Autophagy In H9c2 Cells Research Articles

Isoliquiritigenin attenuates myocardial ischemia reperfusion through autophagy activation mediated by AMPK/mTOR/ULK1 signaling

BackgroundIschemia reperfusion (IR) causes impaired myocardial function, and autophagy activation ameliorates myocardial IR injury. Isoliquiritigenin (ISO) has been found to protect myocardial tissues via AMPK, with exerting anti-tumor property through autophagy activation. This study aims to investigate ISO capacity to attenuate myocardial IR through autophagy activation mediated by AMPK/mTOR/ULK1 signaling.MethodsISO effects were explored by SD rats and H9c2 cells. IR rats and IR-induced H9c2 cell models were established by ligating left anterior descending (LAD) coronary artery and hypoxia/re-oxygenation, respectively, followed by low, medium and high dosages of ISO intervention (Rats: 10, 20, and 40 mg/kg; H9c2 cells: 1, 10, and 100 μmol/L). Myocardial tissue injury in rats was assessed by myocardial function-related index, HE staining, Masson trichrome staining, TTC staining, and ELISA. Autophagy of H9c2 cells was detected by transmission electron microscopy (TEM) and immunofluorescence. Autophagy-related and AMPK/mTOR/ULK1 pathway-related protein expressions were detected with western blot.ResultsISO treatment caused myocardial function improvement, and inhibition of myocardial inflammatory infiltration, fibrosis, infarct area, oxidative stress, CK-MB, cTnI, and cTnT expression in IR rats. In IR-modeled H9c2 cells, ISO treatment lowered apoptosis rate and activated autophagy and LC3 fluorescence expression. In vivo and in vitro, ISO intervention exhibited enhanced Beclin1, LC3II/LC3I, and p-AMPK/AMPK levels, whereas inhibited P62, p-mTOR/mTOR and p-ULK1(S757)/ULK1 protein expression, activating autophagy and protecting myocardial tissues from IR injury.ConclusionISO treatment may induce autophagy by regulating AMPK/mTOR/ULK1 signaling, thereby improving myocardial IR injury, as a potential candidate for treatment of myocardial IR injury.

Ischemia-induced cardiac dysfunction is exacerbated in adiponectin-knockout mice due to impaired autophagy flux.

Strategies to enhance autophagy flux have been suggested to improve outcomes in cardiac ischemic models. We explored the role of adiponectin in mediating cardiac autophagy under ischemic conditions induced by permanent coronary artery ligation. We studied the molecular mechanisms underlying adiponectin's cardio-protective effects in adiponectin knockout (Ad-KO) compared with wild-type (WT) mice subjected to ischemia by coronary artery ligation and H9c2 cardiomyocyte cell line exposed to hypoxia. Systemic infusion of a cathepsin-B activatable near-infrared probe as a biomarker for autophagy and detection via noninvasive three-dimensional fluorescence molecular tomography combined with computerized tomography to quantitate temporal changes, indicated increased activity in the myocardium of WT mice after myocardial infarction which was attenuated in Ad-KO. Seven days of ischemia increased myocardial adiponectin accumulation and elevated ULK1/AMPK phosphorylation and autophagy assessed by Western blotting for LC3 and p62, an outcome not observed in Ad-KO mice. Cell death, assessed by TUNEL analysis and the ratio of Bcl-2:Bax, plus cardiac dysfunction, measured using echocardiography with strain analysis, were exacerbated in Ad-KO mice. Using cellular models, we observed that adiponectin stimulated autophagy flux in isolated primary adult cardiomyocytes and increased basal and hypoxia-induced autophagy in H9c2 cells. Real-time temporal analysis of caspase-3/7 activation and caspase-3 Western blot indicated that adiponectin suppressed activation by hypoxia. Hypoxia-induced mitochondrial reactive oxygen species production and cell death were also attenuated by adiponectin. Importantly, the ability of adiponectin to reduce caspase-3/7 activation and cell death was not observed in autophagy-deficient cells generated by CRISPR-mediated deletion of Atg7. Collectively, our data indicate that adiponectin acts in an autophagy-dependent manner to attenuate cardiomyocyte caspase-3/7 activation and cell death in response to hypoxia invitro and ischemia in mice.

Semaphorin‑3A alleviates cardiac hypertrophy by regulating autophagy.

Cardiac hypertrophy, characterized by cardiomyocyte enlargement, is an adaptive response of the heart to certain hypertrophic stimuli; however, prolonged hypertrophy results in cardiac dysfunction and can ultimately cause heart failure. The present study evaluated the role of semaphorin-3A (Sema3A), a neurochemical inhibitor, in cardiac hypertrophy, utilizing an isoproterenol (ISO) induced H9c2 cell model. Cells were stained with rhodamine-phalloidin to assess the cell surface area and reverse transcription-quantitative PCR was performed to quantify mRNA expression levels of Sema3A, brain natriuretic factor (BNF) and β-myosin heavy chain (β-MHC). The protein expression levels of the autophagy-related proteins light chain 3 (LC3), p62 and Beclin-1, and the Akt/mTOR signaling pathway associated proteins Akt, phosphorylated (p)-Akt, mTOR, p-mTOR, 4E-binding protein 1 (4EBP1) and p-4EBP1 were semi-quantified using western blotting. Rapamycin, a canonical autophagy inducer, was administered to H9c2 cells to elucidate the regulatory mechanism of Sema3A. The results indicated significantly increased cell surface area and elevated BNF and β-MHC mRNA expression levels, increased LC3II/I ratio and Beclin-1 protein expression levels and significantly decreased p62 protein expression levels after treatment of H9c2 cardiomyocytes with ISO for 24 h. Sema3A overexpression improved ISO-induced hypertrophy in H9c2 cells, indicated by decreased cell surface area and reduced BNF and β-MHC mRNA expression levels. Moreover, Sema3A overexpression inhibited ISO-induced autophagy in H9c2 cells, indicated by decreased LC3II/I ratio and Beclin-1 protein expression levels and increased p62 protein expression levels. The autophagy activator rapamycin partially inhibited the protective effect of Sema3A on ISO-induced hypertrophy. Sema3A overexpression suppressed the decrease of the protein expression levels of p-Akt, mTOR and their downstream target 4EBP1, which is induced by ISO. Collectively, these results suggested Sema3A prevented ISO-induced cardiac hypertrophy by inhibiting autophagy via the Akt/mTOR signaling pathway.

Catalpol Ameliorates Myocardial I/R Injury Through PI3k/Akt/P53 Signaling-mediated Anti-autophagy and Anti-apoptosis

Background Myocardial infarction (MI) is a leading cardiovascular disease worldwide, with high mortality. Purpose The study aimed to observe the mechanism of catalpol inhibiting apoptosis and autophagy in H9c2 cells after ischemia/reperfusion (I/R) injury. Materials and Methods We grouped H9c2 cells into 4 groups: control, I/R injury, catalpol (I/R injury +1 ug/mL catalpol treatment), and wortmannin (I/R injury + 1 ug/mL catalpol +150 nM wortmannin treatment) groups. Both catalpol and wortmannin group cells were given drug treatment 30 min before I/R injury. At 2h post-I/R insult, we used flow cytometry for detecting cellular apoptosis and reactive oxygen species (ROS) levels. We identified p-PI3K, p-Akt, PI3K, Akt, p53, Bcl-2, Caspase-3, Bax, beclin1, LC3II, and LC3I protein expression levels. Results I/R significantly increased the apoptosis rate and ROS level of H9c2 cells, increased expression levels of p53, LC3II/LC3I, and Caspase-3, and decreased p-Akt/Akt, beclin1, Bcl-2/Bax, and p-PI3K/PI3K expression levels. Catalpol can reduce the expressions of p53, Caspase-3, and LC3II/LC3I ( p < 0.5). Catalpol can increase the expression levels of Akt/ p-Akt, Bax/ Bcl-2, and beclin1. Also, it can inhibit apoptosis and autophagy levels of H9c2 cells ( p < 0.5). Wortmannin, a PI3 K-specific inhibitor, was able partially to block the catalpol’s and anti-autophagic and anti-apoptotic effects. Conclusion Catalpol can inhibit apoptosis, reduces excessive autophagy, and alleviates the effects of myocardial ischemia-reperfusion through PI3K/Akt/p53 pathway.

Arsenic trioxide-induced cardiotoxicity triggers ferroptosis in cardiomyoblast cells.

Arsenic trioxide (ATO) has been found to be effective in acute promyelocytic leukemia. However, ATO-induced severe cardiotoxicity limits its clinical application. To date, the mechanisms of ATO-induced cardiotoxicity remain unclear. It is hypothesized that ferroptosis may trigger ATO-induced cardiotoxicity; however, this has not yet been investigated. To clarify this hypothesis, rat cardiomyocyte H9c2 cells were treated with ATO with or without ferrostain-1 (Fer-1). The results indicated that ATO exposure induced H9c2 cell death and apoptosis, and the ferroptosis inhibitor Fer-1, administered for 24h before ATO exposure, suppressed ATO-induced cell death, and apoptosis, as determined by Annexin V-APC/7-AAD apoptosis assay. Furthermore, Fer-1 displayed a cardioprotective effect through inhibiting the ATO-induced production of intracellular reactive oxygen species, improving the ATO-induced loss of the mitochondrial membrane potential, alleviating hyperactive endoplasmic reticulum stress, and alleviating the ATO-induced impairment in autophagy in H9c2 cells. Overall, the cardioprotective effect of Fer-1 against ATO-induced cell injury implies that ATO may trigger ferroptosis to induce cardiotoxicity. These findings lay the foundation for exploring the potential value of ferroptosis inhibitors against ATO-induced cardiotoxicity in the future.

Dexmedetomidine improves myocardial ischemia-reperfusion injury by increasing autophagy via PINK1/PRKN pathway

Ischemic heart disease poses a great threat to human life with its high morbidity and mortality. Timely reperfusion is considered as the most effective intervention for clinical treatment of myocardial ischemia. Reperfusion can commonly lead to cell death and myocardial injury. There has already been evidence that dexmedetomidine (DEX) can protect the injury of myocardial ischemia reperfusion (MI/R). The aims of this research are to explain DEX’s functions in protecting MI/R injury as well as probing into its inner mechanisms. H9c2 cells was adopted to generate the injury of MI/R cell as a result of hypoxia/reoxygenation (H/R). MTT test was used to examine cell activity and flow cytometry was utilized to check cell apoptosis rate. Western blot test was adopted to appraise the protein expressions of Parkin RBR E3 ubiquitin protein ligase (PRKN or Parkin), PTEN-induced kinase-1 (PINK1) and markers associate with autophagy (Light Chain 3-II/I (LC3-II/I)). Immunofluorescence was applied to estimate the LC3 level. Next, the function of DEX in protecting the injury of H/R induced cell by PINK1/PRKN pathway was confirmed. DEX treatment significantly promoted viability, improved apoptotic rate and increased autophagy in H9c2 cells processed by H/R. Moreover, the protein expressions of PINK1 and PRKN were boosted by DEX, suggesting that DEX induced autophagy through the activation of PINK1/PRKN signaling pathway. In later tests, PINK1 teardown weakened DEX’s effect the injury of myocardial cell induced by H/R. DEX improves MI/R injury by inducing autophagy through activating PINK1/PRKN pathway.

Ginkgolide B Protects Cardiomyocytes from Angiotensin II-Induced Hypertrophy via Regulation of Autophagy through SIRT1-FoxO1.

Ginkgolide B (GB) is an active ingredient extracted from Ginkgo biloba leaves. However, the effects of GB on cardiac hypertrophy remain unclear. The study is aimed at determining whether GB could alleviate cardiac hypertrophy and exploring its underlying molecular mechanism. Rat cardiomyocyte cell line H9c2 cells were pretreated with GB and incubated with angiotensin II (Ang II) to simulate an in vitro cardiac hypertrophy model. Cell viability, cell size, hypertrophy markers, and autophagy were determined in H9c2 cells after Ang II treatment. Proteins involved in autophagy and the SIRT1 pathway were determined by western blot. Our data demonstrated that GB attenuated Ang II-induced cardiac hypertrophy and reduced the mRNA expressions of hypertrophy marker, atrial natriuretic peptide (ANP), and β-myosin heavy chain (β-MHC). GB further increased Ang II-induced autophagy in H9c2 cells and modulated expressions of autophagy-related proteins Beclin1 and P62. Modulation of autophagy using autophagy inhibitor 3-methyladenine (3-MA) could abrogate GB-downregulated transcription of NPPA. We then showed that GB attenuated Ang II-induced oxidative stress and reduction in SIRT1 and FoxO1 protein expression. Finally, the effect of GB on autophagy and cardiac hypertrophy could be reversed by SIRT1 inhibitor EX-527. GB inhibits Ang II-induced cardiac hypertrophy by enhancing autophagy via the SIRT1-FoxO1 signaling pathway and might be a potential agent in treating pathological cardiac hypertrophy.

Potential cytotoxicity of silver nanoparticles: Stimulation of autophagy and mitochondrial dysfunction in cardiac cells.

In the present study, we elucidated the potential cytotoxicity of AgNPs in H9c2 rat cardiomyoblasts and assessed the underlying toxicological manifestations responsible for their toxicity thereof. The results indicated that the exposure of AgNPs to H9c2 cardiac cells decreased cell viability in a dose-dependent manner and caused cell cycle arrest followed by induction of apoptosis. The AgNPs treated cardiac cells showed a generation of reactive oxygen species (ROS) and mitochondrial dysfunction where mitochondrial ATP was reduced and the expression of AMPK1α increased. AgNPs also induced ROS-mediated autophagy in H9c2 cells. There was a significant time-dependent increase in intracellular levels of Atg5, Beclin1, and LC3BII after exposure to AgNPs, signifying the autophagic response in H9c2 cells. More importantly, the addition of N-acetyl-L-cysteine (NAC) inhibited autophagy and significantly reduced the cytotoxicity of AgNPs in H9c2 cells. The study highlights the prospective toxicity of AgNPs on cardiac cells, collectively signifying a potential health risk.

Pretreatment of Huoxue Jiedu Formula Ameliorates Myocardial Ischaemia/Reperfusion Injury by Decreasing Autophagy via Activation of the PI3K/AKT/mTOR Pathway.

Background: Myocardial ischaemia/reperfusion (I/R) results in myocardial injury via excessive autophagy. Huoxue Jiedu Formula (HJF) has been widely applied in China for the treatment of ischaemic heart disease. However, the mechanisms of HJF are still poorly understood. Thus, the present experiment was designed to observe the effects of HJF on myocardial I/R injury and explore the possible mechanism. Methods: Myocardial injury in rats subjected to myocardial I/R was reflected by nitrotetrazolium blue chloride staining, thioflavin S staining, serum creatine kinase-MB (CK-MB) and cardiac troponin T (cTnT). Autophagy was determined by electron microscopy, laser confocal microscopy, Q-PCR and western blot. The possible pathway was predicted by network pharmacology and validated in vivo and in vitro. Results: Pretreatment of HJF decreased the no-reflow area, infarcted area, serum CK-MB levels and serum cTnT levels in I/R rat model. In addition, pretreatment of HJF decreased autophagy in heart tissues (decrease in Beclin-1 and LC3-II, and increase in Bcl-2, p62 and ratio of LC3-I/LC3-II). In the vivo study, pretreatment of HJF significantly decreased hypoxia/reoxygenation (H/R)-induced autophagy in H9C2 cells. Network pharmacology was applied to predict the possible mechanism by which HJF affects cardiac autophagy, and the PI3K/AKT/mTOR signalling pathway was the most significantly enriched pathway. And experimental studies demonstrated that pretreatment of HJF increased the phosphorylation of AKT and mTOR, and the effects of HJF on autophagy would be offset by PI3K inhibitor LY294002. Conclusion: Pretreatment of HJF ameliorates myocardial I/R injury by decreasing autophagy through activating PI3K/AKT/mTOR pathway.

A novel danshensu/tetramethypyrazine derivative attenuates oxidative stress-induced autophagy injury via the AMPK-mTOR-Ulk1 signaling pathway in cardiomyocytes.

Myocardial ischemia/reperfusion injury (MIRI) is an inevitable and unsolved clinical problem in the treatment of ischemic heart diseases. Compound DT-010 is a novel danshensu/tetramethylpyrazine derivative and was examined as a candidate for treating MIRI. In the present study, MTT, lactate dehydrogenase assay and Hoechst staining data indicated that DT-010 attenuated tert-butylhydroperoxide (t-BHP)-induced oxidative damage by increasing cell survival, reducing cell damage and decreasing apoptosis in H9c2 cardiomyocytes. Autophagy was assessed by western blotting for microtubule-associated protein 1A/1B-light chain 3 (LC3-II and LC3-I) expression, acridine orange and monodansylcadaverine staining for autophagosome formation and the monomeric red fluorescent protein-green fluorescent protein-LC3 assay for autophagic flow. t-BHP-induced cell damage was aggravated by the autophagy agonist rapamycin and alleviated by the autophagy blocker hydroxy-chloroquine, suggesting that autophagy was involved in t-BHP-induced cardiomyocyte injury. DT-010 pretreatment significantly prevented t-BHP-induced cell damage, which was partially but significantly abolished by rapamycin and significantly improved by hydroxy-chloroquine treatment. DT-010 treatment inhibited t-BHP-induced autophagy in H9c2 cells, reduced phosphorylation of 5'-AMP-activated protein kinase (AMPK) and promoted the phosphorylation of mTOR and unc-51 like autophagy activating kinase 1 (Ulk1). To conclude, DT-010 can serve as a potential candidate for myocardial ischemia-reperfusion injury therapy. The cardioprotective effects of DT-010 could be partially attributed to its inhibition of autophagy via the AMPK-mTOR-Ulk1 signaling pathway.

The MicroRNA-210/Casp8ap2 Pathway Alleviates Hypoxia-Induced Injury in Myocardial Cells by Regulating Apoptosis and Autophagy.

This study was conducted to investigate the role of the miR-210/Caspase8ap2 pathway in apoptosis and autophagy in hypoxic myocardial cells. The miR-control, miR-210 mimic, and miR-210 inhibitor were transfected into rat myocardial H9C2 cells. The transfection efficiency of exogenous miR-210 was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). H9C2 cells were then treated with CoCl2 for 24, 48, and 72 h to generate a myocardial injury model. The apoptosis of H9C2 cells was assessed by flow cytometry. Additionally, a western blot assay was used to determine the expression of the autophagy-associated proteins light chain 3 (LC3), p62 and Beclin-1, and apoptosis-associated proteins Caspase8ap2, cleaved caspase 8, and cleaved caspase 3. We determined that a 48 h hypoxia treatment duration in H9C2 cardiomyocytes induced myocardial injury. Additionally, the overexpression of miR-210 significantly inhibited cell apoptosis. MiR-210 suppressed autophagy by upregulating p62 and downregulating LC3II/I in hypoxic H9C2 cells. Caspase8ap2 was a putative target of miR-210, miR-210 mediated apoptosis, and autophagy of H9C2 cells via suppressing Caspase8ap2. Furthermore, the expression of caspase 8, caspase 3, and Beclin-1 were decreased in response to miR-210. miR-210 exhibits anti-apoptosis and anti-autophagy effects, which alleviate myocardial injury in response to hypoxia.

Ambra1 Alleviates Hypoxia/Reoxygenation Injury in H9C2 Cells by Regulating Autophagy and Reactive Oxygen Species.

Reperfusion therapy is the most important method for treating acute myocardial infarction. However, myocardial ischemia reperfusion injury (MIRI) can offset the benefit of reperfusion therapy and worsen the outcome. In both ischemia and reperfusion, autophagy remains problematic. Activating molecule in Beclin1-regulated autophagy (Ambra1) is an important protein in autophagy regulation, and its function in MIRI remains unclear. Thus, we used H9C2 cells to investigate the function of Ambra1 in MIRI and the underlying mechanisms involved. Hypoxia and reoxygenation of H9C2 cells were used to mimic MIRI in vitro. During hypoxia, autophagy flux was blocked, then recovered in reoxygenation. Ambra1 overexpression increased autophagy in the H9C2 cells, as the LC3B II/I ratio increased, and alleviated cellular necrosis and apoptosis during hypoxia and reoxygenation. This effect was counteracted by an autophagy inhibitor. Knocking down Ambra1 can block autophagy which P62 sediment/supernatant ratio increased while the ratio of LC3B II/I decreased, and worsen outcomes. Ambra1 enhances autophagy in H9C2 cells by improving the stability and activity of the ULK1 complex. Reactive oxygen species (ROS) are an important cause of MIRI. ROS were reduced when Ambra1 was overexpressed and increased when Ambra1 was knocked down, indicating that Ambra1 can protect against hypoxia and reoxygenation injury in H9C2 cells by promoting autophagy and reducing ROS.

The MicroRNA-210/Casp8ap2 Pathway Alleviates Hypoxia-Induced Injury in Myocardial Cells by Regulating Apoptosis and Autophagy

Aim: This study was conducted to investigate the role of the miR-210/Caspase8ap2 pathway in apoptosis and autophagy in hypoxic myocardial cells. Methods: The miR-control, miR-210 mimic and miR-210 inhibitor were transfected into rat myocardial H9C2 cells. The transfection efficiency of exogenous miR-210 was determined by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). H9C2 cells were then treated with CoCl2 for 24, 48 and 72 h to generate a myocardial injury model. The apoptosis of H9C2 cells was assessed by flow cytometry. Additionally, a western blot assay was used to determine the expression of the autophagy-associated proteins light chain 3 (LC3), p62 and Beclin-1, and apoptosis-associated proteins Caspase8ap2, cleaved caspase 8 and cleaved caspase 3. Results: We determined that a 48 h hypoxia treatment duration in H9C2 cardiomyocytes induced myocardial injury. Additionally, the overexpression of miR-210 significantly inhibited cell apoptosis. MiR-210 suppressed autophagy by upregulating p62 and downregulating LC3II/I in hypoxic H9C2 cells. Since Caspase8ap2 was a putative target of miR-210, miR-210 mediated apoptosis and autophagy of H9C2 cells via suppressing Caspase8ap2. Furthermore, the expression of caspase 8, caspase 3 and Beclin-1 were decreased in response to miR-210. Conclusions:miR-210 exhibits anti-apoptosis and anti-autophagy effects, which alleviate myocardial injury in response to hypoxia.

(Pro)renin receptor contributes to hypoxia/reoxygenation-induced apoptosis and autophagy in myocardial cells via the beta-catenin signaling pathway.

(Pro)renin receptor (PRR) contributes to regulating many physiological and pathological processes; however, the role of PRR-mediated signaling pathways in myocardial ischemia/reperfusion injury (IRI) remains unclear. In this study, we used an in vitro model of hypoxia/reoxygenation (H/R) to mimic IRI and carried out PRR knockdown by siRNA and PRR overexpression using cDNA in H9c2 cells. Cell proliferation activity was examined by MTT and Cell Counting Kit-8 (CCK-8) assays. Apoptosis-related factors, autophagy markers and beta-catenin pathway activity were assessed by real-time PCR and western blotting. After 24 h of hypoxia followed by 2 h of reoxygenation, the expression levels of PRR, LC3B-I/II, Beclin1, cleaved caspase-3, cleaved caspase-9 and Bax were upregulated, suggesting that apoptosis and autophagy were increased in H9c2 cells. Contrary to the effects of PRR downregulation, the overexpression of PRR inhibited proliferation, induced apoptosis, increased the expression of pro-apoptotic factors and autophagy markers, and promoted activation of the beta-catenin pathway. Furthermore, all these effects were reversed by treatment with the beta-catenin antagonist DKK-1. Thus, we concluded that PRR activation can trigger H/R-induced apoptosis and autophagy in H9c2 cells through the beta-catenin signaling pathway, which may provide new therapeutic targets for the prevention and treatment of myocardial IRI.

Beta carotene protects H9c2 cardiomyocytes from advanced glycation end product-induced endoplasmic reticulum stress, apoptosis, and autophagy via the PI3K/Akt/mTOR signaling pathway.

BackgroundDiabetic cardiomyopathy (DCM), which is associated with many pathological processes, commonly occurs when advanced glycation end products (AGEs) are present. β-carotene (BC) is a well-known vitamin A precursor that is found in many fruits and vegetables. BC can reduce the risk of cancer and cardiovascular disease. This study aimed to investigate the effect of BC on AGE-induced myocardial injury in vitro.MethodsCell viability test was used to select 40 µM concentrations of BC to treat AGE-induced H2c9 cells. The cell apoptosis was detected by flow cytometry. Western blotting was used to measure the protein expression levels of Bcl-2-associated X protein (Bax), B-cell lymphoma-2 (Bcl-2), cleaved caspase-3, activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), beclin 1, p62,microtubule-associated protein 1 light chain 3 (LC3), phosphorylated PI3K (p-PI3K), phosphorylated Akt (p-AKT), and phosphorylated mTOR (p-mTOR). Enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of lactate dehydrogenase (LDH) and cardiac troponin-1 (cTn-I). Reactive oxygen species (ROS) was detected by flow cytometry. The levels of malondialdehyde (MDA), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD) were used to determine MDA kits, SOD assay kit and GSH-Px kit, respectively.ResultsBC significantly inhibited AGE-induced cell death and apoptosis in H9c2 cells. BC had a suppressive effect on intracellular ROS production and antioxidative enzyme reduction. Moreover, BC decreased hyperactive endoplasmic reticulum (ER) stress and autophagy in H9c2 cells. Furthermore, BC exerted a cardioprotective effect in AGE-induced H9c2 cells via the activation of the PI3K/Akt/mTOR signaling pathway.ConclusionsBC exhibited a cardioprotective effect AGE-induced apoptosis. Our study provides a foundation for further study into the potential value of BC for treating DCM or other heart diseases.

Downregulation of miR-26b-5p, miR-204-5p, and miR-497-3p Expression Facilitates Exercise-Induced Physiological Cardiac Hypertrophy by Augmenting Autophagy in Rats.

Exercise-induced autophagy is associated with physiological left ventricular hypertrophy (LVH), and a growing body of evidence suggests that microRNAs (miRNAs) can regulate autophagy-related genes. However, the precise role of miRNAs in exercise induced autophagy in physiological LVH has not been fully defined. In this study, we investigated the microRNA–autophagy axis in physiological LVH and deciphered the underlying mechanism using a rat swimming exercise model. Rats were assigned to sedentary control (CON) and swimming exercise (EX) groups; those in the latter group completed a 10-week swimming exercise without any load. For in vitro studies, H9C2 cardiomyocyte cell line was stimulated with IGF-1 for hypertrophy. We found a significant increase in autophagy activity in the hearts of rats with exercise-induced physiological hypertrophy, and miRNAs showed a high score in the pathway enriched in autophagy. Moreover, the expression levels of miR-26b-5p, miR-204-5p, and miR-497-3p showed an obvious increase in rat hearts. Adenovirus-mediated overexpression of miR-26b-5p, miR-204-5p, and miR-497-3p markedly attenuated IGF-1-induced hypertrophy in H9C2 cells by suppressing autophagy. Furthermore, miR-26b-5p, miR-204-5p, and miR-497-3p attenuated autophagy in H9C2 cells through targeting ULK1, LC3B, and Beclin 1, respectively. Taken together, our results demonstrate that swimming exercise induced physiological LVH, at least in part, by modulating the microRNA–autophagy axis, and that miR-26b-5p, miR-204-5p, and miR-497-3p may help distinguish physiological and pathological LVH.

MicroRNA-1 facilitates hypoxia-induced injury by targeting NOTCH3.

Cell proliferation, apoptosis, and autophagy have been reported to be related to myocardial ischemia injury. MicroRNAs have attracted wide attention on regulating cell proliferation, apoptosis, and autophagy. miR-1 expression has been reported to be dysregulated in cardiac tissue or cells with hypoxia, while the exact roles as well as underlying mechanism remain poorly understood. In this study, we investigated the potential roles of miR-1 in cell proliferation, apoptosis, and autophagy in hypoxia-treated cardiac injury and explored the underlying mechanism using H9c2 cells. Results showed that hypoxic stimulation inhibited cell proliferation and the expression of miR-1 but promoted cell apoptosis in H9c2 cells. Moreover, overexpression of miR-1 promoted cell apoptosis and inhibited cell proliferation and autophagy in H9c2 cells treated with hypoxia, while its knockdown played an opposite effect. In addition, bioinformatics, luciferase reporter, and RNA immunoprecipitation analyses indicated that NOTCH3 was a direct target of miR-1 and its upregulation reversed the effects of miR-1 on cell proliferation, apoptosis, and autophagy in hypoxia-treated H9c2 cells. Taken together, our data suggested that miR-1 promoted hypoxia-induced injury by targeting NOTCH3, indicating novel therapeutic targets for treatment of myocardial ischemia injury.

Astragaloside IV enhances GATA-4 mediated myocardial protection effect in hypoxia/reoxygenation injured H9c2 cells

Background and aimThe transcription factor GATA-4 plays an important role in myocardial protection. Astragaloside IV (Ast-IV) was reported with the effects on improving cardiac function after ischemia. In this study, we explored how Ast-IV interacts with GATA-4 to protect myocardial cells H9c2 against Hypoxia/Reoxygenation (H/R) stress. Methods and resultsH9c2 cells were cultured under the H/R condition. Various cell activity and morphology assays were used to assess the rates of apoptosis and autophagy. In these H/R injured H9c2 cells, increased apoptosis (P < 0.01) and autophagosome number (P < 0.01) were observed, and the addition of Ast-IV ameliorated this tendency. Mechanistically, we used the RT-qPCR and Western blot to evaluate the expressions of various molecules. The results showed that Ast-IV treatment upregulated gene expression of GATA-4 (P < 0.01) and the survival factors (Bcl-2, P < 0.05; p62, P < 0.01), but suppressed apoptosis and autophagy related genes (PARP, Caspase-3, Beclin-1, and LC3-II; All P < 0.01). Furthermore, overexpressing of GATA-4 by its agonist phenylephrine can also protect H/R injured H9c2 cells, and the addition of Ast-IV further enhanced this protection of GATA-4. In contrast, silencing GATA-4 expression abolished the H/R protection of Ast-IV, which demonstrated that the myocardial protection of Ast-IV is mediated by GATA-4. Lastly, along with GATA overexpression, enhanced interactions between Bcl-2 and Beclin-1 were detected by Chromatin immunoprecipitation (P < 0.01). ConclusionAst-IV rescued the H/R injury induced apoptosis and autophagy in H9c2 cells. Ast-IV treatment can stimulate the overexpression of GATA-4, and further enhanced the myocardial protection effect of GATA-4.

Qi-Li-Qiang-Xin Alleviates Isoproterenol-Induced Myocardial Injury by Inhibiting Excessive Autophagy via Activating AKT/mTOR Pathway.

Background: Apoptosis and autophagy are two important patterns of cell death in the process of heart failure. Qi-Li-Qiang-Xin (QLQX), a traditional Chinese medicine, has been frequently used in the treatment of chronic heart failure (CHF) in China. However, the potential effect of QLQX on autophagy has not been reported. In this study, we aimed to investigate whether QLQX alleviated isoproterenol (ISO)-induced myocardial injury through regulating autophagy. Methods: The rapid identification of chemical ingredients of QLQX was performed by UPLC-Q-TOF-MS, and the contents of major constituents in QLQX were also measured by UPLC-Q-TOF-MS. ISO was used to induce myocardial injury in H9c2 cardiomyocytes and SD rats. In vivo, cardiac function was evaluated by echocardiography and cardiac structure was observed by HE and Masson staining. Expressions of Bcl-2, Bax, LC3, P62, AKT, p-AKT, mTOR, and p-mTOR were detected by western blotting. In vitro, H9c2 cells were pretreated with QLQX for 3 h before ISO (80 µM, 48h) addressed. Cell viability, LDH and CK-MB release, apoptosis ratio, and the level of autophagy were measured. Western blotting was also performed to detected related protein expressions. Result: In vivo, treatment by QLQX significantly improved cardiac function and alleviated ISO-induced myocardial structural damage. In addition, QLQX markedly decreased apoptosis and inhibited autophagic activity, accompanied by activating the AKT/mTOR pathway. In vitro, the increased cell apoptosis induced by ISO was paralleling with the gradually increasing level of autophagy. Furthermore, 3-MA, an autophagic inhibitor, could block ISO-induced autophagy in H9c2 cells. Our results suggested that both QLQX and 3-MA treatment could decrease cell death induced by ISO, implying that QLQX protected against ISO-induced myocardial injury possibly by inhibiting excessive autophagy-mediated cell death. In addition, blockage of AKT signaling by an AKT inhibitor, capivasertib, could reduce the effect of QLQX on inhibiting ISO-induced apoptosis and autophagy-mediated cell death. Conclusion: QLQX could alleviate ISO-induced myocardial injury by inhibiting apoptosis and excessive autophagy-mediated cell death via activating the AKT/mTOR pathway.

HIF-1α/BNIP3 signaling pathway-induced-autophagy plays protective role during myocardial ischemia-reperfusion injury

ObjectiveThe study was established to inquire into the protective effect of the HIF-1α (Hypoxia-inducible factor-1α)/ BNIP3(Bcl-2/adenovirus E1B 19-kDa interacting protein) signal path-induced-autophagy during myocardial ischemia/ reperfusion (I/R) and oxygen–glucose deprivation/recovery (OGD/R) injury in heart-derived H9C2 cells as well as its potential underlying mechanism. MethodsImmediate myocardial I/R in SD (Spraque Dawley) rats and cytotoxicity of OGD/R injury on H9C2 cells with and without inhibitors or agonists of HIF-1α and BNIP3 were evaluated. Expression of mitochondrial autophagic protein were detected by Western blot and immunofluorescence. And the mitochondrial autophagosome were detected using Transmission Electron Microscope (TEM). ResultsI/R and OGD/R injury increased the expression level of HIF-1α, activated the downstream BNIP3 and subsequently triggered mitochondria-dependent autophagy. Up-regulation the expression of HIF-1α and BNIP3 may promote the cardiac myocytes of SD rats of I/R injure and OGD/R injury-induced autophagy of H9C2 cells. Moreover, down-regulation the expression of HIF-1α or BNIP3-siRNA decreased H9C2 cells autophagy under OGD/R injury. ConclusionsTogether, our studies indicated that HIF-1α synchronization regulate BNIP3 during OGD/R injury-induced autophagy in H9C2 cells, though BNIP3-induced autophagy acting as a survival mechanism.