AMI). Because the G1-to-S phase transition of the cell cycle is critically regulated by cyclin D proteins, and the expression of cyclin D2 (CCND2) could regenerate injured myocardium, we hypothesized that a transient and exclusive overexpression of CCND2 in cardiomyocytes can result in myocytes proliferation and therefore, remuscularize myocardial infarct. The transient and exclusive myocyte overexpression of CCND2 was achieved by Specific ModRNA Translation system (SMRTs) 1 . In vitro assessments were performed in hiPSC-derived cardiomyocytes (hiPSC-CMs) by immunofluorescence staining of cell proliferation markers (Ki67, BrdU, PH3, and Aurora B) to evaluate whether cardiomyocyte-specific modified RNA CCND2 ( CMS modRNA-CCND2) could reactivate cell cycle. Using a mouse model of AMI, normal and infarcted mice were compared 4 weeks after AMI to assess the structural and functional outcomes of myocardial injection 100μg CMS modRNA-CCND2. In vitro , the, CMS modRNA-CCND2 resulted in significantly higher CMs cell cycle activity as evidenced by significantly higher percentage of hiPSC-CMs that expressed Ki67 (8.68 ± 1.64 VS 36.68 ± 2.61 , p<0.01), BrdU (8.44 ± 1.14% VS 17.59 ± 0.84%, p<0.01), PH3 (0.51 ± 0.04% VS 1.68 ± 0.09%, p<0.01), and Aurora B (0.47 ± 0.03% VS 1.59 ± 0.07%, P<0.01), CMS modRNA-Luciferase vs CMS modRNA-CCND2, respectively. In vivo, CMs cell cycle activity was significantly increased in SMRT treated hearts 4 weeks post AMI as evidenced by: Ki67 (0.41 ± 0.04% VS 2.00 ± 0.17%, p<0.01), PH3 (0.039 ± 0.004% VS 0.30 ± 0.006%, p<0.01), AMI VS AMI + SMRT, respectively. LV Infarct size also decreased significantly 4 weeks after AMI in AMI + SMRT group of hearts (50.04 ± 1.42% VS 31.28 ± 1.28%, p<0.01). Thus, CMS modRNA-CCND2 could induce myocardial remuscularization by re-activating CM cell- cycle in hearts with AMI. *, equal contribution first author

Electroacupuncture Treats Myocardial Infarction by Influencing the Regulation of Substance P in the Neurovascular to Modulate PGI2/TXA2 Metabolic Homeostasis via PI3K/AKT Pathway: A Bioinformatics-Based Multiomics and Experimental Study.

Acute myocardial infarction (AMI) is the most severe form of coronary heart disease caused by ischemia and hypoxia. The study is aimed at investigating the role of neuropeptides and the mechanism of electroacupuncture (EA) in acute myocardial infarction (AMI) treatment. Compared with the normal population, a significant increase in substance P (SP) was observed in the serum of patients with AMI. PGI2 expression was increased in the SP-treated AMI mouse model, and TXA2 expression was decreased. And PI3K pathway-related genes, including Pik3ca, Akt, and Mtor, were upregulated in myocardial tissue of SP-treated AMI patients. Human cardiomyocyte cell lines (HCM) treated with SP increased mRNA and protein expression of PI3K pathway-related genes (Pik3ca, Pik3cb, Akt, and Mtor). Compared to MI control and EA-treated MI rat models, Myd88, MTOR, Akt1, Sp, and Irak1 were differentially expressed, consistent with in vivo and in vitro studies. EA treatment significantly enriched PI3K/AKT signaling pathway genes within MI-associated differentially expressed genes (DEGs) according to Kyoto Encyclopedia of Genes and Genomes (KEGG). Furthermore, it was confirmed by molecular docking analysis that PIK3CA, AKT1, and mTOR form stable dockings with neuropeptide SP. PI3K/AKT pathway activity may be affected directly or indirectly by EA via SP, which corrects the PGI2/TXA2 metabolic imbalance in AMI. MI treatment is now better understood as a result of this finding.

MiR-208 inhibits myocardial tissues apoptosis in mice with acute myocardial infarction by targeting inhibition of PDCD4.

This article aimed to investigate the role of miR-208 in the apoptosis of myocardial tissues in acute myocardial infarction (AMI) mice. The AMI mouse model was constructed. Then, miR-208 expression in AMI mice was regulated by transfection. The mouse myocardial tissues were subject to hematoxylin-eosin (HE) staining, TUNEL assay, and immunofluorescence analysis. H9c2 cell transfection and hypoxia induction were then completed, and cell apoptosis and cytokine levels were tested. Additionally, RNA pull-down and dual luciferase reporter gene assays were conducted for exploring the relation of miR-208 with programmed cell death 4 (PDCD4). Additionally, fluorescence in situ hybridization (FISH) was conducted for investigating miR-208 and PDCD4 colocalization within H9c2 cells. AMI mice had severe damage, apoptosis, decreased miR-208 expression, increased IL-1β, IL-6, IL-8 levels, whereas reduced IL-10 level within myocardial tissues. H9c2 cells under hypoxia induction exhibited decreased miR-208 expression, promoted apoptosis, increased protein expression of Bax and cleaved-caspase-3, decreased protein expression of Bcl-2 and caspase-3, elevated IL-1β, IL-6, IL-8 levels and decreased IL-10 level. miR-208 upregulation alleviated the damage and apoptosis of myocardial tissues in AMI mice. AMI mice with miR-208 upregulation showed decreased expression of Bax and cleaved-caspase-3, increased expression of Bcl-2 and caspase-3, reduced levels of IL-1β, IL-6, IL-8, whereas an increased level of IL-10. miR-208 showed direct inhibition of PDCD4. PDCD4 and miR-208 were mainly co-expressed in the cytoplasm. The upregulated PDCD4 expression abolished miR-208's suppression of H9c2 cell apoptosis induced by hypoxia. Besides this, miR-208 inhibited myocardial tissue apoptosis in AMI mice by inhibiting PDCD4 expression.

Sulfonated Thermoresponsive Injectable Gel for Sequential Release of Therapeutic Proteins to Protect Cardiac Function after Myocardial Infarction.

Myocardial infarction causes cardiomyocyte death and persistent inflammatory responses, which generate adverse pathological remodeling. Delivering therapeutic proteins from injectable materials in a controlled-release manner may present an effective biomedical approach for treating this disease. A thermoresponsive injectable gel composed of chitosan, conjugated with poly(N-isopropylacrylamide) and sulfonate groups, was developed for spatiotemporal protein delivery to protect cardiac function after myocardial infarction. The thermoresponsive gel delivered vascular endothelial growth factor (VEGF), interleukin-10 (IL-10), and platelet-derived growth factor (PDGF) in a sequential and sustained manner in vitro. An acute myocardial infarction mouse model was used to evaluate polymer biocompatibility and to determine therapeutic effects from the delivery system on cardiac function. Immunohistochemistry showed biocompatibility of the hydrogel, while the controlled delivery of the proteins reduced macrophage infiltration and increased vascularization. Echocardiography showed an improvement in ejection fraction and fractional shortening after injecting the thermal gel and proteins. A factorial design of experimental study was implemented to optimize the delivery system for the best combination and doses of proteins for further increasing stable vascularization and reducing inflammation using a subcutaneous injection mouse model. The results showed that VEGF, IL-10, and FGF-2 demonstrated significant contributions toward promoting long-term vascularization, while PDGF's effect was minimal.

Palmatine Alleviates Acute Myocardial Infarction Through Activating pAMPK/Nrf2 Signaling Pathway in Mouse Model

Acute myocardial infarction is one of the major leading causes for heart failure, which can lead to the irreversible loss of cardiomyocytes and impaired cardiac function. Hence, the efficient therapeutic agents are still urgent. Our study aimed to explore the role of a natural isoquinoline alkaloid, palmatine, in an acute myocardial infarction mouse model. In this study, intragastric administrated palmatine significantly enhanced left ventricle ejection fraction and left ventricle end-systolic of infarcted mice heart. Meanwhile, palmatine administration partially recovered myocardial structure and attenuated the cardiac fibrosis and infiltration of inflammatory cells. In addition, the usage of palmatine further enhanced the increased transforming growth factor (TGF)-beta1 level, reduced the elevated tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta level in the myocardium of acute myocardial infarction–induced mice, as well as elevated the reduced superoxide dismutase production and inhibited the increased malondialdehyde secretion in infarcted myocardium of mice. Meanwhile, acute myocardial infarction led the significant upregulation of Bcl-2-associated X and downregulation of B-cell lymphoma-2 in the myocardium, and palmatine administration statistically enabled to recover the expression changes of these two apoptosis-related proteins. Moreover, palmatine administration obviously elevated the expression levels of phosphorylated AMP-activated protein kinase and nuclear factor erythroid 2–related factor 2 in the myocardium of acute myocardial infarction–induced mice. In a word, our study indicated that palmatine could protect infarcted myocardium of mice from apoptosis, inflammation, and oxidative stress. Our results suggested that palmatine might be a novel therapeutic agent for acute myocardial infarction.Graphical

Blocking the A2B adenosine receptor alleviates myocardial damage by inhibiting spleen-derived MDSC mobilisation after acute myocardial infarction

Background Myeloid-derived suppressor cell (MDSC) mobilisation is an important immune event in acute myocardial infarction (AMI). The A2B adenosine receptor (A2BAR) plays key role in regulating MDSC function, but its specific involvement in MDSC mobilisation in AMI remains unclear. Methods In AMI patients, the circulating MDSC ratio and A2BAR mRNA expression were measured. A mouse AMI model was established by left anterior descending coronary artery (LADCA) ligation. MDSCs were analysed by FACS and immunofluorescence staining (of heart tissue). A2BAR mRNA expression was assessed by qRT-PCR. Myocardial injury was detected by HE staining. Myocardial cell apoptosis was analysed by immunohistochemistry. Cardiac systolic function was evaluated by transthoracic echocardiography. Results In AMI patients, the circulating MDSC ratio was increased and positively correlated with A2BAR mRNA expression (r = 0.86, p < 0.01). In AMI model mice, the percentage of MDSCs was increased in the circulation and infarcted heart and decreased in the spleen. MRS-1754-mediated A2BAR inhibition decreased the MDSC ratio in the circulation and infarcted heart and prevented the decrease in MDSC number in the spleens of mice with AMI. A2BAR blockade inhibited myocardial cell apoptosis, alleviated myocardial inflammatory injury, and improved myocardial systolic function in the AMI mouse model. Similar results were found in mice after splenectomy. Additionally, spleen-derived MDSC injection increased the MDSC ratio in the infarcted heart, increased myocardial cell apoptosis, aggravated myocardial injury, and decreased cardiac systolic function in mice with AMI. Conclusion Blocking A2BAR alleviates myocardial damage and improves myocardial systolic function through inhibition of spleen-derived MDSC mobilisation after AMI. Key Messages Spleen-derived MDSC mobilisation aggravates myocardial inflammatory injury within 24 h of AMI. A2BAR promotes spleen-derived MDSC mobilisation within 24 h of AMI. Blocking A2BAR improves myocardial systolic function through inhibition of spleen-derived MDSC mobilisation.

Identification of Immune-Related Genes in Patients with Acute Myocardial Infarction Using Machine Learning Methods.

ObjectiveThis study aimed to analyze immune-related genes and immune cell components in the peripheral blood of patients with acute myocardial infarction (AMI).MethodsSix datasets were obtained from the GEO repository comprising 88 healthy samples and 215 AMI samples. We performed the weighted gene co-expression analysis (WGCNA) and five machine learning (ML) methods to identify immune-related genes and construct diagnostic models. CIBERSORT algorithm was adopted for the assessment of the degree of immune infiltration. Finally, RT-PCR, immunofluorescence double and immunohistochemistry were conducted to analyze the expression level of the identification of featured immune-related genes and localization relationship in heart tissue of AMI mouse model.ResultsA total of 496 immune-related DEGs were obtained between AMI and normal samples. WGCNA finally determined the co-expression modules that showed the most significantly positively associated with AMI (r=0.41; P<0.001). Among the five ML models, XGBoost had the highest AUC (0.849) and accuracy (0.812) to discriminate patients with AMI from normal in the validation sets. Furthermore, we found that the proportion of chemokine receptor (CCR), macrophages, neutrophils, and Treg cells in the AMI groups was significantly higher than that in the normal groups. In vitro RT-PCR verification revealed that SOCS3, MMP9, and AQP9 expression increased significantly in the AMI mouse model. Among the 22 immune cells, AQP9, MMP9, and SOCS3 displayed the strongest positive correlation with neutrophils. In MI-mice, MPO stained strongly along the lateral cardiomyocytes, whereas it was weaker in sham mice. Combined immunofluorescence was observed in same parts of the cytoplasm of cardiomyocytes in myocardial infarction area, indicating co-localization of MPO with MMP9 and SOCS3 in these areas, respectively.ConclusionImmune-related genes and immune cells are intimately related to AMI. Constructing different ML models based on these biomarkers could be a valuable approach to diagnosing AMI in clinical practice.

LncRNA NORAD promotes the progression of myocardial infarction by targeting the miR-22-3p/PTEN axis.

NORAD is a newly identified long non-coding RNA (lncRNA) that plays an important role in cancers. NORAD has been found to be highly expressed in the mouse model of acute myocardial infarction (AMI). However, the role of NORAD in the regulation of AMI remains unknown. In the present study, we aimed to investigate the function of NORAD in AMI and explore the potential regulatory mechanisms. A mouse model of AMI was established and NORAD was knocked-down. The infarcted size of heart tissues and the cardiac function were evaluated. In addition, two cardiomyocyte cell lines were treated with hypoxia/re-oxygenation (H/R) to mimic AMI . Luciferase reporter assay, RNA pull-down assay, fluorescence hybridization, qRT-PCR, and western blot analysis were performed. Apoptotic cells and the levels of L-lactate dehydrogenase (LDH) and malondialdehyde (MDA) were detected. Our results show that downregulation of NORAD efficiently attenuates heart damage in the AMI mouse model. NORAD interacts with miR-22-3p. Knock-down of NORAD inhibits H/R-induced cell apoptosis and reduces LDH and MDA levels, while its effects are abolished by miR-22-3p inhibitor. MiR-22-3p interacts with PTEN and inhibits its expression. Overexpression of miR-22-3p inhibits H/R-induced cell apoptosis and reduces LDH and MDA levels, while its effects are abolished by overexpression of PTEN. Finally, overexpression of NORAD inhibits the AKT/mTOR signaling pathway, and its effects are attenuated by overexpression of miR-22-3p. Taken together, our study reveals that NORAD promotes the progression of AMI by regulating the miR-22-3p/PTEN axis, and the AKT/mTOR signaling may also be involved in the regulatory processes.

LncRNA-HOTAIR Inhibits H9c2 Apoptosis After Acute Myocardial Infarction via miR-206/FN1 Axis.

Although previous studies showed that long non-coding RNAs (lncRNAs) have critical roles in the pathogenesis of acute myocardial infarction (AMI), the underlying molecular mechanism that lncRNAs participate in MI remains unclear. Herein, we explored the expression of lncRNA HOX antisense non-coding RNA (HOTAIR) in the serum of MI patients and mouse model of AMI. Biological functions of HOTAIR in hypoxic H9c2 cells, the in vitro model of MI, were also assessed. RT-qPCR results showed that HOTAIR expression was downregulated in the serum of AMI patients and AMI mice. HOTAIR overexpression promoted H9c2 cell viability and inhibited cell apoptosis under hypoxic conditions. Mechanically, HOTAIR was regulated by miR-206 and FN1 was the direct target of miR-206. More importantly, miR-206 overexpression or FN1 knockdown reversed the effect of HOTAIR overexpression on H9c2 cell viability and apoptosis under hypoxic conditions. Therefore, targeting the HOTAIR/miR-206/FN1 axis may be a promising therapeutic method for MI.

Gasdermin D inhibition confers antineutrophil-mediated cardioprotection in acute myocardial infarction.

Acute myocardial infarction (AMI) induces blood leukocytosis, which correlates inversely with patient survival. The molecular mechanisms leading to leukocytosis in the infarcted heart remain poorly understood. Using an AMI mouse model, we identified gasdermin D (GSDMD) in activated leukocytes early in AMI. We demonstrated that GSDMD is required for enhanced early mobilization of neutrophils to the infarcted heart. Loss of GSDMD resulted in attenuated IL-1β release from neutrophils and subsequent decreased neutrophils and monocytes in the infarcted heart. Knockout of GSDMD in mice significantly reduced infarct size, improved cardiac function, and increased post-AMI survival. Through a series of bone marrow transplantation studies and leukocyte depletion experiments, we further clarified that excessive bone marrow–derived and GSDMD-dependent early neutrophil production and mobilization (24 hours after AMI) contributed to the detrimental immunopathology after AMI. Pharmacological inhibition of GSDMD also conferred cardioprotection after AMI through a reduction in scar size and enhancement of heart function. Our study provides mechanistic insights into molecular regulation of neutrophil generation and mobilization after AMI, and supports GSDMD as a new target for improved ventricular remodeling and reduced heart failure after AMI.

LncRNA Nuclear Factor of Activated T Cells Knockdown Alleviates Hypoxia/Reoxygenation-induced Cardiomyocyte Apoptosis by Upregulating HIF-1α Expression

Acute myocardial infarction (AMI) has become the most common cause of death in the developed countries. However, its pathogenesis is poorly understood. Increasing studies have revealed that lncRNAs are important modulators of AMI development. This study aimed to explore the function of lncRNA noncoding repressor of nuclear factor of activated T cells (NRON) in hypoxia/reoxygenation (H/R)-stimulated H9c2 cells. NRON expression in peripheral blood of AMI patients and H/R-stimulated H9c2 cells was measured by quantitative real-time polymerase chain reaction. H9c2 cells were transfected with si-NRON or cotransfected with si-NRON and si-hypoxia-inducible factor-1 alpha (HIF-1α). The viability and apoptosis of these cells were evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay and flow cytometer, respectively. In addition, HIF-1α, AKT/mTOR signal pathways and ERK1/2 were detected by western blot. NRON knockdown in the myocardial infarction mouse model was conducted through adeno-associated virus injection, and cardiac function was evaluated by motion-mode echocardiography. The results showed that NRON was highly expressed in peripheral blood of AMI patients and H/R-stimulated H9c2 cells. NRON knockdown promoted cell viability and inhibited cell apoptosis of H/R-stimulated H9c2 cells. Meanwhile, NRON knockdown also significantly attenuated heart damage and improved cardiac function in an AMI mouse model. Furthermore, compared with si-normal control, NRON knockdown increased the levels of HIF-1α, p-AKT, p-mTOR, and p-ERK1/2. HIF-1α knockdown reversed the effects of NRON knockdown in H/R-stimulated-H9c2 cells damage. Overall, our study revealed that NRON knockdown alleviated H/R-induced cardiomyocyte apoptosis by upregulating HIF-1α expression, suggesting that NRON might be a novel therapeutic target for AMI.

Ginsenoside Re inhibits myocardial fibrosis by regulating miR-489/myd88/NF-κB pathway

BackgroundMyocardial fibrosis (MF) is an advanced pathological manifestation of many cardiovascular diseases, which can induce heart failure and malignant arrhythmias. However, the current treatment of MF lacks specific drugs. Ginsenoside Re has anti-MF effect in rat, but its mechanism is still not clear. Therefore, we investigated the anti-MF effect of ginsenoside Re by constructing mouse acute myocardial infarction (AMI) model and AngⅡ induced cardiac fibroblasts (CFs) model. MethodsThe anti-MF effect of miR-489 was investigated by transfection of miR-489 mimic and inhibitor in CFs. Effect of ginsenoside Re on MF and its related mechanisms were investigated by ultrasonographic, ELISA, histopathologic staining, transwell test, immunofluorescence, Western blot and qPCR in the mouse model of AMI and the AngⅡ-induced CFs model. ResultsMiR-489 decreased the expression of α-SMA, collagenⅠ, collagen Ⅲ and myd88, and inhibited the phosphorylation of NF-κB p65 in normal CFs and CFs treated with AngⅡ. Ginsenoside Re could improve cardiac function, inhibit collagen deposition and CFs migration, promote the transcription of miR-489, and reduce the expression of myd88 and the phosphorylation of NF-κB p65. ConclusionMiR-489 can effectively inhibit the pathological process of MF, and the mechanism is at least partly related to the regulation of myd88/NF-κB pathway. Ginsenoside Re can ameliorate AMI and AngⅡ induced MF, and the mechanism is at least partially related to the regulation of miR-489/myd88/NF-κB signaling pathway. Therefore, miR-489 may be a potential target of anti-MF and ginsenoside Re may be an effective drug for the treatment of MF.

Genetic deletion of aryl hydrocarbon receptor activates cardiac NLRP3-inflammasome and suppresses apoptosis in mice with acute myocardial infarction

Abstract Background and purpose There is great interest in understanding the role of immune cells in the inflammatory mechanisms associated with acute myocardial infarction (AMI). Resident cardiac immune cells are activated by damage-associated molecular patterns interacting with specific pattern recognition receptors (PRRs), triggering inflammatory responses. Activated immune cells and fibroblasts then promote prohypertrophic and profibrotic signalling, which induces cardiac hypertrophy, fibrosis and remodelling after AMI. Nucleotide-binding domain leucine-rich repeat-containing proteins (NLRs) are PRRs that have emerged as key therapeutic targets in cardiovascular disease. NLRP3 assembles the inflammasome, which serves as molecular signalling platform to activate caspase-1 and regulate maturation of the potent proinflammatory cytokine IL-1β. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor of the basic Helix-Loop-Helix-PER-ARNT-SIM superfamily that mediates responses against environmental pollutants. Beyond its role in metabolising exogenous toxins as part of an adaptive chemical response, there is growing evidence that AhR is activated by endogenous ligands under physiological conditions. AhR has been proposed as a negative regulator of NLRP3-inflammasome activation. Here we studied the effects of defective AhR signalling on apoptosis, NLRP3-inflammasome activation and survival in a mouse model of AMI. Methods and results AMI was induced in 8–10-weeks-old AhR+/+ and AhR−/− mice by ligation of the left anterior descending coronary artery. Immunofluorescence analysis revealed that the number of TUNEL-positive nuclei and the expression of the apoptotic effector cleaved caspase-3 was higher in hearts from infarcted AhR+/+ mice than from sham-operated AhR+/+ mice 24 h after surgery. A similar analysis in AhR−/− mice revealed the absence of apoptotic markers after AMI. Gene analysis showed that cardiac levels of Nlrp3, apoptosis-associated speck-like protein (Asc) and Il1b were all significantly higher in AhR−/− mice than in AhR+/+ mice 24 h after AMI. Finally, survival analysis showed that 25% of AhR+/+ mice died 72 h after AMI surgery whereas the mortality in AhR−/− mice was 100%. Conclusion Our results show that loss of AhR inhibits cardiac apoptosis and increases NLRP3-mediated inflammation 24 h after AMI, highlighting a possible role for this nuclear receptor in cardiac remodelling post-AMI. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund

Cardioprotective Role of SIRT5 in Response to Acute Ischemia Through a Novel Liver-Cardiac Crosstalk Mechanism.

Protein posttranslational modifications play important roles in cardiovascular diseases. The authors’ previous report showed that the abundance of succinylated and glutarylated proteins was significantly lower in the serum of patients with acute myocardial infarction (AMI) than in that of healthy volunteers, suggesting a potential relationship between protein acylation and AMI. Sirtuin 5 (SIRT5) facilitates the removal of malonyl, succinyl, and glutaryl modification; however, its effects on AMI remain unknown. In this study, the levels of SIRT5 in AMI mouse model was compared. Results showed elevated hepatic SIRT5 after myocardial infarction. Hepatocyte-specific SIRT5 overexpressing mice (liver SIRT5 OE) were generated to address the possible involvement of hepatic SIRT5 in AMI. The areas of myocardial infarction, myocardial fibrosis, and cardiac function in a model of experimental myocardial infarction were compared between liver SIRT5 OE mice and wild-type (WT) mice. The liver SIRT5 OE mice showed a significantly smaller area of myocardial infarction and myocardial fibrosis than the WT mice. The fibroblast growth factor 21 (FGF21) in the blood and myocardium of liver SIRT5 OE mice after AMI was markedly elevated compared with that in WT mice. The results of mass spectrometry showed increased levels of proteins regulating tricarboxylic acid cycle, oxidative phosphorylation, and fatty acid β-oxidation pathways in the liver mitochondria of liver SIRT5 OE mice. These findings showed that SIRT5 may exhibit a cardioprotective effect in response to acute ischemia through a liver-cardiac crosstalk mechanism, probably by increasing the secretion of FGF21 and the improvement of energy metabolism.