Autophagy In H9c2 Cardiomyocytes Research Articles

Dapagliflozin attenuates high glucose-and hypoxia/reoxygenation-induced injury via activating AMPK/mTOR-OPA1-mediated mitochondrial autophagy in H9c2 cardiomyocytes

This study investigated the protective effect of dapagliflozin on H9c2 cardiomyocyte function under high glucose and hypoxia/reoxygenation (HG-H/R) conditions and identified the underlying molecular mechanisms. Dapagliflozin reduced the level of lactate dehydrogenase and reactive oxygen species in cardiomyocytes under HG-H/R conditions and was accompanied by a decrease in caspase-3/9 activity. In addition, Dapagliflozin significantly reduced mitochondrial permeability transition pore opening and increased ATP content, accompanied by upregulation of OPA1 with autophagy-related protein molecules and activation of the AMPK/mTOR signalling pathway in HG-H/R treated cardiomyocytes. OPA1 knockdown or compound C treatment attenuated the protective effects of dapagliflozin on the cardiomyocytes under HG-H/R conditions. Downregulation of OPA1 expression increased mitochondrial intolerance in cardiomyocytes during HG-H/R injury and the AMPK-mTOR-autophagy signalling is a key mechanism for protecting mitochondrial function and reducing cardiomyocyte apoptosis. Collectively, dapagliflozin exerted protective effects on the cardiomyocytes under HG-H/R conditions. Dapagliflozin attenuated myocardial HG-H/R injury by activating AMPK/mTOR-OPA1-mediated mitochondrial autophagy.

MicroRNA‐17‐3p protects against excessive posthypoxic autophagy in H9C2 cardiomyocytes via PTEN–Akt–mTOR signaling pathway

The activity of phosphatase and tensin homolog (PTEN) can be inhibited by miR-17-3p, which results in attenuating myocardial ischemia/reperfusion injury (IRI), however, the mechanism behind this phenomenon is still elusive. Suppression of PTEN leads to augmented protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling strength and constrained autophagy activation, which might be the one mechanism for the ameliorated myocardial IRI. Thus, we tested the hypothesis that miR-17-3p attenuated hypoxia/reoxygenation (H/R)-mediated damage in cardiomyocytes by downregulating excessive autophagy via the PTEN-Akt-mTOR axis. The expression of miR-17-3p was remarkably increased after H/R treatment (6-h hypoxia followed by 6-h reoxygenation; H6/R6), which was concomitant with the increase of the release of lactic acid dehydrogenase (cell injury marker) and the enhancement LC3II/I ratio (autophagy markers) in H9C2 cardiomyocytes. Ectoexpression of miR-17-3p agomir led to remarkable augmentation of miR-17-3p expression and evidently attenuated H/R-mediated cell damage and excessive autophagy. Furthermore, an increase in miR-17-3p expression elicited constrained phosphorylation of PTEN (Ser380 ) while enhanced the phosphorylation of Akt (Thr308 , Ser473 ) and mTOR (Ser536 ) after H/R stimulation. In addition, pretreatment with LY-294002 (an Akt selective inhibitor) and rapamycin (an mTOR selective inhibitor) significantly abrogated the protective function of miR-17-3p on H/R-mediated cell damage and autophagy in H9C2 cardiomyocytes. Taken together, these observations indicated that the enhancement of the PTEN/Akt/mTOR axis and the consequent suppression of autophagy overactivation might represent an underlying mechanism by which miR-17-3p attenuated H/R-mediated damage in H9C2 cells.

Aconitine induces autophagy via activating oxidative DNA damage-mediated AMPK/ULK1 signaling pathway in H9c2 cells

Ethnopharmacological relevanceAconitum species, with a medicinal history of 2000 years, was traditionally used in the treatment of rheumatism, arthritis, bruises, and pains. However, many studies have reported that Aconitum species can cause arrhythmia in experimental animals, resulting in myocardial fibrosis and cardiomyocyte damage. Cardiotoxicity is the main toxic effect of aconitine, but the detailed mechanism remains unclear. Aim of the studyThis study aimed to explore the effects and underlying mechanism of autophagy in H9c2 cardiomyocytes induced by aconitine. Materials and methodsH9c2 cells were incubated with different concentrations of aconitine for 24 h, and the intervention sections were pretreated with various inhibitors for 1 h. The effects of aconitine on the oxidative DNA damage, autophagy and viability of H9c2 cells were evaluated by flow cytometry, confocal microscopy, enzyme-linked immunosorbent assay and Western blot. ResultsIn H9c2 cells, the cell viability declined, LDH release rate, the number of autophagosomes, protein expression levels of LC3 and Beclin-1 increased significantly after 24 h of aconitine incubation. The pretreatment of autophagy inhibitor 3-MA decreased markedly autophagosomes and protein expression levels of LC3 and Beclin-1, which suggested that aconitine could induce cell autophagy. The significant increase of ROS and 8-OHdG showed that aconitine could cause oxidative DNA damage through ROS accumulation. Meanwhile, treatment of aconitine dramatically increased AMPKThr172 and ULK1Ser317 phosphorylation, and Compound C inhibited AMPKThr172 and ULK1Ser317 phosphorylation, which proved that aconitine induced autophagy via AMPK activation mediated ULK1 phosphorylation. Antioxidant NAC significantly reduced LDH, ROS and 8-OHdG, inhibited the phosphorylation of AMPKThr172 and ULK1Ser317, and down-regulated autophagosomes and proteins expression levels of LC3 and Beclin-1. Consequently, the inhibition of oxidative DNA damage and AMPK/ULK1 signaling pathway alleviated the aconitine-induced autophagic death of H9c2 cells. ConclusionsThese results showed that aconitine induces autophagy of H9c2 cardiomyocytes by activating AMPK/ULK1 signaling pathway mediated by oxidative DNA damage. The autophagy induced by aconitine in cardiomyocytes is dependent on the activation of the AMPK pathway, which may provide novel insights into the prevention of aconitine-related toxicity.

Dysregulation of miR-342-3p in plasma exosomes derived from convalescent AMI patients and its consequences on cardiac repair

Plasma exosomes derived from healthy people have been shown to be beneficial in terms of protecting against ischemia-reperfusion injury or acute myocardial infarction (AMI). However, a pathological condition may severely affect the constitution and biological activity of exosomes. In our study, we isolated plasma exosomes from healthy volunteers and convalescent AMI patients (3–7 d after onset). Compared to exosomes from healthy controls (Nor-Exo), exosomes from convalescent AMI patients (AMI-Exo) exhibited an impaired ability to repair damaged cardiomyocytes both in vitro and in vivo. miRNA sequencing and PCR analysis indicated that miR-342-3p was significantly downregulated in AMI-Exo. Moreover, miR-342-3p alleviated H2O2-induced injury and reduced apoptosis and autophagy in H9c2 cardiomyocytes, while in vivo restoration of miR-342-3p expression enhanced the reparative function of AMI-Exo. Further mechanistic studies revealed that the SOX6 and TFEB genes were two direct and functional targets of miR-342-3p. Taken together, during the early convalescent phase after AMI, dysregulated miR-342-3p in plasma exosomes might be responsible for their impaired cardioprotective potential. miR-342-3p contributed to exosome-mediated heart repair by inhibiting cardiomyocyte apoptosis and autophagy through targeting SOX6 and TFEB, respectively. Our work provided novel insights on the role of plasma exosomes in the natural process of cardiac repair after AMI and suggestions for therapy development.

Selective inhibition of PKCβ2 improves Caveolin-3/eNOS signaling and attenuates lipopolysaccharide-induced injury by inhibiting autophagy in H9C2 cardiomyocytes.

Lipopolysaccharide (LPS)-induced autophagy is involved in sepsis-associated myocardial injury with increased PKCβ2 activation. We previously found hyperglycemia-induced PKCβ2 activation impaired the expression of caveolin-3 (Cav-3), the dominant isoform to form cardiomyocytes caveolae which modulate eNOS signaling to confer cardioprotection in diabetes. However, little is known about the roles of PKCβ2 in autophagy and Cav-3/eNOS signaling in cardiomyocytes during LPS exposure. We hypothesize LPS-induced PKCβ2 activation promotes autophagy and impairs Cav-3/eNOS signaling in LPS-treated cardiomyocytes. H9C2 cardiomyocytes were treated with LPS (10µg/mL) in the presence or absence of PKCβ2 inhibitor CGP53353 (CGP, 1µM) or autophagy inhibitor 3-methyladenine (3-MA, 10µM). LPS stimulation induced cytotoxicity overtime in H9C2 cardiomyocytes, accompanied with excessive PKCβ2 activation. Selective inhibition of PKCβ2 with CGP significantly reduced LPS-induced cytotoxicity and autophagy (measured by LC-3II, Beclin-1, p62 and autophagic flux). In addition, CGP significantly attenuated LPS-induced oxidative injury, and improved Cav-3 expression and eNOS activation, similar effects were shown by the treatment of autophagy inhibitor 3-MA. LPS-induced myocardial injury is associated with excessive PKCβ2 activation, which contributes to elevated autophagy and impaired Cav-3/eNOS signaling. Selective inhibition of PKCβ2 improves Cav-3/eNOS signaling and attenuates LPS-induced injury through inhibiting autophagy in H9C2 cardiomyocytes.

Activation of Nrf2/HO-1 signaling pathway attenuates ROS-mediated autophagy induced by silica nanoparticles in H9c2 cells.

Silica nanoparticles (SiNPs) as one of the most productive nano-powder, has been extensively applied in various fields. There has been increasing concern about the adverse effects of SiNPs on the health of ecological organisms and human. The potential cardiovascular toxicity of SiNPs and involved mechanisms remain elusive. Hence, in this study, we investigated the cardiovascular toxicity of SiNPs (60 nm) and explored the underlying mechanisms using H9c2 cardiomyocytes. Results showed that SiNPs induced oxidative stress and activated the Nrf2/HO-1 antioxidant pathway. Autophagy was also activated by SiNPs. Interestingly, N-acetyl-L-cysteine (NAC）attenuated autophagy after inhibiting reactive oxygen species (ROS). Meanwhile, down-regulation of Nrf2 enhanced autophagy. In summary, these data indicated that SiNPs induce autophagy in H9c2 cardiomyocytes through oxidative stress, and the Nrf2/HO-1 pathway has a negative regulatory effect on autophagy. This study provides new evidence for the cardiovascular toxicity of SiNPs and provides a reference for the safe use of nanomaterials in the future.

MiR-129-5p attenuates hypoxia-induced apoptosis in rat H9c2 cardiomyocytes by activating autophagy.

Autophagy is closely associated with apoptosis in H9c2 cardiomyocytes as a result of hypoxia. The present study aimed to determine whether microRNAs (miRs) mediated apoptosis and autophagy in hypoxia-stimulated H9c2 cardiomyocytes. miR microarrays and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays were used to detect differentially expressed miRs in H9c2 cardiomyocytes following hypoxia stimulation. Annexin V-fluorescein isothiocyanate double staining was performed to evaluate hypoxia-induced cell apoptosis, and the protein expression levels of autophagy-associated genes were detected using western blotting. miR microarrays and qRT-PCR assays showed that the expression of miR-129-5p is significantly decreased in hypoxia-exposed H9c2 cardiomyocytes. Under hypoxic stimulation, miR-129-5p mimics alleviated hypoxia-induced cell apoptosis and also restored autophagy in H9c2 cardiomyocytes. However, transfection with miR-129-5p inhibitors accelerated hypoxia-induced cell apoptosis and autophagy deficiency in H9c2 cardiomyocytes. Furthermore, overexpression of miR-129-5p enhanced cell viability and reduced the release of lactate dehydrogenase in hypoxia-stimulated H9c2 cardiomyocytes. These findings highlight the protective effect of miR-129-5p against hypoxia-induced apoptosis in H9c2 cardiomyocytes through the activation of autophagy.

Ginsenoside Re enhances the survival of H9c2 cardiac muscle cells through regulation of autophagy

We examined the effect of ginsenoside Re (G-Re) on autophagy in H9c2 cardiomyocytes cultured in glucose deprivation (GD). Levels of the membrane-bound autophagy-related microtubule-associated protein 1A/1B-light chain 3 (LC3) B-2 were measured via immunoblotting and immunofluorescence was conducted to assess autophagosome formation. GD H9c2 cells were treated with 100 μmol/l G-Re. Cell viability was determined in culture medium. Phosphorylated 5′ AMP-activated protein kinase (AMPK)-α and mammalian target of rapamycin (mTOR) levels were measured to explore the mechanisms underlying the effects of G-Re on autophagy in GD cells. G-Re treatment inhibited autophagosome formation and may be beneficial to GD cardiomyocytes.

Remifentanil attenuates lipopolysaccharide-induced oxidative injury by downregulating PKCβ2 activation and inhibiting autophagy in H9C2 cardiomyocytes

AimLipopolysaccharide (LPS)-induced myocardial injury is a leading cause of death in patients with sepsis, which is associated with excessive activation of PKCβ (especially PKCβ2) and autophagy. Remifentanil, a μ-opioid receptor agonist, is well demonstrated to have beneficial effects during sepsis, but the underlying mechanisms are still unknown. The present study was designed to investigate the roles of remifentanil in PKCβ2 and autophagy in LPS-treated cardiomyocytes. Main methodsH9C2 cardiomyocytes were treated with or without remifentanil (2.5 μM), PKCβ2 inhibitor CGP53353 (CGP, 1 μM) or autophagy inhibitor 3-methyladenine (3-MA, 10 μM) in the presence or absence of LPS (10 μg/mL). Key findingsLPS exposure for 24 h led to a significant increase in cell death, LDH release and MDA production in H9C2 cardiomyocytes, accompanied with decreased SOD activity and excessive PKCβ2 activation and autophagy indicated by enhanced Beclin-1 and LC-3II expression and decreased p62 expression. All these changes were attenuated by remifentanil intervention. In addition, inhibition of LPS-induced PKCβ2 activation by CGP or autophagy inhibitor 3-MA has similar effects to remifentanil. SignificanceRemifentanil protects H9C2 cardiomyocytes against LPS-induced oxidative injury, as a result of downregulating PKCβ2 activation and inhibiting autophagy, partially.

MicroRNA‐17‐3p inhibits excessive post‐hypoxic autophagy and attenuates H9C2 cardiomyocytes reoxygenation injury via PTEN‐Akt‐mTOR signaling

BackgroundThe microRNA (miR)‐17~92 is one of the best‐characterized polycistronic miRNA clusters, which encodes six individual miRNAs and contributes to various cellular and biological processes. In particular, miR‐17‐3p (a passenger miRNA of miR‐17) has been shown to indirectly inhibit Phosphatase and tensin homolog (PTEN) and attenuate myocardial ischemia/reperfusion (I/R) injury, but the underneath mechanism is unclear. Inhibition of PTEN can upregulate Akt/mTOR signaling and therefore suppresses the excessive autophagy, which may be a mechanism that underlies myocardial I/R injury. We, thus, hypothesized that miR‐17‐3p may protect against hypoxia/reoxygenation (H/R) induced cell injury by inhibiting post‐hypoxic excessive autophagy in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.Methods and resultsIn rat H9C2 cardiomyocytes, H/R (6 hours hypoxia followed by 6 hours reoxygenation) significantly enhanced the expression of miR‐17‐3p (P <0.05 vs. Control), which was concomitant by increased lactic acid dehydrogenase (LDH) leakage (cell injury marker, P<0.05 vs. Control) and the levels of p62 and the ratio of LC3II/I (autophagy markers, P<0.05 vs. Control), suggesting that miR‐17‐3p may be involved in the pathogenesis of H/R induced autophagic cell death. To explore the potential role of miR‐17‐3p in H/R‐induced cell injury, H9C2 cells were transfected with the miR‐17‐3p agomir or its negative control. The overexpression of miR‐17‐3p can significantly attenuate H/R‐induced cell injury (reduced LDH level, P <0.05 vs. H/R) and inhibit H/R‐induced excessive autophagy (decreased levels of p62 and LC3II/I, P <0.05 vs. H/R). Given the important role of PTEN‐Akt‐mTOR signaling in autophagy, the effects of miR‐17‐3p on the key events in the PTEN‐Akt‐mTOR signaling cascade during H/R were examined. As anticipated, miR‐17‐3p overexpression significantly down‐regulated PTEN expression and up‐regulated the levels of phosphorylated Akt1 (Thr308) and mTOR (P<0.05 vs. H/R), indicating that the miR‐17‐3p may protect against excessive post‐hypoxic autophagic cell death in H9C2 cardiomyocytes via PTEN‐Akt‐mTOR signaling pathway.ConclusionUpregulating PTEN‐Akt‐mTOR axis and the subsequent inhibition of excessive autophagy may represent the major mechanism whereby miR‐17‐3p attenuates H/R injury in H9C2 cardiomyocytes.Support or Funding InformationStudied supported by RGC/GRF grants (17158616M, 17117217 M)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Inhibition of microRNA-101 attenuates hypoxia/reoxygenation-induced apoptosis through induction of autophagy in H9c2 cardiomyocytes

Autophagy is a cellular self‑catabolic process responsible for the degradation of proteins and organelles. Autophagy is able to promote cell survival in response to stress, and increased autophagy amongst cardiomyocytes has been identified in conditions of heart failure, starvation and ischemia/reperfusion. However, the detailed regulatory mechanisms underlying autophagy in heart disease have remained elusive. MicroRNAs (miRNAs) have been implicated in the regulation of autophagy in cells under stress. In the present study, the protective effect of miRNA (miR)‑101 on hypoxia/reoxygenation (H/R)‑induced cardiomyocyte apoptosis was investigated. It was revealed that H/R induced apoptosis in H9c2 cardiomyocytes, accompanied by a downregulation of miR‑101 expression. Further investigation identified Ras‑related protein Rab‑5A (RAB5A) as a direct target of miR‑101. RAB5A was previously reported to be involved in autophagy; therefore, the present study further focused on the role of miR‑101 in the regulation of autophagy under H/R and found that the inhibition of miR‑101 attenuated H/R‑induced apoptosis, at least partially, via the induction of autophagy. In conclusion, the results of the present study revealed a beneficial effect of miR‑101 inhibition on H/R‑induced apoptosis in cardiomyocytes, indicating that miR‑101 inhibition may present a potential therapeutic agent in the treatment or prevention of heart diseases.

Reduction of autophagy: Another potential mechanism of cardioprotective effect of mild hypothermia?

We read with great interest the recent article “Hypothermia may attenuate ischemia/reperfusion-induced cardiomyocyte death by reducing autophagy” by Cheng et al. [1] in the International Journal of Cardiology. The authors demonstrated that application of mild hypothermia could reduce the cell autophagy in H9c2 cardiomyocytes subjected to ischemia/reperfusion (I/R). To the best of our knowledge, autophagy, as one of the forms of programmed cell death, was firstly reported by the authors to be a potential mechanism for the cardioprotection provided by mild hypothermia.

Ginsenoside Rg1 inhibits autophagy in H9c2 cardiomyocytes exposed to hypoxia/reoxygenation

Ginsenoside Rg1 promotes antioxidative protection and intracellular calcium homeostasis in cardiomyocytes hypoxia/reoxygenation (H/R) model. However, the pharmacological effects of G-Rg1 on autophagy in cardiomyocytes have not been reported. In this study, we employed H9c2 cardiomyocytes as a model to investigate the effects of G-Rg1 on autophagy in cardiomyocytes under H/R stress. Our results showed that H/R induced increased level of LC3B-2, an autophagy marker, in a time-dependent manner in association with decreased cell viability and cellular ATP content. H/R-induced autophagy and apoptosis were further confirmed by morphological examination. 100 μmol/l Rg1-inhibited H/R induced autophagy and apoptosis, and this was associated with the increase of cellular ATP content and the relief of oxidative stress in the cells. Mechanistically, we found that Rg1 inhibited the activation of AMPKα, promoted the activation of mTOR, and decreased the levels of LC3B-2 and Beclin-1. In conclusion, our data suggest that H/R induces autophagy in H9c2 cells leading to cell injury. Rg1 inhibits autophagosomal formation and apoptosis in the cells, which may be beneficial to the survival of cardiomyocytes under H/R.