Overexpression Of MiR-223 Tips The Balance Of Pro-and Anti-Hypertrophic Signaling Cascades Toward Physiologic Cardiac Hypertrophy

Objective:Cardiac hypertrophy is an adaptive response to different stress.Pathological cardiac hypertrophy is induced by hypertension,valve disease,myocardial infarction which is associated with fetal genes reexpression interstitial fibrosis,increased myocyte apoptosis,leading to heart failure.Physiological cardiac hypertrophy induced by postnatal development,pregnancy,exercise training is characterized by normal cardiac structure and function,presenting an adaptive beneficial response.microRNAs are small endogenous,noncoding RNAs that function primarily as post-transcriptional regulators by complementary to 3’-UTR of target mRNAs.microRNAs may play a critical role in the development of cardiac hypertrophy.In this study we generated a transgenic mouse model with cardiac specific overexpression of miR-223 to detect the mechanism of physiological cardiac hypertrophy induced by miR-223.Methods:1.To induce physiological cardiac hypertrophy mouse model using exercise training on treadmill,then detect the expression level of miR-223 in the hearts of exercised mice or sedentary control by RT-PCR.2.To generate the transgenic mouse model with cardiac specific overexpression of miR-223,then determine the expression level of miR-223 in TG mice hearts.HW/BW(heart weight to body weight)was determined as marker of cardiac hypertrophy.Mice hearts were fixed,embedded,and sections were cut in 5μm thickness.WGA(wheat germ agglutinin)staining was performed for cell size.Masson trichrome staining was proceeded to detect fibrosis.RT-PCR was carried out to determine the expression level of fibrosis gene markers and pathological cardiac hypertrophy markers.To measure LVPWd,LVPWs,IVSd,IVSs,EF and FS by echocardiography.3.To isolate and culture the NRCM(neonatal rat cardiomyocyte),the transfect cells with Ad.miR-223 or Ad.GFP(as control).Cells transfected were fixed,followed by immunofluorescence staining,then imaged using confocal microscope and cell size was analyzed with ImageJ.4.RNAseq was performed to identify gene expression profiles in miR-223 TG hearts.Then we validated expression levels of those dys-regulated genes in TG hearts.5.Total proteins were extracted from hearts or cultured NRCMs.Western blot was performed to determine the expression levels of target proteins.GAPDH was as control.6.Luciferase reporter assay was used to validate target mRNA of miR-223.Results:1.After exercise training on treadmill for 4 weeks,trained mice displayed enlarged hearts with a significant HW/BW,and expression levels of miR-223 were increased in the mice hearts after treadmill training.2.RT-PCR showed that,in the hearts of miR-223 transgenic mice models controlled by ɑ-MHC promoter,the expression level of mi R-223 were 1300-fole(line6)and 600-fold(line10)than WT hearts.3.3-months TG mice exhibited larger hearts than WTs,and HW/BW in TGs were significantly higher than WTs.Cell size in cross-section was increased in miR-223 TG mice compared with WTs.The results of Masson staining revealed no detectable fibrosis in either TG or WT hearts.The expression levels of fibrosis gene markers and pathological cardiac hypertrophy markers displayed no difference between WT and TG hearts.The results showed that,compared to WTs,IVSd,IVSs,EF and FS were increased in TGs.4.NRCMs were isolated and transfected with Ad.miR-223 or Ad.GFP.The expression level of miR-223 was increased by 3.4-fold at 24 h post-transfection compared with Ad.GFPcells,and this did not lead to any change in cardiomyocyte size.At the 48-h time point,miR-223 expression level was further increased by about 71-fold compared with GFP-cells,which significantly enlarged the cardiomyocyte size by 1.5-fold,comparing to Ad.GFP-infected cells.5.The results of RNAseq showed that there were a total of 81 up-regulated and 27 down-regulated genes in miR-223 TG hearts compared with WT hearts.The data of RTPCR analysis showed that the alterations of the selected genes,8 up-regulated and 3 downregulated,except Atf3 in two lines of miR-223 TG hearts were consistent with the RNAseq results.The validation of genes expression in NRCM observed in TG hearts showed that Postn,Rxfp1 and Egln3 were consistent with the alterations in TG hearts at 24-h time point after transfection,and at 48-h time point the altered pattern of the dys-regulated genes was similar to the results from TG hearts.6.The results of luciferase reporter assays indicated that ACVR2 A and FBXW7 transcripts contain binding motifs,which represent targets of miR-223.7.By western blotting analysis,the results showed that Ad.miR-223-transfected NRCM displayed lower levels of ACVR2 A,FBXW7 and Egln3 and higher levels of Hif-1α and Postn compared with control GFP-cells.miR-223 TG hearts exhibited a similar change pattern as observed in ex vivo myocytes.Conclusion:1.Treadmill-trained mice displayed enlarged hearts after 4 weeks and mi R-223 level was significantly increased in the heart after treadmill training.2.Cardiac-specific overexpression of miR-223 in vivo could induce physiological hypertrophy.Physiological cardiac hypertrophy triggered by elevation of miR-223 is largely dependent on the activation of Akt.3.Physiological cardiac hypertrophy induced by miR-223 is associated with direct regulation of FBXW7-Hif-1α-Postn and ACVR2a-Egln3-Hif-1α signaling cascades.