Long noncoding RNA TALNEC2 regulates myocardial ischemic injury in H9c2 cells by regulating miR-21/PDCD4-medited activation of Wnt/β-catenin pathway

The goal of this study was to explore the role of tumor associated long noncoding RNA expressed on chromosome 2 (TALNEC2) in protecting against myocardial ischemic injury, as well as its underlying molecular mechanism. We established a cell model of myocardial injury through treating H9c2 cells with hypoxia, and the expression level of TALNEC2 was analyzed. Further, in vitro studies investigated the functional role of TALNEC2 dysregulation in hypoxia injury by assessing cell proliferation, migration, invasion, and apoptosis. Moreover, the expression of miR-21 was determined after dysregulation of TALNEC2, and whether TALNEC2-regulated hypoxia injury in H9c2 cells via regulating miR-21 expression were explored. Furthermore, the regulatory relationship between TALNEC2 and Wnt/β-catenin pathway was also investigated. TALNEC2 was highly expressed in the serum from patients with myocardial ischemic compared with that in healthy persons. Hypoxia-induced injury in H9c2 cells. Overexpression of TALNEC2 aggravated hypoxia injury in H9c2 cells. TALNEC2 could negative regulate the miR-21 expression, and overexpression of TALNEC2 aggravated hypoxia injury by downregulation of miR-21. Moreover, miR-21 negatively regulated the PDCD4 expression, and PDCD4 was a target of miR-21. Further studies disclosed that the overexpression of TALNEC2 further activated the Wnt/β-catenin pathway in hypoxia-treated H9c2 cells, implying that the Wnt/β-catenin pathway was a downstream mechanism mediating the role of TALNEC2 in regulating hypoxia injury in H9c2 cells. These findings confirmed the key functions of TALNEC2 in regulating myocardial ischemic injury. Upregulation of TALNEC2 may aggravate hypoxia injury in H9c2 cells via regulating miR-21/PDCD4-medited activation of the Wnt/β-catenin pathway. TALNEC2 may serve as a promising therapeutic target in myocardial ischemia.     

MiR-29b-3p aggravates cardiac hypoxia/reoxygenation injury via targeting PTX3

Our current research aimed to decipher the role and underlying mechanism with regard to miR-29b-3p involving in myocardial ischemia/reperfusion (I/R) injury. In the present study, cardiomyocyte H9c2 cell was used, and hypoxia/reoxygenation (H/R) model was established to mimic the myocardial I/R injury. The expressions of miR-29b-3p and pentraxin 3 (PTX3) were quantified deploying qRT-PCR and Western blot, respectively. The levels of LDH, TNF-α, IL-1β and IL-6 were detected to evaluate cardiomyocyte apoptosis and inflammatory response. Cardiomyocyte viability and apoptosis were examined employing CCK-8 assay and flow cytometry, respectively. Verification of the targeting relationship between miR-29b-3p and PTX3 was conducted using a dual-luciferase reporter gene assay. It was found that miR-29b-3p expression in H9c2 cells was up-regulated by H/R, and a remarkable down-regulation of PTX3 expression was demonstrated. MiR-29b-3p significantly promoted of release of inflammatory cytokines of H9c2 cells, and it also constrained the proliferation and promoted the apoptosis of H9c2 cells. Additionally, PTX3 was inhibited by miR-29b-3p at both mRNA and protein levels, and it was identified as a direct target of miR-29b-3p. PTX3 overexpression could reduce the inflammatory response, increase the viability of H9c2 cells, and inhibit apoptosis. Additionally, PTX3 counteracted the function of miR-29b-3p during the injury of H9c2 cells induced by H/R. In summary, miR-29b-3p was capable of aggravating the H/R injury of H9c2 cells by repressing the expression of PTX3.     

Long noncoding RNA AK088388 regulates autophagy through miR-30a to affect cardiomyocyte injury

Finding ways to reduce myocardial ischemia/reperfusion injury in the process of myocardial infarction has been an area of intense study in the field of heart disease. Recent studies have shown that long noncoding RNA (lncRNA) and autophagy play important roles in cardiovascular diseases. In our study, software analysis and dual-luciferase reporter assays have shown that miR-30a has binding sites on both AK088388 and Beclin-1. Continuing experiments found that miR-30a expression is downregulated, while the expressions of AK088388, Beclin-1, and LC3-II are upregulated in hypoxia/reoxygenation (H/R) cardiomyocytes; miR-30a inhibits the expression of AK088388, Beclin-1, and LC3-II in H/R cardiomyocytes, while AK088388 promotes the expression of Beclin-1 and LC3-II and inhibits miR-30a expression. AK088388 small interfering RNA and miR-30a mimics can promote the viability of H/R cardiomyocytes, reduce lactate dehydrogenase release, and reduce apoptosis. Mutations of the miR-30a binding site in AK088388 could not block the effects of miR-30a mentioned above. Therefore, AK088388 can competitively bind to miR-30a, promoting the expression of Beclin-1 and LC3-II, autophagy, and eventually cell damage. This finding provides new evidence for understanding the role of lncRNA in myocardial ischemia/reperfusion injury.     

GRP78 effectively protect hypoxia/reperfusion‐induced myocardial apoptosis via promotion of the Nrf2/HO‐1 signaling pathway

Myocardial infarction is a major cause of death worldwide. Despite our understanding of the pathophysiology of myocardial infarction and the therapeutic options for treatment have improved substantially, acute myocardial infarction remains a leading cause of morbidity and mortality. Recent findings revealed that GRP78 could protect myocardial cells against ischemia reperfusion injury‐induced apoptosis, but the exact function and molecular mechanism remains unclear. In this study, we aimed to explore the effects of GRP78 on hypoxia/reperfusion (H/R)‐induced cardiomyocyte injury. Intriguingly, we first observed that GRP78 overexpression significantly protected myocytes from H/R‐induced apoptosis. On mechanism, our work revealed that GRP78 protected myocardial cells from hypoxia/reperfusion‐induced apoptosis via the activation of the Nrf2/HO‐1 signaling pathway. We observed the enhanced expression of Nrf2/HO‐1 in GRP78 overexpressed H9c2 cell, while GRP78 deficiency dramatically antagonized the expression of Nrf2/HO‐1. Furthermore, we found that blocked the Nrf2/HO‐1 signaling by the HO‐1 inhibitor zinc protoporphyrin IX (Znpp) significantly retrieved H9c2 cells apoptosis that inhibited by GRP78 overexpression. Taken together, our findings revealed a new mechanism by which GRP78 alleviated H/R‐induced cardiomyocyte apoptosis in H9c2 cells via the promotion of the Nrf2/HO‐1 signaling pathway.     

Bone marrow mesenchymal stem cells-derived conditioned medium protects cardiomyocytes from hypoxia/reoxygenation-induced injury through Notch2/mTOR/autophagy signaling

Bone marrow mesenchymal stem cells (BMSC) can ameliorate ischemic injury of various tissues. However, the molecular mechanisms involved remain to be clarified. In this study, we intend to investigate the effects of BMSC-derived conditioned medium (BMSC-CM) on hypoxia/reoxygenation (H/R)-induced injury of H9c2 myocardial cells, and the potential mechanisms. Cell injury was determined through level of cell viability, lactate dehydrogenase (LDH) release, total intracellular reactive oxygen species (ROS), mitochondrial membrane potential (Δψm), and cell apoptosis. Autophagic activity of cells was detected through levels of the autophagy-associated proteins and autophagic flux. Results showed that BMSC-CM alleviated H/R-induced injury in H9c2 cells, as demonstrated by increased cell viability and Δψm, decreased ROS production, LDH release, and cell apoptosis. Furthermore, the H/R treatment induced a decrease in autophagic activity and an increase in Notch2 signaling activation in H9c2 cells. In the presence of BMSC-CM, the autophagic activity impaired by the H/R treatment was upregulated with decreased phosphorylation of mTOR, and the activation of Notch2 signaling was downregulated. These effects of BMSC-CM could be replicated by Notch signaling inhibitor. In contrast, inhibitors of cell autophagy including chloroquine (CQ) and 3-methyladenine, diminished the protective effects of BMSC-CM. Taken together results, our study showed that BMSC-CM could protect H9c2 cells from H/R-induced injury potentially through regulating Notch2/mTOR/autophagy signaling. These findings may provide a novel insight into the mechanisms of BMSC-CM in therapy of myocardial ischemia/reperfusion injury as well as other ischemic diseases.     

MicroRNA‐21 protects against cardiac hypoxia/reoxygenation injury by inhibiting excessive autophagy in H9c2 cells via the Akt/mTOR pathway

MicroRNAs and autophagy play critical roles in cardiac hypoxia/reoxygenation (H/R)‐induced injury. Here, we investigated the function of miR‐21 in regulating autophagy and identified the potential molecular mechanisms involved. To determine the role of miR‐21 in regulating autophagy, H9c2 cells were divided into the following six groups: control group, H/R group, (miR‐21+ H/R) group, (miR‐21‐negative control + H/R) group, (BEZ235+ H/R) group and (miR‐21+ BEZ235+ H/R) group. The cells underwent hypoxia for 1 hr and reoxygenation for 3 hrs. Cell count kit‐8 was used to evaluate cell function and apoptosis was analysed by Western blotting. Western blotting and transmission electron microscopy were used to investigate autophagy. We found that miR‐21 expression was down‐regulated, and autophagy was remarkably increased in H9c2 cells during H/R injury. Overexpression of miR‐21 with a miR‐21 precursor significantly inhibited autophagic activity and decreased apoptosis, accompanied by the activation of the AKT/mTOR pathway. In addition, treatment with BEZ235, a novel dual Akt/mTOR inhibitor, resulted in a significant increase in autophagy and apoptosis. However, we found that miR‐21‐mediated inhibition of apoptosis and autophagy was partly independent of Akt/mTOR activation, as demonstrated in cells treated with both miR‐21 and BEZ235. We showed that miR‐21 could inhibit H/R‐induced autophagy and apoptosis, which may be at least partially mediated by the Akt/mTOR signalling pathway.     

MiR-17-5p-mediated endoplasmic reticulum stress promotes acute myocardial ischemia injury through targeting Tsg101

Cardiovascular diseases are the leading cause of death globally, among which acute myocardial infarction (AMI) frequently occurs in the heart and proceeds from myocardium ischemia and endoplasmic reticulum (ER) stress-induced cell death. Numerous studies on miRNAs indicated their potential as diagnostic biomarkers and treatment targets for heart diseases. Our study investigated the role of miR-17-5p and its regulatory mechanisms during AMI. Echocardiography, MTT, flow cytometry assay, evaluation of caspase-3 and lactate dehydrogenase (LDH) activity were conducted to assess cell viability, apoptosis in an MI/R mice model, and an H₂O₂-induced H9c2 hypoxia cell model, respectively. The expression levels of ER stress response-related biomarkers were detected using qRT-PCR, IHC, and western blotting assays. The binding site of miR-17-5p on Tsg101 mRNA was determined by bioinformatic prediction and luciferase reporter assay. The expression levels of miR-17-5p were notably elevated in MI/R mice and hypoxia cell models, accompanied by enhanced cell apoptosis. Inhibition of miR-17-5p led to decreased apoptosis related to ER stress response in the hypoxia model, which could be counteracted by knockdown of Tsg101 (tumor susceptibility gene 101). Transfection with miR-17-5p mimics downregulated the expression of Tsg101 in H9c2 cells. Luciferase assay demonstrated the binding between miR-17-5p and Tsg101. Moreover, 4-PBA, the inhibitor of the ER stress response, abolished shTsg101 elevated apoptosis in hypoxic H9c2 cells. Our findings investigated the pro-apoptotic role of miR-17-5p during MI/R, disclosed the specific mechanism of miR-17-5p/Tsg101 regulatory axis in ER stress-induced myocardium injury and cardiomyocytes apoptosis, and presented a promising diagnostic biomarker and potential target for therapy of AMI.     

Protective Role of miR-34c in Hypoxia by Activating Autophagy through BCL2 Repression

Hypoxia leads to significant cellular stress that has diverse pathological consequences such as cardiovascular diseases and cancers. MicroRNAs (miRNAs) are one of regulators of the adaptive pathway in hypoxia. We identified a hypoxia-induced miRNA, miR-34c, that was significantly upregulated in hypoxic human umbilical cord vein endothelial cells (HUVECs) and in murine blood vessels on day 3 of hindlimb ischemia (HLI). miR-34c directly inhibited BCL2 expression, acting as a toggle switch between apoptosis and autophagy in vitro and in vivo. BCL2 repression by miR-34c activated autophagy, which was evaluated by the expression of LC3-II. Overexpression of miR-34c inhibited apoptosis in HUVEC as well as in a murine model of HLI, and increased cell viability in HUVEC. Importantly, the number of viable cells in the blood vessels following HLI was increased by miR-34c overexpression. Collectively, our findings show that miR-34c plays a protective role in hypoxia, suggesting a novel therapeutic target for hypoxic and ischemic diseases in the blood vessels.     

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.     

Retracted: MicroRNA-132 protects H9c2 cells against oxygen and glucose deprivation-evoked injury by targeting FOXO3A

Myocardial ischemia/reperfusion injury (MIRI) is a clinically familiar disease, which possesses a great negative impact on human health. But, the effective treatment is still absent. MicroRNAs (miRNAs) have been testified to play a momentous role in MIRI. The purpose of the study aimed to probe the functions of miR-132 in oxygen and glucose deprivation (OGD)-evoked injury in H9c2 cells. miR-132 expression in H9c2 cells accompanied by OGD disposition was evaluated via real-time quantitative polymerase chain reaction. After miR-132 mimic and inhibitor transfections, the impacts of miR-132 on OGD-affected H9c2 cell viability, apoptosis, cell cycle, and the interrelated factors were appraised by exploiting cell counting kit-8, flow cytometry, and western blot analysis. FOXO3A expression was estimated in above-transfected cells, meanwhile, the correlation between miR-132 and FOXO3A was probed by dual-luciferase report assay. Ultimately, above mentioned cell processes were reassessed in H9c2 cells after preprocessing OGD administration and transfection with si-FOXO3A and si-NC plasmids. We got that OGD disposition obviously enhanced miR-132 expression in H9c2 cells. Overexpressed miR-132 evidently reversed OGD-evoked cell viability repression and apoptosis induction in H9c2 cells. In addition, overexpressed miR-132 mitigated OGD-evoked G0/G1 cell arrest by mediating p21, p27, and cyclin D1 expression. Repression of FOXO3A was observed in miR-132 mimic-transfected cells, which was also predicated as a direct gene of miR-132. We discovered that silenced FOXO3A alleviated OGD-evoked cell injury in H9c2 cells via facilitating cell viability, hindering apoptosis and restraining cell arrest at G0/G1 phase. In conclusion, these investigations corroborated that miR-132 exhibited the protective impacts on H9c2 cells against OGD-evoked injury via targeting FOXO3A.     

Dietary methionine restriction attenuates renal ischaemia/reperfusion‐induced myocardial injury by activating the CSE/H2S/ERS pathway in diabetic mice

Methionine restrictive diet may alleviate ischaemia/reperfusion (I/R)‐induced myocardial injury, but its underlying mechanism remains unclear. HE staining was performed to evaluate the myocardial injury caused by I/R and the effect of methionine‐restricted diet (MRD) in I/R mice. IHC and Western blot were carried out to analyse the expression of CSE, CHOP and active caspase3 in I/R mice and hypoxia/reoxygenation (H/R) cells. TUNEL assay and flow cytometry were used to assess the apoptotic status of I/R mice and H/R cells. MTT was performed to analyse the proliferation of H/R cells. H2S assay was used to evaluate the concentration of H2S in the myocardial tissues and peripheral blood of I/R mice. I/R‐induced mediated myocardial injury and apoptosis were partially reversed by methionine‐restricted diet (MRD) via the down‐regulation of CSE expression and up‐regulation of CHOP and active caspase3 expression. The decreased H2S concentration in myocardial tissues and peripheral blood of I/R mice was increased by MRD. Accordingly, in a cellular model of I/R injury established with H9C2 cells, cell proliferation was inhibited, cell apoptosis was increased, and the expressions of CSE, CHOP and active caspase3 were dysregulated, whereas NaHS treatment alleviated the effect of I/R injury in H9C2 cells in a dose‐dependent manner. This study provided a deep insight into the mechanism underlying the role of MRD in I/R‐induced myocardial injury.     

M3 subtype of muscarinic acetylcholine receptor promotes cardioprotection via the suppression of miR-376b-5p

The M(3) subtype of muscarinic acetylcholine receptors (M(3)-mAChR) plays a protective role in myocardial ischemia and microRNAs (miRNAs) participate in many cardiac pathophysiological processes, including ischemia-induced cardiac injury. However, the role of miRNAs in M(3)-mAChR mediated cardioprotection remains unexplored. The present study was designed to identify miRNAs that are involved in cardioprotective effects of M(3)-mAChR against myocardial ischemia and elucidate the underlying mechanisms. We established rat model of myocardial ischemia and performed miRNA microarray analysis to identify miRNAs involved in the cardioprotection of M(3)-mAChR. In H9c2 cells, the viability, intracellular free Ca(2+) concentration ([Ca(2+)]i), intracellular reactive oxygen species (ROS), miR-376b-5p expression level, brain derived neurophic factor (BDNF) and nuclear factor kappa-B (NF-κB) levels were measured. Our results demonstrated that M(3)-mAChR protected myocardial ischemia injury. Microarray analysis and qRT-PCR revealed that miR-376b-5p was significantly up-regulated in ischemic heart tissue and the M(3)-mAChRs agonist choline reversed its up-regulation. In vitro, miR-376b-5p promoted H(2)O(2)-induced H9c2 cell injuries measured by cells viability, [Ca(2+)]i and ROS. Western blot and luciferase assay identified BDNF as a direct target of miR-376b-5p. M(3)-mAChR activated NF-κB and thereby inhibited miR-376b-5p expression. Our data show that a novel M(3)-mAChR/NF-κB/miR-376b-5p/BDNF axis plays an important role in modulating cardioprotection. MiR-376b-5p promotes myocardial ischemia injury possibly by inhibiting BDNF expression and M(3)-mAChR provides cardioprotection at least partially mediated by the downregulation of miR-376b-5p through NF-κB. These findings provide new insight into the potential mechanism by which M(3)-mAChR provides cardioprotection against myocardial ischemia injury.     

Alpha-lipoic acid protects cardiomyocytes against hypoxia/reoxygenation injury by inhibiting autophagy

Hypoxia/reoxygenation (H/R) is an important in vitro model for exploring the molecular mechanisms and functions of autophagy during myocardial ischemia/reperfusion (I/R). Alpha-lipoic acid (LA) plays an important role in the etiology of cardiovascular disease. Autophagy is widely implicated in myocardial I/R injury. We assessed the degree of autophagy by pretreatment with LA exposed to H/R in H9c2 cell based on the expression levels of Beclin-1, LC3II/LC3I, and green fluorescent protein-labeled LC3 fusion proteins. Autophagic vacuoles were confirmed in H9c2 cells exposed to H/R using transmission electron microscopy. Our findings indicated that pretreatment with LA inhibited the degree of autophagy in parallel to the enhanced cell survival and decreased total cell death in H9c2 cells exposed to H/R. We conclude that LA protects cardiomyocytes against H/R injury by inhibiting autophagy.