Hypoxia-induced Cardiomyocyte Injury Research Articles

Identification of a novel native peptide derived from 60S ribosomal protein L23a that translationally regulates p53 to reduce myocardial ischemia-reperfusion

Myocardial ischemia-reperfusion (I/R) is a severe disease，but its underlying mechanism is not fully elucidated and no effective clinical treatment is available. Utilizing intracellular peptidomics, we identified a novel native peptide PDRL23A (Peptide Derived from RPL23A), that is intimately related to hypoxic stress. We further show that PDRL23A effectively alleviates hypoxia-induced cardiomyocyte injury in vitro, along with improvements in mitochondrial function and redox homeostasis, including ROS accumulation, oxidative phosphorylation, and mitochondrial membrane potential. Strikingly, the in vivo results indicate that, short-term pretreatment with PDRL23A could effectively inhibit I/R-induced cardiomyocyte death, myocardial fibrosis and decreased cardiac function. Interestingly, PDRL23A was found to interact with 60 S ribosomal protein L26 (RPL26), hampering RPL26-governed p53 translation, and resulting in a reduction in the level of p53 protein, which in turn reduced p53-mediated apoptosis under hypoxic conditions. Collectively, a native peptide, PDRL23A, which translationally regulates p53 to protect against myocardial I/R injury, has been identified for the first time. Our findings provide insight into the adaptive mechanisms of hypoxia and present a potential new treatment for myocardial I/R.

LncRNA PVT1 Promotes Hypoxia-Induced Cardiomyocyte Injury by Inhibiting miR-214-3p.

Objective To explore the effect and related mechanism of LncRNA PVT1 on hypoxia-induced cardiomyocyte injury. Methods PVT1RNA and miR-214-3p levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Cell vitality and apoptosis were, respectively, evaluated by Cell Counting Kit-8 (CCK-8) and flow cytometry analysis. Starbase and Dual luciferase reporter (DLR) gene assay was employed to validate the interaction between miR-214-3p and PVT1. Results PVT1 was statistically upregulated, and miR-214-3p was statistically downregulated in hypoxia-induced H9c2 cells. The survival rate of H9c2 cells induced by hypoxia decreased statistically, while the apoptosis rate increased statistically (P < 0.05). PVT1 knockdown upregulated the hypoxia-induced H9c2 cell viability and inhibited apoptosis. DLR assay verified the targeting relationship between PVT1 and miR-214-3p. In addition, miR-214-3p inhibitors reversed the viability of H9c2 cells with PVT1 knockout and promoted apoptosis. Conclusion Silencing PVT1 can enhance the hypoxia-induced H9c2 cell viability and inhibit apoptosis, providing a potential target for the treatment of cardiovascular diseases.

Inhibition of GARS1-DT Protects Against Hypoxic Injury in H9C2 Cardiomyocytes via Sponging miR-212-5p.

The present study aimed to elucidate the function of long noncoding RNA GARS1-DT in hypoxia-induced injury in ex-vivo cardiomyocytes and explore its underlying mechanism. Hypoxic injury was confirmed in H9C2 cells by the determination of cell viability, migration, invasion, and apoptosis. GARS1-DT expression was estimated in H9C2 cells after hypoxia. We then measured the effects of GARS1-DT knockdown on hypoxia-induced H9C2 cells. The interaction between GARS1-DT and miR-212-5p was also investigated. Hypoxia treatment led to cell damage in H9C2 cardiomyocytes, accompanied with the upregulation of GARS1-DT expression. Transfection of GARS1-DT small interfering RNA remarkably attenuated hypoxia-induced injury by enhancing cell viability, migration, and invasion, and reducing apoptosis. Furthermore, GARS1-DT served as an endogenous sponge for miR-212-5p, and its expression was negatively regulated by GARS1-DT. The effects of GARS1-DT knockdown on hypoxia-induced injury were significantly abrogated by miR-212-5p silence. Besides, suppression of GARS1-DT activated PI3K/AKT pathway in hypoxia-treated H9C2 cells, which were reversed by inhibition of miR-212-5p. Our findings demonstrated the novel molecular mechanism of GARS1-DT/miR-212-5p/PI3K/AKT axis on the regulation of hypoxia-induced myocardial injury in H9C2 cells, which may provide potential therapeutic targets for acute myocardial infarction treatment.

VEGF121 como Mediador de Efeitos Cardioprotetores Pós-Hipóxia via CaSR e via da Protease Dependente de Mitocôndria

Resumo Fundamento: A doença cardiovascular é a principal causa de morte em todo o mundo. A apoptose mediada por hipóxia em cardiomiócitos é uma das principais causas de distúrbios cardiovasculares. O tratamento com a proteína do fator de crescimento endotelial vascular (VEGF, do inglês vascular endothelial growth factor) foi testado, mas as dificuldades operacionais limitaram seu uso. Entretanto, com os avanços da terapia gênica, aumentou o interesse na terapia gênica baseada no VEGF em doenças cardiovasculares. No entanto, o mecanismo preciso pelo qual a reposição de VEGF resgata os danos pós-hipóxia em cardiomiócitos não é conhecido. Objetivos: Investigar o efeito da expressão de VEGF121 pós-hipóxia utilizando cardiomiócitos de ratos neonatos. Métodos: Cardiomiócitos isolados de ratos neonatos foram utilizados para estabelecer um modelo in vitro de lesão cardíaca induzida por hipóxia. O efeito da superexpressão de VEGF, isolado ou em conjunto com inibidores de moléculas pequenas que têm como alvo os canais de cálcio, receptores sensíveis ao cálcio (CaSR, do inglês calcium-sensitive receptors) e calpaína, no crescimento e proliferação celular em lesão de cardiomiócitos induzidos por hipóxia, foram determinados com ensaio de MTT, coloração TUNEL, coloração com Anexina V/PI, lactato desidrogenase e atividade da caspase. Para análise estatística, um valor de p<0,05 foi considerado significativo. Resultados: Verificou-se que o efeito do VEGF121 foi mediado por CaSR e calpaína, mas não foi dependente dos canais de cálcio. Conclusões: Nossos resultados, mesmo em um ambiente in vitro, estabelecem as bases para uma validação futura e testes pré-clínicos da terapia gênica baseada em VEGF em doenças cardiovasculares.

Circ_0124644 Serves as a ceRNA for miR-590-3p to Promote Hypoxia-Induced Cardiomyocytes Injury via Regulating SOX4.

Circular RNA (circRNA) is an important factor for regulating the progression of many cardiovascular diseases, including acute myocardial infarction (AMI). However, the role of circ_0124644 in AMI progression remains unclear. Hypoxia was used to induce cardiomyocytes injury. The expression of circ_0124644, microRNA (miR)-590-3p, and SRY-box transcription factor 4 (SOX4) mRNA was measured by qRT-PCR. Cell counting kit 8 (CCK8) assay and flow cytometry were utilized to detect cell viability, cell cycle progression, and apoptosis. The protein levels of apoptosis markers and SOX4 were determined by western blot (WB) analysis, and the levels of oxidative stress markers were assessed using commercial Assay Kits. Dual-luciferase reporter assay, RIP assay, and RNA pull-down assay were employed to confirm the interaction between miR-590-3p and circ_0124644 or SOX4. Circ_0124644 was upregulated in AMI patients and hypoxia-induced cardiomyocytes. Hypoxia could inhibit cardiomyocytes viability, cell cycle process, and promote apoptosis and oxidative stress, while silencing circ_0124644 could alleviate hypoxia-induced cardiomyocytes injury. In terms of mechanism, circ_0124644 could target miR-590-3p. MiR-590-3p overexpression could relieve hypoxia-induced cardiomyocytes injury. Also, the suppressive effect of circ_0124644 knockdown on hypoxia-induced cardiomyocytes injury could be reversed by miR-590-3p inhibitor. Moreover, SOX4 was found to be a target of miR-590-3p, and its overexpression also could reverse the regulation of miR-590-3p on hypoxia-induced cardiomyocytes injury. Circ_0124644 silencing could alleviate hypoxia-induced cardiomyocytes injury by regulating the miR-590-3p/SOX4 axis, suggesting that it might be a target for alleviating AMI.

CircRNA 010567 plays a significant role in myocardial infarction via the regulation of the miRNA-141/DAPK1 axis.

BackgroundMyocardial infarction (MI), caused by temporary or permanent coronary artery occlusion, poses a serious threat to patients’ lives. Circular RNAs (circRNAs), a new kind of endogenous noncoding RNAs, have been widely studied recently. This study was designed to illustrate and potential molecular mechanisms of circRNA 010567 in hypoxia-induced cardiomyocyte injury in vitro, so as to provide new strategies for the therapy of MI.MethodsH9c2 cells were cultured in anoxic conditions with 94% N2, 5% CO2, and 1% O2 to establish the in vitro MI model. Cell viability and apoptosis were checked using MTT and flow cytometry assay, respectively, Moreover, the levels of circRNA 010567, miR-141, and DAPK1 was determined using qRT-PCR. The putative targets of circRNA 010567 and miR-141 were confirmed by dual-luciferase reporter system and the RNA immunoprecipitation (RIP) assay. The release of creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and the viability of mitochondria were detected using assay kits.ResultsThe current study revealed that circRNA 010567 and DAPK1 were over-expressed, and miR-141 was low-expressed in hypoxia-induced MI. circRNA 010567 sponges miR-141 and DAPK1 was a direct target of miR-141. Mechanistic investigations revealed that circRNA 010567-siRNA impaired the release of CK-MB and cTnI, and promoted the viability of mitochondria in hypoxia-induced H9c2 cells, while these findings were reversed by the miR-141 inhibitor. In addition, the miR-141 mimic markedly reduced the release of CK-MB and cTnI, and promoted the viability of mitochondria, and these results were reversed by the DAPK1-plasmid. Subsequently, functional experiments revealed that hypoxia-stimulated decreases in H9c2 cell viability, as well as increases in apoptosis and caspase-3 activity, were induced by the miR-141 mimic and circRNA 010567-siRNA. However, these results were reversed by the miR-141 inhibitor and DAPK1-plasmid.ConclusionsOur results demonstrated that circRNA 010567-siRNA played a protective role in hypoxia-induced cardiomyocyte damage via regulating the miR-141/DAPK1 axis, indicating that circRNA 010567-siRNA may be a promising target for MI therapy.

Retraction Note to: Downregulation of miR-483-5p decreases hypoxia-induced injury in human cardiomyocytes by targeting MAPK3

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s11658-021-00252-1

MiR-3574 ameliorates intermittent hypoxia-induced cardiomyocyte injury through inhibiting Axin1.

Objective: miRNAs play critical roles in the regulation of many cardiovascular diseases. However, its role and potential mechanism in cardiac injury caused by obstructive sleep apnea (OSA) remain poorly elucidated. In the present study, we aimed to investigate the effects of miR-3574 on cardiomyocyte injury under intermittent hypoxia (IH).Results: We confirmed that IH inhibited cell viability, induced cell apoptosis and suppressed miR-3574 expression in the H9c2. miR-3574 overexpression could ameliorate the effects of IH on the cell viability and cell apoptosis in the H9c2. Axin1 was a target gene of miR-3574, and miR-3574 overexpression reduced the expression of Axin1. miR-3574 could inhibit the IH-induced cardiomyocyte injury via downregulating Axin1. However, Axin1 could partially reverse these effects of miR-3574.Conclusion: Our study first reveals that miR-3574 could alleviate IH-induced cardiomyocyte injury by targeting Axin1, which may function as a novel and promising therapy target for OSA-associated cardiovascular diseases.Methods: H9c2 were exposed to IH condition. CCK-8 assay was applied to determine cell viability of H9c2. qRT-PCR was conducted to measure the expression level of mRNA and miRNA. Western blot assay was then performed to detect the protein levels. Finally, we used dual-luciferase reporter assay identify the potential target of miR-3574.

MicroRNA-9 facilitates hypoxia-induced injury and apoptosis in H9c2 cells via targeting CDK8

Hypoxia plays an important role in many heart diseases. MicroRNA-9 (miR-9) has been reported to be involved in hypoxia-induced cell proliferation, injury and apoptosis in cardiomyocytes. However, the underlying mechanism still remains poorly understood. The expression levels of miR-9 and cyclin-dependent kinase 8 (CDK8) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The relative protein expression was measured by Western blot. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), lactate dehydrogenase (LDH) measurement, flow cytometry assays were conducted to detect cell proliferation, the release of LDH and cell apoptosis, respectively. The potential relationship between miR-9 and CDK8 was predicted by online database, and confirmed by dual-luciferase reporter assay. We found that miR-9 was increased, while CDK8 was decreased in hypoxia-treated H9c2 cells. miR-9 down-regulation or CDK8 up-regulation promoted cell proliferation, while repressed cell damage and apoptosis in hypoxia-induced H9c2 cells. Moreover, CDK8 was identified to be target of miR-9, and CDK8 knockdown could reverse the effects of miR-9 inhibitor on cell proliferation, damage and apoptosis in hypoxia-treated H9c2 cells. Besides, miR-9 could regulate the Wnt/b-catenin pathway by targeting CDK8 in hypoxic-induced H9c2 cells. In conclusion, miR-9 repressed cell proliferation and promoted cell damage and apoptosis by binding to CDK8 through the Wnt/ β-catenin pathway in hypoxic-induced H9c2 cells, which provided a new direction for further studying the treatment of hypoxia-aroused heart diseases.

MiR-22 Host Gene Enhances Nuclear Factor-kappa B Activation to Aggravate Hypoxia-induced Injury in AC16 Cardiomyocytes

Myocardial infarction (MI) is a severe life-threatening disease caused by acute and persistent ischemia and hypoxia and eventually leads to heart failure and sudden death. Long noncoding RNAs (lncRNAs) play significant roles in the pathology, diagnosis, and development of various cardiovascular diseases, including MI. This study aimed to explore the effect and molecular mechanism of lncRNA miR-22 host gene (MIR22HG) on hypoxia-induced injury in AC16 cardiomyocytes. The expression of MIR22HG and miR-24 in hypoxia-treated AC16 cardiomyocytes was detected by quantitative real-time polymerase chain reaction. Cell viability, lactate dehydrogenase release, levels of aspartate aminotransferase (AST) and creatine kinase-MB (CK-MB), and apoptosis were detected by Cell Counting Kit-8, lactate dehydrogenase (LDH) release assay, commercial enzyme-linked immune sorbent assay kits, and flow cytometry analysis, respectively. The protein levels of nuclear factor-kappa B (NF-κB) p65 and cytoplasmic inhibitor of kappa B alpha (IκBα) and phosphorylated IκBα were detected by western blot. Results showed that hypoxia treatment decreased viability and increased MIR22HG expression in AC16 cardiomyocytes. MIR22HG overexpression aggravated hypoxia-induced viability reduction, leakage of myocardial injury markers LDH, AST, and CK-MB, and apoptosis in AC16 cardiomyocytes, while MIR22HG knockdown elicited the reverse effects. MIR22HG overexpression enhanced NF-κB activation in hypoxia-treated AC16 cardiomyocytes. Inhibition of NF-κB pathway impaired the effects of MIR22HG overexpression on hypoxia-induced injury in AC16 cardiomyocytes. Moreover, MIR22HG knockdown inhibited the NF-κB pathway by upregulating miR-24 in AC16 cardiomyocytes. Inhibition of miR-24 resisted the effects of MIR22HG silencing on hypoxia-induced injury in AC16 cardiomyocytes. In conclusion, MIR22HG overexpression aggravated hypoxia-induced injury in AC16 cardiomyocytes via enhancing NF-κB activation by targeting miR-24.

Long non-coding RNA plasmacytoma variant translocation 1 linked to hypoxia-induced cardiomyocyte injury of H9c2 cells by targeting miR-135a-5p/forkhead box O1 axis.

BackgroundMyocardial infarction occurs due to insufficient (ischemia) blood supply to heart for long time; plasmacytoma variant translocation 1 (PVT1) is a long non-coding RNAs (lncRNAs) involved in the pathogenesis of various diseases, including heart disease; However, few studies have explored its role. The present study evaluated the effects of lncRNA PVT1 on hypoxic rat H9c2 cells.MethodsHypoxic injury was examined by measuring cell viability and apoptosis by using cell counting kit-8 activity and flow cytometry assays. Gene expressions after hypoxia were estimated by quantitative real time polymerase chain reaction and the signaling pathway were explored by Western blot analysis. RNA immunoprecipitation and luciferase reporter assays were applied to examine the interactions among genes. Data were analyzed using t-test with one-way or two-way analysis of variance.ResultsThe lncRNA PVT1 is up-regulated in hypoxia-stressed H9c2 cells and knockdown of PVT1 mitigates hypoxia-induced injury in H9c2 cells. PVT1 acts as a sponge for miR-135a-5p and knockdown of PVT1 attenuated the increased hypoxia-induced injury by up-regulating miR-135a-5p. Forkhead box O1 (FOXO1) was identified as a target of miR-135a-5p, and the expression was negatively regulated by miR-135a-5p. The exploration of the underlying mechanism demonstrated that knockdown of FOXO1 reversed PVT1/miR-135a-5p mediated hypoxia-induced injury in H9c2 cells.ConclusionsPVT1 plays a crucial role in hypoxia-injured H9c2 cells through sponging miR-135a-5p and then positively regulating FOXO1.

Metabolomic Analysis of the Ameliorative Effect of Enhanced Proline Metabolism on Hypoxia-Induced Injury in Cardiomyocytes.

Background Coronary heart disease is currently the leading cause of death in humans. Its poor prognosis and high mortality are associated with myocardial ischemia, which leads to metabolic disorder-related cardiomyocyte apoptosis and reactive oxygen species (ROS) production. Previous cardiovascular metabolomics studies in humans and mice have shown that proline metabolism is severely altered after cardiomyocyte hypoxia. Proline dehydrogenase (PRODH) is located on the inner mitochondrial membrane and is an enzyme that catalyzes the first step of proline catabolism, which plays an important role in improving the cellular redox state. In vitro oxygen-glucose deprivation can mimic in vivo myocardial ischemic injury. This study is aimed at investigating whether enhancing proline metabolism by overexpressing PRODH can ameliorate hypoxia-induced injury in cardiomyocytes and to reveal the related altered metabolites and mechanistic pathway via untargeted metabolomics analysis. Methods and Results First, through public database analysis and RT-qPCR and western blot analyses in a cardiomyocyte hypoxia model, we found that the expression of the proline-degrading enzyme PRODH was downregulated after myocardial infarction and hypoxia exposure. Second, LDH assays, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), DHE staining, flow cytometric apoptosis analysis with DCFH and Annexin V-FITC/PI, and western blot analysis were used to assess the injury level in cardiomyocytes. Enhanced proline metabolism induced by PRODH overexpression reduced the levels of reactive oxidative stress and apoptosis, whereas PRODH knockdown had the opposite effects. Third, untargeted metabolomics analysis revealed that the protective effect was associated with significant changes in metabolism linked to sphingolipid signaling pathways, unsaturated fatty acid biosynthesis, phosphocreatine, glutathione disulfide, aminoacyl-tRNA biosynthesis, and ABC transporters. Conclusions Our study demonstrated a protective effect of enhanced proline metabolism in cardiomyocytes under hypoxia, providing a novel strategy for exploring new treatments for coronary heart disease.

Downregulation of FOXO6 alleviates hypoxia-induced apoptosis and oxidative stress in cardiomyocytes by enhancing Nrf2 activation via upregulation of SIRT6.

Forkhead box protein O6 (FOXO6) has been recently identified as a novel regulator of oxidative stress in multiple pathological processes. However, whether FOXO6 participates in the regulation of oxidative stress of myocardial infarction is unclear. The present study was performed to evaluate the potential role of FOXO6 in regulating hypoxia-induced apoptosis and oxidative stress in cardiomyocytes in vitro. Our results demonstrated that FOXO6 expression was highly elevated in cardiomyocytes exposed to hypoxia. Downregulation of FOXO6 expression by the siRNA-mediated gene knockdown in hypoxia-exposed cardiomyocytes increased cell viability, while repressing apoptosis and reactive oxygen species (ROS) production. In contrast, overexpression of FOXO6 enhanced the sensitivity of cardiomyocytes to hypoxia-induced injury. Further, in-depth research revealed that knockdown of FOXO6 promoted the expression of sirtuin6 (SIRT6) and enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2)-mediated antioxidant signaling. Moreover, SIRT6 inhibition markedly blocked the FOXO6 knockdown-induced promotion effect on Nrf2 activation. In addition, Nrf2 inhibition partially reversed the FOXO6 knockdown-mediated protective effect against hypoxia-induced cardiomyocyte injury. Taken together, the findings of our study demonstrate that knockdown of FOXO6 is capable of protecting cardiomyocytes from hypoxia-induced apoptosis and oxidative stress by enhancing Nrf2 activation via upregulation of SIRT6. Our study highlights a potential role of FOXO6 in myocardial infarction and suggests it as an attractive target for cardioprotection.

Inhibition of sphingomyelin synthase 2 relieves hypoxia-induced cardiomyocyte injury by reinforcing Nrf2/ARE activation via modulation of GSK-3β.

Sphingomyelin synthase 2 (SMS2) is a vital contributor to tissue injury and affects various pathological processes. However, whether SMS2 participates in the modulation of cardiac injury in myocardial infarction has not been determined. This study aimed to evaluate the potential role of SMS2 in the regulation of cardiomyocyte injury induced by hypoxia, an in vitro model for studying myocardial infarction. Our data revealed that SMS2 expression was significantly upregulated in cardiomyocytes in response to hypoxia. Loss-of-function experiments revealed that knockdown of SMS2 markedly restored the viability of cardiomyocytes impaired by hypoxia, and attenuated hypoxia-evoked apoptosis and reactive oxygen species (ROS) generation. In contrast, cardiomyocytes that highly expressed SMS2 were more sensitive to hypoxia-induced injury. Moreover, SMS2 deficiency enhanced the activation of nuclear factor erythroid 2-related factor 2 (Nrf2) signaling through inactivation of glycogen synthase kinase-3β. Notably, suppression of Nrf2 markedly abrogated SMS2 knockdown-mediated cardioprotective effects on hypoxia-exposed cardiomyocytes. Our results illustrate that downregulation of SMS2 exerts a cardioprotective function by protecting cardiomyocytes from hypoxia-induced apoptosis and oxidative stress through enhancement of Nrf2 activation. Our study indicates a potential role of SMS2 in the modulation of cardiac injury, which may contribute to the progression of myocardial infarction.

FOXD3-AS1 Knockdown Suppresses Hypoxia-Induced Cardiomyocyte Injury by Increasing Cell Survival and Inhibiting Apoptosis via Upregulating Cardioprotective Molecule miR-150-5p In Vitro.

Congenital heart disease (CHD) is the most common type of human innate malformation in fetuses. LncRNAs have been pointed to play critical regulatory roles in various types of cardiac development and diseases including CHD. Our study aimed to explore the effects of lncRNA forkhead box D3 antisense RNA 1 (FOXD3-AS1) on hypoxia-induced injury in AC16 cardiomyocytes and the related molecular mechanism. In vitro cell model of CHD was established by stimulating AC16 cells with hypoxia (1% O2). Expression of FOXD3-AS1 and miR-150-5p was detected by qRT-PCR. Hypoxia-induced injury was evaluated by detecting cell survival, lactate dehydrogenase (LDH) release, apoptosis, and caspase-3/7 activity using MTT, LDH assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. The regulatory relationship between FOXD3-AS1 and miR-150-5p was explored by luciferase reporter assay, RNA immunoprecipitation (RIP), and qRT-PCR. Results showed that hypoxia exposure caused an upregulation of FOXD3-AS1 and a downregulation of miR-150-5p in AC16 cells. Knockdown of FOXD3-AS1 attenuated reduction of cell survival and increase of LDH release, apoptosis, caspase-3/7 activity, and Bcl-2 associated X (Bax) expression induced by hypoxia in AC16 cells. Notably, we demonstrated that FOXD3-AS1 directly interacted with miR-150-5p to inhibit its expression. miR-150-5p knockdown reinforced the reduction of survival and induction of apoptosis by hypoxia and attenuated the effects of FOXD3-AS1 silencing on the same parameters in AC16 cells. In conclusion, FOXD3-AS1 knockdown protected AC16 cardiomyocytes from hypoxia-induced injury by increasing cell survival and inhibiting apoptosis through upregulating miR-150-5p.

Hypoxia exacerbates cardiomyocyte injury via upregulation of Wnt3a and inhibition of Sirt3

Ischemic injury is a major cause of several cardiovascular diseases, such as myocardial infarction, cardiac hypertrophy, and ventricular remodeling. Using an in vitro hypoxia model to mimic ischemia, we found that hypoxia stimulated Wnt3a expression. A mechanistic study showed that hypoxia-inducible factor 1α (HIF-1α) was directly recruited to the Wnt3a promoter. Wnt3a overexpression significantly decreased cell viability, promoted the generation of apoptotic cells, and enhanced hypoxia-induced injury in neonatal rat cardiomyocytes. This was partially through the upregulation of Caspase-3 mRNA levels and cleaved PARP-1 protein levels. In addition, we observed that Wnt3a exacerbated hypoxia-induced mitochondrial dysfunction and cytosolic release of cytochrome C. Furthermore, we found that Sirt3, a mitochondrial NAD+-dependent deacetylase that modulates mitochondrial metabolism and homeostasis, was negatively regulated by Wnt3a. Conversely, Sirt3 overexpression repressed Wnt3a expression and ameliorated the hypoxia-induced mitochondrial dysfunction. Overall, our findings suggest that the hypoxia–Wnt3a–Sirt3 regulatory axis might be a potential target for cell protection in cardiac ischemia and hypoxia.

Long Non-Coding RNA SNHG8 Plays a Key Role in Myocardial Infarction Through Affecting Hypoxia-Induced Cardiomyocyte Injury.

BackgroundThe objective of the study was to explore the role of long non-coding RNA SNHG8 (lncRNA SNHG8) in myocardial infarction (MI) and the related mechanism of action.Material/MethodsIn vitro model of MI was established by hypoxia induction in cardiomyocyte line H9c2 cells. H9c2 cells were transfected with control-plasmid, SNHG8-plasmid, control-shRNA and SNHG8-shRNA. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed to measure transfection efficiency. Creatine kinase-muscle/brain (CK-MB) release, cardiac troponin 1 (cTnI) release and mitochondria viability were detected by using related detection kits. MTT (3-(45)-dimethylthiahiazo (-z-y 1)-35-diphenytetrazoliumromide) assay was used to detect cell viability and flow cytometry analysis was used to detect cell apoptosis. Western blot assay was performed to measure protein expression of cleaved-Caspase3, p-p65 and p65. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR assay were performed to detect expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α and IL-6.ResultsLncRNA SNHG8 was overexpressed in hypoxia-induced cardiomyocytes. SNHG8-plasmid increased lncRNA SNHG8 expression, CK-MB release, cTnI release, and mitochondria viability in hypoxia-induced H9c2 cells. In addition, SNHG8-plasmid reduced cell viability, induced cell apoptosis, and increased expression of cleaved-caspase3, IL-1β, TNF-α, IL-6, and p-p65 in hypoxia-induced H9c2 cells, while the effects of SNHG8-shRNA were opposite.ConclusionsWe demonstrated that lncRNA SNHG8 affected myocardial infarction by affecting hypoxia-induced cardiomyocyte injury via regulation of the NF-κB pathway.

6-Gingerol protects cardiomyocytes against hypoxia-induced injury by regulating the KCNQ1OT1/miR-340-5p/PI3K/AKT pathway.

Hypoxia could induce cardiomyocytes injury and lead to heart disease. Studies have shown that 6-Gingerol has a protective effect on cardiomyocytes injury, but its molecular mechanism is still unclear. Cell counting kit 8 (CCK8) and flow cytometry assays were used to measure the viability and apoptosis of cardiomyocytes. Western blot (WB) analysis was performed to assess the levels of proliferation, apoptosis, and phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling pathway-related proteins. The reactive oxygen species (ROS) level, superoxide dismutase (SOD) activity and malondialdehyde (MDA) level were detected by their corresponding Assay Kits. Besides, the expression levels of potassium voltage-gated channel subfamily Q member 1 opposite strand 1 (KCNQ1OT1) and microRNA-340-5p (miR-340-5p) were determined by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to verify the interaction between KCNQ1OT1 and miR-340-5p. Hypoxia could inhibit the viability and enhance the apoptosis and oxidative stress of cardiomyocytes to induce cardiomyocytes injury, while 6-Gingerol could alleviate this effect. Overexpression of KCNQ1OT1 aggravated hypoxia-induced cardiomyocytes injury and reversed the protective effect of 6-Gingerol on cardiomyocytes injury. Besides, miR-340-5p could be sponged by KCNQ1OT1, and its overexpression could invert the promotion effect of KCNQ1OT1 overexpression on hypoxia-induced cardiomyocytes injury. Moreover, miR-340-5p expression was regulated by 6-Gingerol and KCNQ1OT1. In addition, hypoxia inactivated the PI3K/AKT signaling pathway, whereas 6-Gingerol and miR-340-5p could reverse this effect. 6-Gingerol could hinder the expression of KCNQ1OT1 to protect cardiomyocytes from hypoxia-induced injury through regulation of the miR-340-5p/ PI3K/AKT pathway, providing a new mechanism of 6-Gingerol protecting cardiomyocytes from injury.

MiR-134-5p inhibition reduces infarct-induced cardiomyocyte apoptosis via Creb1 upregulation

BackgroundFollowing the recent discovery that microRNA-134-5p (miR-134-5p) is elevated in the early stages of acute myocardial infarction (AMI), we examined the specific role of miR-134-5p in cardiomyocytes during AMI. MethodsTo study miR-134-5p's role in the context of AMI, we used a combination of in vitro experiments in H2O2–treated or hypoxic cardiomyocyte cell cultures as well as in vivo experiments in a murine model of AMI. ResultsH2O2- and hypoxia-induced cardiomyocyte injury upregulated miR-134-5p expression. miR-134-5p overexpression increased cardiomyocyte apoptosis, whereas miR-134-5p inhibition reduced cardiomyocyte apoptosis. We discovered that the transcription factor cAMP-responsive element binding protein 1 (Creb1) is a functional target of miR-134-5p responsible for regulating cardiomyocyte apoptosis. In vivo AMI resulted in the upregulation and downregulation of miR-134-5p and Creb1 in the infarct area, respectively. Circulating miR-134-5p levels were also increased at days 1 and 2 post-AMI. Modulation of myocardial miR-124-5p expression by intramyocardial injection of antagomiR-134-5p or agomiR-134-5p significantly affected cardiomyocyte apoptosis, infarct size, and cardiac function in vivo. ConclusionsmiR-134-5p/Creb1 axis dysregulation plays a role in hypoxia- or oxidative stress-induced cardiomyocyte apoptosis as well as AMI. Circulating miR-134-5p may show promise as a biomarker for AMI or post-AMI cardiac dysfunction. Manipulating the miR-134-5p/Creb1 axis through either inhibition of miR-134-5p or overexpression of Creb1 may show promise as a novel therapeutic strategy to attenuate cardiac dysfunction following AMI.

LncRNA Kcnq1ot1 renders cardiomyocytes apoptosis in acute myocardial infarction model by up-regulating Tead1

Acute myocardial infarction (AMI) is a major cardiovascular disease with high mortality worldwide. Hypoxia is a key inducing factor for AMI. We aimed to examine the expression and functions of Kcnq1ot1 (KCNQ1 overlapping transcript 1) in hypoxia-induced cardiomyocytes in the process of AMI. The left anterior descending coronary artery ligation (LAD) was used for inducing in-vivo AMI model and the primary cardiomyocytes were extracted; in-vitro H9c2 cell model was simulated by hypoxia treatment. TUNEL, flow cytometry and JC-1 assay were carried out to evaluate cell apoptosis. Mechanism assays including luciferase reporter assay and RIP assay revealed interplays between RNAs. To begin with, Kcnq1ot1 was revealed to be conspicuously upregulated in myocardium infracted zone and border zone within 2 days since establishment of the model. Moreover, inhibition of Kcnq1ot1 protected cardiomyocytes against hypoxia-triggered cell apoptosis during the process of AMI. Then, miR-466k and miR-466i-5p were proved to bind with Kcnq1ot1 and participated in Kcnq1ot1-mediated cardiomyocyte injury under hypoxia. Subsequently, Kcnq1ot1 was found to elevate Tead1 (TEA domain transcription factor 1) expression via sponging miR-466k and miR-466i-5p. Finally, it was verified that Kcnq1ot1 regulated hypoxia-induced cardiomyocyte injury dependent on Tead1. In conclusion, Kcnq1ot1 sponged miR-466k and miR-466i-5p to up-regulate Tead1, thus triggering cardiomyocyte injury in the process of AMI.