The Role And Underlying Mechanisms Of N~6-Methyladenosine Methyltransferase In Myocardial Hypoxic Preconditioning

PartⅠHypoxic preconditioning protects cardiomyocytes from oxidative stress injury induced by H₂O₂Objective:To isolate and culture the primary cardiomyocytes（CMs）and H9c2 cells,to establish the H₂O₂-induced oxidative stress injury models of CMs and H9c2 cells,and to examine the protective effect of hypoxic preconditioning（HPC）.Methods:The hearts of newborn Sprague Dawley（SD）rats aged 1 to 3 days were excised in the sterile environment.CMs were isolated and cultured by enzymatic digestion and differential adhesion method.The morphology of CMs was observed under the inverted phase contrast microscope.α-Actinin and vimentin were detected by immunofluorescent staining.H9c2 cells and CMs were divided into the normoxia and HPC groups.HPC treatment was performed at 1%oxygen concentration for30min in the hypoxic incubator and then recovered in an aerobic incubator under 21%O₂ and 5%CO₂ for 30min.Subsequently,the oxidative stress injury was induced by adding different concentrations of H₂O₂（0μM,50μM,100μM,200μM,400μM）to the cells.Cell viability was measured using the cell counting kit-8（CCK-8）assay.Western blot（WB）assay was performed to detect the expression of cleaved caspase-3.The apoptosis of H9c2 cells and CMs was observed by the flow cytometry and TUNEL staining.Results:Under the inverted phase contrast microscope,the freshly extracted CMs were circular and freely suspended in the culture medium.After 12 hours,the CMs possessing the large nuclei and strong refractivity were entirely attached to the cell culture dishes and demonstrated fusiform,rod-shaped or polygonal morphology.Three days later,the pseudopodia of CMs gradually appeared.Some of the CMs were cross-linked.The proportion of spontaneous pulsation was more than 90%.According to the results of immunofluorescent staining,the CMs were positive forα-actinin but negative for the vimentin.The results of CCK-8 assay showed that HPC treatment could increase the viability of H9c2 cells and CMs.H₂O₂ reduced the cell viability in a dose-dependent manner.Compared with their respective baselines,the viabilities of H9c2 cells and CMs were significantly inhibited at 200μM H₂O₂ in both the normoxia group and the HPC group（P<0.001）.The cleaved caspase-3 protein level in the HPC group was significantly lower than that in the normoxia group when exposed to200μM H₂O₂.The results of flow cytometry and TUNEL staining showed that HPC treatment could significantly reduce the apoptosis induced by H₂O₂.Conclusion:CMs can be isolated and cultured successfully by enzymatic digestion integrated with differential adhesion method.The H₂O₂ concentration of 200μM can be utilized to establish the oxidative stress injury models of CMs and H9c2 cells.HPC treatment increases the cell viability of cardiomyocytes,alleviates their apoptosis,and could protect them from the oxidative stress injury induced by H₂O₂.Part Ⅱ The role of N6-methyladenosine methyltransferase in the HPC-mediated myocardial protective effectObjective: To explore the influence of HPC treatment on the overall level of N6-methyladenosine（m6A）modification in total RNA,and to investigate the role of m6 A methyltransferase in the HPC-mediated myocardial protective effect.Methods: H9c2 cells and CMs were divided into the normoxia and HPC groups and subsequently exposed to the H2O2 injury.The overall level of m6 A in total RNA was measured by colorimetric assay.Quantitative real-time PCR（q RT-PCR）and WB analysis were carried out to detect methyltransferase like 3（METTL3）,methyltransferase like 14（METTL14）and Wilms tumor 1-associating protein（WTAP）.The experimental SD rats were divided into sham-operated,ischemic preconditioning（IPC）,ischemia/reperfusion injury（I/R）and IPC+I/R groups.The myocardial injury and cardiomyocyte apoptosis were detected by HE staining and TUNEL staining.The overall level of m6 A modification in total RNA of myocardial tissue was detected by colorimetric assay.The expressions of METTL3 and METTL14 in heart tissues were assessed by q RT-PCR and WB analysis.Short hairpin RNA（sh RNA）mediated loss-of-function assay was performed using lentiviral vectors.CCK-8 assay,WB analysis and TUNEL staining were used to evaluate the influence of METTL3 or METTL14 knockdown on the HPC-mediated myocardial protective effect.Results: Compared with the normoxia group,the m6 A modification levels in total RNA of H9c2 cells and CMs were significantly increased in the HPC group（P<0.001）.The METTL3 and METTL14 in H9c2 cells and CMs were significantly up-regulated in m RNA and protein levels（P<0.05）,while the expression of WTAP was not significantly changed in vitro.Compared with the I/R group,the apoptosis and interstitial swelling in IPC+I/R group were significantly decreased.In agreement with the in vitro data,IPC treatment enhanced the m6 A abundance in total RNA and the expressions of METTL3 and METTL14 in heart tissues（P<0.05）.When METTL3 or METTL14 were silenced,the levels of m6 A modification in total RNA of H9c2 cells and CMs decreased remarkably（P<0.001）.The results of CCK-8 assay,WB analysis,and TUNEL staining showed that knockdown of METTL3 or METTL14 markedly reversed the HPC-induced increase of cell viability and the decline in cleaved caspase-3 and apoptosis.Conclusion: The HPC treatment in vitro and IPC treatment in vivo increase the overall levels of m6 A modification in total RNA and the expressions of METTL3 and METTL14 in cardiomyocytes.Since knockdown of METTL3 or METTL14 reverses the HPC-induced increase of cell viability and the decrease of apoptosis,both METTL3 and METTL14 are involved in the myocardial protective effect of HPC treatment.Part Ⅲ The role and underlying mechanisms of lnc RNA H19 in the HPC-mediated myocardial protective effect regulated by METTL3/14Objective: To investigate the role of long non-coding RNA（lnc RNA）H19 in the HPC-mediated myocardial protective effect and the underlying mechanisms regarding METTL3/14 on H19.Methods: The expressions of lnc RNA H19 in H9c2 cells and rat models were detected by q RT-PCR analysis.The knockdown and overexpression models of H19 in H9c2 cells were constructed using lentiviral vectors.The influences of H19 knockdown and overexpression on the viability and apoptosis of H9c2 cells were evaluated by CCK-8 assay,WB analysis and TUNEL staining.The quantification of m6 A abundance in H19 was based on m6 A RNA immunoprecipitation（Me RIP）assay to gather the methylated RNA fragments,which was followed by q RT-PCR analysis.The recovery experiments were performed to investigate the influences of H19 overexpression on the decreased HPC effect which was induced by METTL3 or METTL14 knockdown.The RNA-binding protein immunoprecipitation（RIP）assay was performed to detect the binding of METTL3 and METTL14 to H19.Results: The expressions of H19 markedly decreased in the H2O2 and I/R injury environments respectively.Compared with the normoxia group,the expression of H19 in H9c2 cells was significantly increased in HPC group（P<0.01）.The contents of H19 in heart tissues after IPC treatment were also significantly up-regulated（P<0.05）.Compared with the negative control（NC）group,knockdown of H19 reduced the viability of H9c2 cells（P<0.01）,while enhancing the protein expression of cleaved caspase-3 under the HPC plus H2O2-treated condition.The results of TUNEL staining also indicated that ablation of H19 enhanced the apoptosis of H9c2 cells exposed to H2O2.In addition,the enforced expression of H19 was found to enhance cell viability and inhibit apoptosis in H9c2 cells exposed to H2O2.The results of Me RIP assay demonstrated that the m6 A levels of H19 were notably enhanced in the HPC groups（P<0.05）.Compared with the NC group,the m6 A levels of H19 were markedly decreased in the METTL3 knockdown and METTL14 knockdown groups（P<0.001）.Knockdown of METTL3 or METTL14 significantly decreased the H19 expression（P<0.01）.H19 overexpression could partially rescue the decreased cell viability and the increased apoptosis in HPC-treated H9c2 cells under H2O2 injury environment,which was mediated by METTL3 or METTL14 knockdown.The results of RIP assay showed that the H19 contents were significantly increased in the METTL3-antibody and METTL14-antibody groups compared with the Ig G group（P<0.01）.Conclusion: Lnc RNA H19 is up-regulated after HPC treatment and plays a role in HPC-mediated myocardial protective effect.METTL3 and METTL14 can bind to H19,contribute to its level of m6 A modification,and regulate the expression of H19 in HPC treatment.Part Ⅳ The role and underlying mechanisms of METTL3/micro RNA-210 axis in the HPC-mediated myocardial protective effectObjective: To investigate the role of micro RNA-210（mi R-210）in the HPC-mediated myocardial protective effect and the underlying mechanisms regarding METTL3 on mi R-210.Methods: The mi RNA profiles of CMs under hypoxic environment were retrieved from the Gene Expression Omnibus（GEO）database.The microarray data of GSE24954 were downloaded and reanalyzed.The expressions of mi R-210 of CMs in the normoxia and HPC groups were detected by q RT-PCR analysis.The mimics and inhibitors were utilized to construct the overexpression and inhibition models of mi R-210.The influences of mi R-210 overexpression on the viability and apoptosis of CMs were evaluated by CCK-8 assay,WB analysis and TUNEL staining.The influences of METTL3 knockdown and overexpression on the expression of mi R-210 and primary micro RNA-210（pri-mi R-210）were detected by q RT-PCR analysis.The quantification of m6 A abundance in pri-mi R-210 was based on Me RIP assay followed by q RT-PCR analysis.RIP assay was performed to evaluate the influence of METTL3 overexpression on the binding of Di George syndrome critical region gene 8（DGCR8）protein to pri-mi R-210.The inhibitor-mediated loss-of-function assays were performed to analyze the influences of down-regulation of mi R-210 on the enhanced HPC myocardial protective effect induced by METTL3 overexpression.Results: The microarray data of GSE24954 indicated that mi R-210 in rat CMs was sensitive to hypoxia concentration.The results of q RT-PCR analysis confirmed that the expressions of mi R-210 in CMs were notably increased after HPC treatment.With the increase of H2O2-induced injury,the level of mi R-210 in CMs decreased gradually.Micro RNA-210 mimics increased the viability and decreased the expression of cleaved caspase-3 protein and apoptosis of CMs exposed to H2O2.METTL3 inhibition decreased the expression of mi R-210 while significantly increasing the expression of pri-mi R-210.In addition,METTL3 overexpression up-regulated the expression of mi R-210 while decreasing the expression of pri-mi R-210.The results of Me RIP assays demonstrated that the m6 A abundances of pri-mi R-210 were markedly enhanced after METTL3 overexpression（P<0.05）.The results of RIP experiments showed that METTL3 overexpression increased the contents of DGCR8-bound pri-mi R-210 significantly（P<0.01）.The data of CCK-8 assay,WB analysis and TUNEL staining showed that inhibition of mi R-210 significantly reversed the enhanced cell viability and the decline in apoptosis which were mediated by METTL3 overexpression.Conclusion: Micro RNA-210 is up-regulated after HPC treatment,protects CMs from oxidative stress injury induced by H2O2 and plays a role in the HPC-mediated myocardial protective effect.METTL3 methylates pri-mi R-210,increases its m6 A abundance and promotes the maturation of mi R-210 in HPC treatment.