Treated H9c2 Cells Research Articles

Naringin protects H9C2 cardiomyocytes from chemical hypoxia‑induced injury by promoting the autophagic flux via the activation of the HIF‑1α/BNIP3 signaling pathway.

Naringin, a natural bioflavonoid, has been shown to exert protective effects in multiple cardiovascular diseases; however, the protective effects of naringin against hypoxic/ischemia‑induced myocardial are not yet fully understood. Autophagy is a vital factor involved in the pathogenesis of myocardial injury. The aim of the present study was to investigate the protective effects of naringin on H9c2 cells against chemical hypoxia [cobalt chloride (CoCl2)]‑induced injury. The role of autophagy and the hypoxia‑inducible factor‑1α (HIF‑1α)/Bcl‑2/BCL2 interacting protein3(BNIP3) signaling pathway in the protective effects of naringin were also assessed. The results revealed that naringin pre‑treatment significantly attenuated the CoCl2‑induced cytotoxicity and apoptosis, and also decreased caspase‑3 activity, which had been increased by CoCl2. In addition, CoCl2 increased Beclin‑1 expression, enhanced the IL3B‑II/IL3B‑I ratio and increased p62 expression in the H9C2 cells. Treatment with 3‑methyladenine (3‑MA), a selective inhibitor of autophagy, also blocked CoCl2‑induced cytotoxicity and apoptosis. Notably, treatment with bafilomycin A1 (Baf A1), an inhibitor of the vacuolar H+ ATPase of lysosomes, resulted in an increase in the upregulation of the LC3B‑II/LC3B‑I ratio, but did not further increase the LC3B‑II/LC3B‑I ratio compared with CoCl2 treatment. These results suggested that CoCl2 inhibited the autophagic flux, which resulted in myocardial cell damage. Furthermore, naringin pre‑treatment exacerbated Beclin 1 expression and the increased IL3B‑II/IL3B‑I ratio, and reduced p62 expression in CoCl2‑treated H9C2 cells. 3‑MA and Baf A1 both reversed the protective effects of naringin against CoCl2‑induced injury, indicating that naringin attenuated CoCl2‑induced myocardial cell injury by the increasing autophagic flux. Moreover, naringin treatment resulted in upregulated expression levels of HIF‑1α and BNIP3 in the CoCl2‑treated H9C2 cells. The inhibition of the HIF‑1α/BNIP3 signaling pathway using 3‑(5'‑hydroxymethyl‑2'‑furyl)‑1‑benzylindazole (an inhibitor of HIF‑1α) prevented the effects of naringin on the autophagic flux and reversed its protective effects against CoCl2‑induced injury. Taken together, these results suggest that naringin protects the H9C2 cells against CoCl2‑induced injury by enhancing the autophagic flux via the activation of the HIF‑1α/BNIP3 signaling pathway.

Inhibition of adenosine kinase attenuates myocardial ischaemia/reperfusion injury.

Increased adenosine helps limit infarct size in ischaemia/reperfusion‐injured hearts. In cardiomyocytes, 90% of adenosine is catalysed by adenosine kinase (ADK) and ADK inhibition leads to higher concentrations of both intracellular adenosine and extracellular adenosine. However, the role of ADK inhibition in myocardial ischaemia/reperfusion (I/R) injury remains less obvious. We explored the role of ADK inhibition in myocardial I/R injury using mouse left anterior ligation model. To inhibit ADK, the inhibitor ABT‐702 was intraperitoneally injected or AAV9 (adeno‐associated virus)—ADK—shRNA was introduced via tail vein injection. H9c2 cells were exposed to hypoxia/reoxygenation (H/R) to elucidate the underlying mechanisms. ADK was transiently increased after myocardial I/R injury. Pharmacological or genetic ADK inhibition reduced infarct size, improved cardiac function and prevented cell apoptosis and necroptosis in I/R‐injured mouse hearts. In vitro, ADK inhibition also prevented cell apoptosis and cell necroptosis in H/R‐treated H9c2 cells. Cleaved caspase‐9, cleaved caspase‐8, cleaved caspase‐3, MLKL and the phosphorylation of MLKL and CaMKII were decreased by ADK inhibition in reperfusion‐injured cardiomyocytes. X‐linked inhibitor of apoptosis protein (XIAP), which is phosphorylated and stabilized via the adenosine receptors A2B and A1/Akt pathways, should play a central role in the effects of ADK inhibition on cell apoptosis and necroptosis. These data suggest that ADK plays an important role in myocardial I/R injury by regulating cell apoptosis and necroptosis.

MiR-484 targeting of Yap1-induced LPS-inhibited proliferation, and promoted apoptosis and inflammation in cardiomyocyte.

Apoptosis and inflammation were the main hallmarks of sepsis-induced cardiomyopathy (SIC). Yes-associated protein isoform 1 (Yap1) and miR-484 were involved in mitochondrial fission and apoptosis, especially proapoptotic roles in SIC. Here, we investigated the role of Yap1 and miR-484 in lipopolysaccharide (LPS)-treated H9c2 cells. Yap1 was downregulated, while miR-484 was elevated by LPS treatment. Cell counting kit-8, flow cytometry, western blotting, and ELISA showed that miR-484 inhibitor significantly improved cell viability, decreased apoptosis, suppressed NLRP3 inflammasome formation, and reduced secretion of inflammatory cytokines TNF-α, IL-1β, and IL-6. Yap1, directly targeted by miR-484 shown in the luciferase assay, was more like a compensatory regulator of LPS stimulation. Knockdown of Yap1 inverted the effects of miR-484 inhibitor, including decreased cell viability, and promoted apoptosis and inflammation. These revealed miR-484 directly targeted mRNA of Yap1 to inhibit cell viability, and promote apoptosis and inflammation in LPS-treated H9c2 cells.

Long Noncoding RNA OIP5-AS1 Overexpression Promotes Viability and Inhibits High Glucose-Induced Oxidative Stress of Cardiomyocytes by Targeting MicroRNA-34a/SIRT1 Axis in Diabetic Cardiomyopathy.

Diabetic cardiomyopathy (DCM) is an important complication of diabetes. This study was attempted to discover the effects of long noncoding RNA OIP5-AS1 (OIP5-AS1) on the viability and oxidative stress of cardiomyocyte in DCM. The expression of OIP5-AS1 and microRNA-34a (miR-34a) in DCM was detected by qRT-PCR. In vitro, DCM was simulated by high glucose (HG, 30 mM) treatment in H9c2 cells. The viability of HG (30 mM)-treated H9c2 cells was examined by MTT assay. The reactive oxygen species (ROS), superoxide dismutase (SOD) and malondialdehyde (MDA) levels were used to evaluate the oxidative stress of HG (30 mM)-treated H9c2 cells. Dual-luciferase reporter assay was used to confirm the interactions among OIP5-AS1, miR-34a and SIRT1. Western blot was applied to analyze the protein expression of SIRT1. The expression of OIP5-AS1 was down-regulated in DCM, but miR-34a was up-regulated. The functional experiment stated that OIP5-AS1 overexpression increased the viability and SOD level while decreased the ROS and MDA levels in HG (30 mM)-treated H9c2 cells. The mechanical experiment confirmed that OIP5-AS1 and SIRT1 were both targeted by miR-34a with the complementary binding sites at 3'UTR. MiR-34a overexpression inhibited the protein expression of SIRT1. In the feedback experiments, miR-34a overexpression or SIRT1 inhibition weakened the promoting effect on viability and mitigated the reduction effect on oxidative stress caused by OIP5-AS1 overexpression in HG (30 mM)-treated H9c2 cells. OIP5-AS1 overexpression enhanced viability and attenuated oxidative stress of cardiomyocyte via regulating miR-34a/SIRT1 axis in DCM, providing a new therapeutic target for DCM.

Betulinic Acid Protects DOX-Triggered Cardiomyocyte Hypertrophy Response through the GATA-4/Calcineurin/NFAT Pathway.

Cardiac hypertrophy is a major risk factor for heart failure and leads to cardiovascular morbidity and mortality. Doxorubicin (DOX) is regarded as one of the most potent anthracycline antibiotic agents; however, its clinical usage has some limitations because it has serious cardiotoxic side effects such as dilated cardiomyopathy and congestive heart failure. Betulinic acid (BA) is a pentacyclic-cyclic lupane-type triterpene that has been reported to have anti-bacterial, anti-inflammatory, anti-vascular neogenesis, and anti-fibrotic effects. However, there is no study about its direct effect on DOX induced cardiac hypertrophy and apoptosis. The present study aims to investigate the effect of BA on DOX-induced cardiomyocyte hypertrophy and apoptosis in vitro in H9c2 cells. The H9c2 cells were stimulated with DOX (1 µM) in the presence or absence of BA (0.1–1 μM) and incubated for 24 h. The results of the present study indicated that DOX induces the increase cell surface area and the upregulation of hypertrophy markers including atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), beta-myosin heavy chain (β-MHC), and Myosin Light Chain-2 (MLC2) in H9c2 cells. However, the pathological hypertrophic responses were downregulated after BA treatment. Moreover, phosphorylation of JNK, ERK, and p38 in DOX treated H9c2 cells was blocked by BA. As a result of measuring the change in ROS generation using DCF-DA, BA significantly inhibited DOX-induced the production of intracellular reactive oxygen species (ROS) when BA was treated at a concentration of over 0.1 µM. DOX-induced activation of GATA-4 and calcineurin/NFAT-3 signaling pathway were remarkably improved by pre-treating of BA to H9c2 cells. In addition, BA treatment significantly reduced DOX-induced cell apoptosis and protein expression levels of Bax and cleaved caspase-3/-9, while the expression of Bcl-2 was increased by BA. Therefore, BA can be a potential treatment for cardiomyocyte hypertrophy and apoptosis that lead to sudden heart failure.

Shikonin Ameliorates LPS-Induced Cardiac Dysfunction by SIRT1-Dependent Inhibition of NLRP3 Inflammasome.

Shikonin (SHI) is an anti-inflammatory agent extracted from natural herbs. It is still unknown whether SHI ameliorates lipopolysaccharide (LPS)-induced cardiac dysfunction. This study aims to explore the protective effects of SHI on LPS-induced myocardial injury and its mechanism. The LPS-induced cardiac dysfunction mouse model was employed to investigate the protective effects of SHI. In the present study, we found that SHI treatment improved the survival rate and cardiac function and remarkably ameliorated the release of inflammatory cytokines and macrophage infiltration in heart tissue of LPS-treated mice. SHI also reduced lactate dehydrogenase (LDH) and cardiac troponin (cTn) release, cell inflammation, and apoptosis in LPS plus adenosine triphosphate (ATP)-treated H9c2 cells. In addition, SHI significantly upregulated silent information regulator 1 (SIRT1) expression and suppressed the upregulation of NOD-like receptor protein 3 (NLRP3), cleaved caspase-1, and caspase-1 activity in heart tissues induced by LPS. Meanwhile, we got the same results in LPS plus ATP-treated H9c2 cells in vitro. Further, SIRT1 inhibitor or siRNA partially blocked SHI-mediated upregulation of SIRT1 expression and downregulation of NLRP3, cleaved caspase-1, and caspase-1 activity in heart tissues induced by LPS. Therefore, we conclude that SHI ameliorates LPS-induced cardiac dysfunction by inhibiting SIRT1-dependent activation of NLRP3 inflammasomes and might be a promising therapeutic strategy for the treatment of LPS-induced cardiac dysfunction.

Critical functions of microRNA-30a-5p-E2F3 in cardiomyocyte apoptosis induced by hypoxia/reoxygenation.

The high-mortality rate of cardiovascular diseases (CVDs) is associated with the myocardial ischemia and reperfusion (I/R). Recent investigations have revealed that microRNAs (miRNAs) exert vital functions in the apoptosis of cardiomyocyte cell. Nevertheless, the potential role of miR-30a-5p in the regulation of cardiomyocyte cell apoptosis needs to be illuminated. In the current study, we observed that hypoxia/reoxygenation (H/R) remarkably raised the level of miR-30a-5p but reduced the expression of E2F transcription factor 3 (E2F3) in H9c2 cardiomyocytes. In vivo, miR-30a-5p was found to be significantly upregulated in the hearts of rats following I/R. Downregulation of miR-30a-5p using anti-miR-30a-5p decreased H9c2 cardiomyocytes apoptosis caused by H/R and promoted the proliferation of H9c2 inhibited by H/R. Moreover, E2F3 was a possible target gene of miR-30a-5p and upregulation of miR-30a-5p reduced the expression level of E2F3 in H9c2 cardiomyocytes. We further identified that E2F3 silencing reversed the effect of anti-miR-30a-5p on the proliferation and apoptosis in H/R treated H9c2 cells. These studies suggested that downregulation of miR-30a-5p attenuated the impact of H/R on H9c2 cardiomyocytes through targeting E2F3.

MicroRNA-338 in MSCs-derived exosomes inhibits cardiomyocyte apoptosis in myocardial infarction.

Myocardial infarction (MI) is caused by myocardial ischemia and hypoxia, which causes irreversible damage to the myocardium and seriously endangers human health. Exosomes are small, monolayer-structured extracellular vesicles that transport proteins, lipids, mRNAs, and miRNAs between cells. Mesenchymal stem cells (MSCs) can secrete a large number of exosomes and play a role in many pathophysiological processes. The purpose of this paper was to investigate the role of exosomal microRNA-338 (miR-338) in MI and its underlying mechanism of action. We transfected rat bone marrow-derived MSCs with miR-338 mimic or negative control and extracted exosomes secreted by MSCs. Expression of miR-338 in MSCs, exosomes, and H9c2 cells co-cultured with exosomes was detected by PCR. Then, we treated H9c2 cells with H2O2. We transfected miR-338 inhibitor into H9c2 cells co-cultured with exosomes to further study the function of miR-338. Apoptosis of H9c2 cells were observed by Western blot, flow cytometry, and cell staining. We also established a MI rat model to study the function in vivo and injected exosomes in the myocardium. Seven days later, we used echocardiography to detect the heart function of rats. MiR-338 was upregulated in MSCs transfected with miR-338 mimic, exosomes, and H9c2 cells co-cultured with exosomes. When H9c2 cells were co-cultured with exosomes overexpressing miR-338, the expression of Bax was decreased while the expression of Bcl-2 was increased, and the apoptosis rate was also decreased as shown in flow cytometry, and the amount of caspase3 fluorescence was also decreased. Cardiac function was markedly improved after intramyocardial injection of exosomes overexpressing miR-338 in rats. It was demonstrated using computational tools, Western blot, and Luciferase reporter gene experiments that miR-338 could regulate JNK pathway via targeting MAP3K2. Exosomal miR-338 can inhibit cardiomyocyte apoptosis and improve cardiac function in rats with myocardial infarction by regulating MAP3K2/JNK signaling pathway.

MiR-181c protects cardiomyocyte injury by preventing cell apoptosis through PI3K/Akt signaling pathway.

Cardiomyocyte apoptosis plays an important role in the development of heart failure, which leads to high mortality in patients with cardiovascular diseases. In this study, we are focused to identify the role of miRNA-181c in the regulating of myocardial tissue apoptosis in the doxorubicin (DOX) or hypoxia/reoxygenation (H/R) induced H9C2 cardiomyocyte injury. DOX-induced heart failure animal model was established using mice. Total RNA was extracted from tissue and cell using Trizol. RT-PCR was conducted for real-time RNA quantification. H9c2 cells were collected and labeled using an Annexin V-PI apoptosis kit. Flow cytometry was conducted to identify the cell apoptosis. Rat cardiomyocyte H9c2 cell was treated by 16 hours' hypoxia and 2 hours' reoxygenation to induce cell apoptosis. TUNEL assay was employed for myocardial tissue apoptosis analysis. It was revealed that miR-181c was suppressed on the heart tissue of DOX-induced heart failure animal model. We observed miR-181c overexpression reduced apoptosis through TUNEL assay, which suggested the inhibitory effect of miR-181c on myocardial tissue apoptosis. Transfection of miR-181c mimic could decrease cell apoptosis in H/R treated H9C2 cells in vitro. Under the stimulation of H/R or DOX, miR-181c could downregulate protein expression of Fas, IL-6 and TNF-α, and upregulated Bcl2 and the phosphorylation of Akt. Our study revealed that miR-181c protected heart failure by impeding cardiomyocyte apoptosis through PI3K/Akt pathway, implying the therapeutic role of miR-181c during the exacerbation of the cardiovascular disease.

Suppression of Netrin-1 attenuates angiotension II-induced cardiac remodeling through the PKC/MAPK signaling pathway

BackgroundMyocardial remodeling caused by angiotensin II (Ang II) is essential for the pathological process of heart failure. Netrin-1, which is an axonal guidance cue, has been shown to be involved in the inflammatory response, tumorigenesis, and angiogenesis in non-neuronal tissues. However, the role of Netrin-1 in cardiac remodeling has not been fully elucidated. MethodsThe rat cardiomyocyte cell line H9c2 and primary neonatal rat cardiomyocytes were treated with Ang II. Cells were transfected with siRNA to silence Netrin-1 expression. Real-time polymerase chain reaction and Western blot analysis were used to detect the markers for fibrosis, apoptosis, and hypertrophy in cardiomyocytes. An Annexin V-EGFP/PI cell apoptosis detection kit was used to measure the level of apoptosis caused by angiotensin II. ResultsWe found that Netrin-1 expression was upregulated in the H9c2 cells and the neonatal rat cardiomyocytes stimulated by Ang II. The increased Netrin-1 expression was decreased by valsartan to block AT1R. Importantly, the application of Netrin-1 siRNA significantly alleviated the degrees of myocardial hypertrophy, fibrosis (reflected by Myhc, collagen I, and TGF-β) and apoptosis (reflected by the level of Caspase 3, Bax, and Bcl-2) induced by Ang II. In addition, the silencing of Netrin-1 substantially decreased the phosphorylation of PKCα, JNK, and P38. We treated H9c2 cells with LY317615, SP600125, and SB203580, inhibitors of PKCα, JNK, and P38, respectively, thereby resulting in a substantial decrease in hypertrophy, fibrosis, and apoptosis. ConclusionsAng II produces cardiac hypertrophy, fibrosis, and apoptosis through the upregulation of Netrin-1 and the activation of the AT1R/PKCα/MAPK (JNK, P38) pathway. Suppression of Netrin-1 can relieve Ang II-induced cardiac remodeling via inhibition of the PKCα/MAPK (JNK and P38) signaling pathway. Thus, Netrin-1 may be a novel therapeutic target for Ang II-mediated cardiac remodeling.

MiR-1247-3p protects rat cardiomyocytes against hypoxia/reoxygenation-induced injury via targeting BCL2L11 and caspase-2

Acute myocardial infarction (AMI) represents a severe coronary heart disease with relatively high rate of mortality and usually can lead to the damage of the myocardial tissues. Reperfusion of the ischemic myocardial tissues can minimize AMI-induced damage. As far as we know, the molecular mechanisms underlying ischemia/reperfusion (I/R)-induced injury remains elusive. This study was undertaken to explore the role of miR-1247-3p in regulating myocardial I/R injury. The hypoxia/reoxygenation (H/R)-treated H9c2 cells showed a decreased cell viability and mitochondrial membrane potential with an increase in the apoptosis; furthermore, miR-1247-3p was down-regulated in these cells. MiR-1247-3p overexpression attenuated H/R-induced H9c2 cell injury; while miR-1247-3p knockdown in H9c2 cells exhibited similar effects being observed in H/R-treated cells. The bioinformatics prediction revealed Bcl-2-like protein 11 (BCL2L11) and caspase-2 were two potential targets for miR-1247-3p, and functional assays confirmed that miR-1247-3p targeted both BCL2L11 and caspase-2 3′ untranslated regions, which lead to the repressed expression of these genes. Silencing of BCL2L11 and caspase-2 both, respectively, counteracted the H9c2 cell injury caused by H/R treatment. Moreover, BCL2L11 and caspase-2 overexpression, respectively, impaired the protective effects of miR-1247-3p overexpression on H/R-treated H9c2 cells. The data in the present investigation revealed that miR-1247-3p restoration exhibited protective effects on H/R-induced cardiomyocyte injury through targeting BCL2L11 and caspase-2, implying that miR-1247-3p along with caspase-2/BCL2L11 signaling may provide novel sight for a better understating of I/R-induced myocardial damage. The role of miR-1247-3p might be further confirmed in animal models and clinical studies.

MicroRNA-147 inhibits myocardial inflammation and apoptosis following myocardial infarction via targeting HIPK2.

The incidence of acute myocardial infarction (AMI) is increasing year by year, and it has become one of the diseases with the highest mortality in humans. MicroRNAs (miRNAs) are involved in the regulation of many diseases, including AMI. This study aims to investigate the function of miR-147 in myocardial infarction (MI) and its underlying mechanism of action. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) was used to detect miR-147, Bcl-2 mRNA, and Bax mRNA expressions. Enzyme-linked immunosorbent assay (ELISA) kits were used to detect the levels of inflammatory factors (TNF-α, IL-6, IL-β) and lactate dehydrogenase (LDH). Besides, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay was performed to detect cell viability. Cell apoptosis was observed using terminal dexynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Moreover, echocardiography was utilized to measure cardiac function of rats. HIPK2 expression was detected by Western blot. MiR-147 expression was significantly decreased in rat MI model and H2O2 treated H9c2 cells. H2O2 treatment increased the expression of inflammatory factors in H9c2 cells and induced apoptosis, while such effects were inhibited by overexpression of miR-147. Overexpression of miR-147 reversed the decrease in Bcl-2 expression and the increase in Bax expression in H9c2 cells caused by H2O2. In addition, overexpression of miR-147 could improve cardiac function and reduce serum LDH levels in myocardial infarction rats. Through TargetScan, we found that HIPK2 might have a binding site for miR-147. Moreover, overexpression of miR-147 could inhibit the expression of HIPK2. In MI, miR-147 expression is decreased in the myocardium and overexpression of miR-147 can inhibit myocardial inflammation and apoptosis and improve cardiac function in rats via targeting HIPK2.

Periostin aggravates NLRP3 inflammasome-mediated pyroptosis in myocardial ischemia-reperfusion injury

Pyroptosis is a form of caspase-1-induced programmed cell death. This study aimed to investigate the effect of periostin (postn) on pyroptosis in myocardial ischemia-reperfusion injury (MIRI). To this end, the differentially expressed genes were obtained from the GSE4105 dataset using the “GEO2R” online tool. Protein-protein interaction networks were constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) database, and Module and Go analysis were conducted using the Cytoscape 3.6 plugs-in MCODE and BINGO, respectively. The analysis showed that postn was a critical gene in the most significant module. Experimental results, including triphenyltetrazolium chloride staining, pathological analysis, TUNEL staining, western blotting, and RT-qPCR assays, showed that MIRI induced caspase-1-mediated pyroptosis by activating the NLRP3 inflammasome. Postn was significantly upregulated in the heart tissues of MIRI rats and in H9C2 cells following hypoxia/reoxygenation (H/R) treatment. In addition, knockdown of postn suppressed the caspase-1-mediated pyroptosis and H/R-mediated NLRP3 inflammasome activation, as evidenced by flow cytometry, CCK8, RT-qPCR, western blotting, and ELISA assays. In contrast, overexpression of postn promoted NLRP3 inflammasome-mediated pyroptosis of H/R-treated H9C2 cells. According to the results of rescue experiments, a caspase-1 inhibitor reduced the increase in NLRP3 inflammasome-mediated pyroptosis induced by overexpression of postn, and the pyroptosis-promoting function of postn overexpression in H/R treated H9C2 cells was reversed by inhibition of NLRP3. In conclusion, postn overexpression promoted the caspase-1-mediated pyroptosis during MIRI by activating the NLRP3.

Semaglutide attenuates excessive exercise-induced myocardial injury through inhibiting oxidative stress and inflammation in rats

AimsTo investigate the protective effects and mechanism of semaglutide on exercise-induced myocardial injury. Main methodsEffects of semaglutide on lipopolysaccharide (LPS)-induced oxidative stress injuries and inflammatory response were assessed in H9c2 cell via MTT assay and Western blot. Quiet control group, over training group and three doses of semaglutide treated overtraining groups were subjected to the swimming training with increasing load for consecutive 10 weeks. Immediately after the last training, the body weight, myocardial morphological changes, injury markers and inflammatory response related proteins of the model rats were analyzed. Key findingsSemaglutide at three concentrations in LPS treated H9c2 cells significantly increased the survival rate and inhibited the apoptosis of cardiomyocytes. Moreover, semaglutide activated AMPK pathway, improve autophagy and inhibited reactive oxygen species production in LPS treated H9C2 cells. In vivo results further revealed that chronic treatment of semaglutide induced significant increase in myocardial injury markers. The pathological histology analysis results showed that semaglutide ameliorated myocardial morphological changes, reduced area of lipid accumulation and significantly decreased the expression levels of NF-κB, TNF-α and IL-1β. SignificanceSemaglutide exert the protective effects on exercise-induced cardiomyopathy by activating AMPK pathway, increasing autophagy, reducing the production of ROS and inflammation-related proteins.

MiR‐26a attenuates cardiac apoptosis and fibrosis by targeting ataxia–telangiectasia mutated in myocardial infarction

Apoptosis and fibrosis play a vital role in myocardial infarction (MI) induced tissue injury. Although microRNAs have been the focus of many studies on cardiac apoptosis and fibrosis in MI, the detailed effects of miR-26a is needed to further understood. The present study demonstrated that miR-26a was downregulated in ST-elevation MI (STEMI) patients and oxygen-glucose deprivation (OGD)-treated H9c2 cells. Downregulation of miR-26a was closely correlated with the increased expression of creatine kinase, creatine kinase-MB and troponin I in STEMI patients. Further analysis identified that ataxia-telangiectasia mutated (ATM) was a target gene for miR-26a based on a bioinformatics analysis. miR-26a overexpression effectively reduced ATM expression, apoptosis, and apoptosis-related proteins in OGD-treated H9c2 cells. In a mouse model of MI, the expression of miR-26a was significantly decreased in the infarct zone of the heart, whereas apoptosis and ATM expression were increased. miR-26a overexpression effectively reduced ATM expression and cardiac apoptosis at Day 1 after MI. Furthermore, we demonstrated that overexpression of miR-26a improved cardiac function and reduced cardiac fibrosis by the reduced expression of collagen type I and connective tissue growth factor (CTGF) in mice at Day 14 after MI. Overexpression of miR-26a or ATM knockdown decreased collagen I and CTGF expression in cultured OGD-treated cardiomyocytes. Taken together, these data demonstrate a prominent role for miR-26a in linking ATM expression to ischemia-induced apoptosis and fibrosis, key features of MI progression. miR-26a reduced MI development by affecting ATM expression and could be targeted in the treatment of MI.

The Effect of Ginsenoside RB1, Diazoxide, and 5-Hydroxydecanoate on Hypoxia-Reoxygenation Injury of H9C2 Cardiomyocytes.

This study was aimed to investigate whether ginsenoside Rb1 (GS-Rb1) from the cardioprotective Chinese medicine ginseng can reduce hypoxia-reoxygenation (HR)-induced damage to cardiomyocytes by protecting the mitochondria. Mitochondria-mediated apoptosis plays a key role during myocardial ischemia-reperfusion injury (MIRI). When MIRI occurs, the continuous opening of the mitochondrial permeability transition pore (mPTP) causes mitochondrial damage and ultimately leads to apoptosis. We treated H9c2 cells, derived from rat embryonic cardiomyoblasts, with GS-Rb1, diazoxide, and 5-hydroxydecanoate (5-HD), using HR to simulate MIRI. We found that GS-Rb1 can reduce mPTP by stabilizing the mitochondrial membrane potential (MMP) and by reducing reactive oxygen species (ROS) during HR. This protects the mitochondria by reducing the release of cytochrome c and the expression of cleaved-caspase-3 in the cytoplasm, ultimately reducing apoptosis. During this process, GS-Rb1 and diazoxide showed similar effects. These findings provide some evidence for a protective effect of GS-Rb1 treatment on MIRI.

Corosolic acid ameliorates cardiac hypertrophy via regulating autophagy.

Aim: In this work, we explored the role of corosolic acid (CRA) during pressure overload-induced cardiac hypertrophy. Methods and results: Cardiac hypertrophy was induced in mice by aortic banding. Four weeks post-surgery, CRA-treated mice developed blunted cardiac hypertrophy, fibrosis, and dysfunction, and showed increased LC3 II and p-AMPK expression. In line with the in vivo studies, CRA also inhibited the hypertrophic response induced by PE stimulation accompanying with increased LC3 II and p-AMPK expression. It was also found that CRA blunted cardiomyocyte hypertrophy and promoted autophagy in Angiotensin II (Ang II)-treated H9c2 cells. Moreover, to further verify whether CRA inhibits cardiac hypertrophy by the activation of autophagy, blockade of autophagy was achieved by CQ (an inhibitor of the fusion between autophagosomes and lysosomes) or 3-MA (an inhibitor of autophagosome formation). It was found that autophagy inhibition counteracts the protective effect of CRA on cardiac hypertrophy. Interestingly, AMPK knockdown with AMPKα2 siRNA-counteracted LC3 II expression increase and the hypertrophic response inhibition caused by CRA in PE-treated H9c2 cells. Conclusion: These results suggest that CRA may protect against cardiac hypertrophy through regulating AMPK-dependent autophagy.

MiR-149 Aggravates Pyroptosis in Myocardial Ischemia-Reperfusion Damage via Silencing FoxO3.

BackgroundMicroRNAs (miRNAs), which modulate the expression of their target genes, are commonly involved in stimulating and adjusting of many processes that result in cardiovascular diseases, contain cardiac ischemia/reperfusion (I/R) damage. However, the expression and role of miR-149 in pyroptosis mediated myocardial I/R damage remains unclear.Material/MethodsReal-time polymerase chain reaction was performed to measure the miR-149 and FoxO3 expression in I/R stimulated H9C2 cells. The cell proliferation, pyroptosis-related inflammatory genes in I/R-treated H9C2 cells transfected miR-149 mimics or miR-149 inhibitor were both explored. We predicted and confirmed miR-149 targets by using bioinformatics analyses and luciferase reporter assay. In addition, the potential relationship between miR-149 and FoxO3 in pyroptosis from I/R treated H9C2 cells was analyzed.ResultsOur results showed that miR-149 was upregulated, while FoxO3 was downregulated in I/R stimulated H9C2 cells. Over-expression of miR-149 inhibited cell viability and promote pyroptosis, however, down-expression of miR-149 had an opposite effect in I/R treated H9C2 cells. Furthermore, miR-149 could negatively regulate FoxO3 expression by binding 3′UTR, whereas silencing of FoxO3 attenuated the effect of miR-149-mimics on cell proliferation and pyroptosis in I/R treated H9C2 cells.ConclusionsOur study found that miR-149 played a critical role in pyroptosis during cardiac I/R injury, and thus, might provide a novel therapeutic target.

Ginsenoside compound-Mc1 attenuates oxidative stress and apoptosis in cardiomyocytes through an AMP-activated protein kinase–dependent mechanism

BackgroundGinsenoside compound-Mc1 (Mc1) is a member of the deglycosylated ginsenosides obtained from ginseng extract. Although several ginsenosides have a cardioprotective effect, this has not been demonstrated in ginsenoside Mc1. MethodsWe treated H9c2 cells with hydrogen peroxide (H2O2) and ginsenoside Mc1 to evaluate the antioxidant effects of Mc1. The levels of antioxidant molecules, catalase, and superoxide dismutase 2 (SOD2) were measured, and cell viability was determined using the Bcl2-associated X protein (Bax):B-cell lymphoma-extra large ratio, a cytotoxicity assay, and flow cytometry. We generated mice with high-fat diet (HFD)–induced obesity using ginsenoside Mc1 and assessed their heart tissues to evaluate the antioxidant effect and the fibrosis-reducing capability of ginsenoside Mc1. ResultsGinsenoside Mc1 significantly increased the level of phosphorylated AMP-activated protein kinase (AMPK) in the H9c2 cells. The expression levels of catalase and SOD2 increased significantly after treatment with ginsenoside Mc1, resulting in a decrease in the production of H2O2-mediated reactive oxygen species. Treatment with ginsenoside Mc1 also significantly reduced the H2O2-mediated elevation of the Bax:Bcl2 ratio and the number of DNA-damaged cells, which was significantly attenuated by treatment with an AMPK inhibitor. Consistent with the in vitro data, ginsenoside Mc1 upregulated the levels of catalase and SOD2 and decreased the Bax:B-cell lymphoma-extra large ratio and caspase-3 activity in the heart tissues of HFD-induced obese mice, resulting in reduced collagen deposition. ConclusionGinsenoside Mc1 decreases oxidative stress and increases cell viability in H9c2 cells and the heart tissue isolated from HFD-fed mice via an AMPK-dependent mechanism, suggesting its potential as a novel therapeutic agent for oxidative stress–related cardiac diseases.

Alprostadil attenuates LPS-induced cardiomyocyte injury by inhibiting the Wnt5a/JNK/NF-κB pathway

BackgroundClinical research has demonstrated that alprostadil has an anti-inflammatory effect; however, to date, its molecular mechanisms remain unclear. This study aimed to examine the anti-inflammatory activity and related mechanisms of alprostadil in lipopolysaccharide (LPS)-treated H9c2 cells.MethodsCell morphology was observed under an inverted light microscope, while cell viability was assessed with the 3‑(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. Enzyme-linked immunosorbent assays (ELISA) were conducted to study biochemical indicators of cellular damage, such as released lactate dehydrase (LDH) and troponin, and inflammatory cytokine levels including interleukin-1β (IL-1β), IL-6, IL-17, and tumor necrosis factor-α (TNF-α). The mRNA expression levels of Wnt5a, c‑jun N‑terminal kinase (JNK), and nuclear factor kappa B (NF-κB) were further investigated by real-time quantitative polymerase chain reaction (RT-PCR). The effects of alprostadil on the Wnt5a/JNK/NF-κB pathway in H9c2 cells was examined by Western blotting.ResultsAlprostadil increased the cell viability of LPS-stimulated H9c2 cells, reduced LDH and troponin production, and attenuated IL-1β, IL-6, IL-17, and TNF-α secretion. Moreover, alprostadil reduced the mRNA expression of Wnt5a, JNK, and NF-κB and decreased the expression of Wnt5a, NF-κB, and the ratio of p‑JNK/JNK in H9c2 cells treated with LPS. The siWnt5a or JNK inhibitor SP600125 significantly augmented the inhibitory effects of alprostadil on the Wnt5a/JNK/NF-κB pathway.ConclusionOur results show that alprostadil has anti-inflammatory effects and could attenuate LPS-induced injury in H9c2 cardiomyocytes via the Wnt5a/JNK/NF-κB pathway.