The Role And Mechanism Of M6A Methyltransferase METTL3 In Ang Ⅱ Induced Myocardial Hypertrophy

Objective:Pathological myocardial hypertrophy is mainly manifested in the damage of heart structure,which leads to heart failure,and finally leads to various adverse cardiovascular events.This abnormal state involves many types of genes and signaling pathways,and the internal mechanism is very complex.Angiotensin Ⅱ(AngⅡ)is recognized as one of the pathogenic factors.At present,the understanding of the internal mechanism of regulating the occurrence and development of pathological myocardial hypertrophy is still not comprehensive.RNA methylation modification m6A is one of the most common forms of RNA modification,and methyltransferase METTL3 is the core catalytic enzyme in this modification process.Studies have shown that METTL3 mediated m6A modification is widely involved in the pathophysiology of various cardiovascular diseases.However,its role in Ang Ⅱinduced cardiac hypertrophy remains unknown.Methods:Section one:Male adult wild-type C57BL/6J mice were continuously perfused with Ang Ⅱ by osmotic pump for 3 weeks to induce myocardial hypertrophy model in vivo;Neonatal rat primary cardiomyocytes(NRCMs)from Sprague Dawley(SD)rats aged 1～3 days were isolated and cultured.Ang Ⅱ intervention for 48 hours induced the formation of cardiomyocyte hypertrophy model in vitro.The heart weight to body weight ratio(HW/BW)was calculated and the cross-sectional area of cardiomyocytes in vivo was evaluated by H&E pathological staining;α-actinin immunofluorescence staining was used to evaluate the cross-sectional area of isolated cardiomyocytes.qRT-PCR and Western blot were used to detect biomarkers of myocardial hypertrophy including ANP and BNP,and METTL3.m6A methylation level was detected by colorimetric method.Section two:Overexpression or knockdown of METTL3 in NRCMs were mediated by lentivirus transfection.Ang Ⅱ was used to intervene for 48 hours to induce hypertrophy of cardiomyocytes.Western blot and qRT-PCR were used to detect the expression of two biomarkers of myocardial hypertrophy and METTL3,and m6A methylation level was evaluated by colorimetric method.α-actinin immunofluorescence staining was used to evaluate the cross-sectional area of cardiomyocytes.Section three:The expression of METTL3 in mouse myocardium was inhibited by adeno-associated virus AAV9 that carrying shMETTL3.Ang Ⅱ was continuously infused by osmotic pump until 3 weeks to induce myocardial hypertrophy in vivo.Western blot was used to detect the level of METTL3,and qRTPCR was used to detect expression of two biomarkers of myocardial hypertrophy.Cardiac structure and function of mice were evaluated by echocardiography,and the cross-sectional area of cardiomyocytes in vivo was evaluated by H&E and WGA pathological staining.Section four:qRT-PCR was used to detect the expression of miR-221/222 and pri-miR-221/222 in Ang Ⅱ induced cardiomyocyte hypertrophy model,the expression of miR-221/222 in METTL3 overexpressed or knockdown cardiomyocytes,and the expression of miR-221/222 in METTL3 knockdown mouse cardiac hypertrophy model.Besides,meRIP and DGCR8-IP were used to evaluate the action of METTL3 on pri-miR-221/222.Furthermore,cardiomyocytes stimulated by Ang Ⅱ were intervented with miR-221/222 inhibitors alone or shMETTL3 combined with miR221/222 mimics.ANP and BNP expression were detected,and α-actinin immunofluorescence staining was used to evaluate the cross-sectional area of cardiomyocytes.Section five:Targetscan database was applied to predict the downstream targets of miR-221/222 and analyze the pathway enrichment,and DKK2/Wnt/β-Catenin signal axis was selected.The Ang Ⅱ induced cardiomyocytes hypertrophy model were intervetened with shMETTL3 or miR-221/222 inhibitors respectively.Subsequently,DKK2,β-catenin and c-Myc were detected by Western blot and qRT-PCR.The expression of β-catenin in METTL3 knockdown mouse cardiac hypertrophy model was detected by Western blot.The direct regulation of DKK2 by miR-221/222 was evaluated via Dual-Luciferase Report Gene System.Furthermore,miR-221/222 inhibitors combined with shDKK2 were used to interfere with the Ang Ⅱ induced cardiomyocytes hypertrophy model,and expression of β-catenin and c-Myc were detected by Western blot and qRT-PCR.Results:Section one:During Ang Ⅱ induced myocardial hypertrophy in mice model,the HW/BW ratio and the cross-sectional area of cardiomyocytes increased.Besides,the expression level of METTL3 was significantly up-regulated.The expression level of ANP and BNP were increased significantly,and the cross-sectional area of cardiomyocytes expanded significantly during Ang Ⅱ induced cardiomyocytes hypertrophy of NRCMs.At the same time,the expression level of METTL3 was significantly up-regulated,accompanied by a significant increase in the level of m6A methylation modification.Section two:Ang Ⅱ could promote the expression of METTL3 and up-regulate the level of m6A modification.The combined application of shMETTL3 not only significantly inhibited the expression of METTL3,but also reduced the level of m6A modification.During the process,Ang Ⅱ induced increasement of ANP and BNP expression,as well as increasement of the cross-sectional area of cardiomyocytes,were all reversely decreased by METTL3 knockdown.Section three:Continuous infusion of Ang Ⅱ in vivo could promote METTL3,ANP and BNP expression,while combined application of shMETTL3 not only inhibited the expression of METTL3 in myocardium,but also down-regulated the levels of two biomarkers of myocardial hypertrophy.Echocardiography showed that Ang Ⅱ intervention for 3 weeks could significantly increase the ventricular wall thickness of mice without affecting the systolic function.After knocking down METTL3,the ventricular wall thickness of mice decreased significantly and the cardiac function did not change.The results of pathological staining showed that AngⅡ stimulation significantly increased the cross-sectional area of mouse cardiomyocytes in vivo,while knockdown of METTL3 in myocardium alleviated the pro-hypertrophic effect of Ang Ⅱ,resulting in the decrease of cross-sectional area of cardiomyocytes.Section four:The expression of miR-221/222 and pri-miR-221/222 were significantly increased and decreased respectively after Ang Ⅱ intervention.Knockdown of METTL3 could down-regulate the expression of miR-221/222 and upregulate the expression of pri-miR-221/222,while overexpression of METTL3 could have the opposite effect.The expression of miR-221/222 was up-regulated during Ang Ⅱ induced in vivo myocardial hypertrophy,while knockdown of METTL3 could decrease their expression levels.Results of meRIP and DGCR8-IP results indicated that METTL3 overexpression could promote m6A modification on pri-mir-221/222 and promote their binding to DGCR8.Inhibition of miR-221/222 could down-regulate the Ang Ⅱ induced increasement of biomarkers including ANP and BNP,as well as the augment of cardiomyocyte cross-sectional area.Knockdown of METTL3 could alleviate the increase of cardiac hypertrophy biomarkers and cardiomyocyte crosssectional area induced by Ang Ⅱ,and overexpression of miR-221/222 could reverse the above protective effect.Section five:Bioinformatics analysis showed that the downstream target genes of miR-221/222 were mostly associated with Wnt/β-catenin pathway.During Ang Ⅱinduced cardiomyocytes hypertrophy,the expression of β-catenin and its downstream c-Myc was significantly up-regulated,while further knockdown expression of METTL3 or miR-221/222 could down-regulate β-catenin and c-Myc.The expression of β-catenin was also up-regulated during Ang Ⅱ induced in vivo myocardial hypertrophy,and knockdown of METTL3 could decrease its expression level.DKK2 was significantly decreased during Ang Ⅱ induced cardiomyocytes hypertrophy,and overexpression/knockdown of miR-221/222 could inhibit and promote the expression of DKK2,respectively.The luciferase reporter system suggested that miR-221/222 could directly inhibit the expression of DKK2.Inhibition of miR-221/222 could alleviate Ang Ⅱ induced up-regulated of β-catenin and c-Myc,and further knockdown DKK2 expression could antagonize the above effects,resulting in re-promoted expression of β-catenin and c-Myc.Conclusion:This study found that Ang Ⅱ can up-regulate the expression of METTL3 in cardiomyocytes.METTL3 promotes the binding of DGCR8 with pri-miR-221/222 in an m6A dependent manner,and promotes the expression of miR-221/222,while miR221/222 can directly inhibit DKK2,resulting in Wnt/β-catenin signaling activation and eventually leads to the formation of myocardial hypertrophy.