The Role Of Long Noncoding RNA Fendrr In Pressure Overload Induced Cardiac Remodeling And Its Mechanisms

Objectives To study the role of long noncoding RNA(lnc RNA)Fenddr in pressure overload induced cardiac remodeling and its underlying mechanismMethods The mice with cardiomyocytes specific knockout of Fendrr(Fendrr-KO)and mice with cardiomyocyte specific overexpression of Fendrr(Fendrr-TG)were constructed by gene editing techniques.Fendrr-KO mice and its Fendrr-Flox control mice,Fendrr-TG mice and its non-transgene littermates(NTG)were subjected to aortic banding(AB)surgery and sham surgery(sham),respectively.8 weeks after AB,echocardiography was used to assess the cardiac function of mice.The end-diastolic ventricular septal thickness(IVSd),end-diastolic left ventricle diameter(LVEDd),end-diastolic left ventricle posterior wall thickness(LVPWd),endsystolic ventricular septal thickness(IVSs),end-systolic left ventricle diameter(LVEDs),end-systolic left ventricle posterior wall thickness(LVPWs)were recorded.The left ventricle ejection fraction(LVEF)and fractional shortening(FS)were calculated according to the results of echocardiography.Heart weight(HW),body weight(BW),lung weight(LW)and tibial length(TL)were collected and the ratios of heart weight/body weight(HW/BW),lung weight/body weight(LW/BW)and heart weight/tibial length(HW/TL)were calculated.After adenovirus with overexpressing Fendrr or sh RNA of Fenddr were used to overexpress or knockdown Fendrr in neonatal rat cardiac myocytes(NRCMs)respectively,the NRCMs were challenged with(R)-(-)-phenylephrine(PE)at a final concentration of 50 m M for 24 h to explore the effect of Fendrr on myocardial hypertrophy in NRCMs.Real time quantitative PCR(q PCR)was performed to detect the levels of cardiac hypertrophy markers(Anp、Bnp、α-MHC、β-MHC、SERCA2α),fibrosis markers(Tgf-β、CollagenⅠ、CollagenⅢ)and the underlying targets of Fendrr(Gata4、Gata6、Foxf1、Nkx2-5、Foxo1、Foxo3a).Western Blot was carried out to determine the changes of Gata6 protein in heart tissues.Hematoxylin-eosin staining(HE)was used to evaluate the cross-sectional area(CSA)of cardiomyocytes.Picric sirius red staining(PSR)was performed to ascertain the degree of myocardial interstitial fibrosis.Immunofluorescence was used to determine the effect of Fendrr on cardiac hypertrophy in NRCMs.In NRCMs with Fendrr knockdown,the Gata6 was silenced by small interfering RNA for confirming that Fendrr regulated cardiac hypertrophy through Gata6.The chromatin immunoprecipitation-q PCR(CHIP-q PCR)was performed to investigate the effects of Fenddr on levels of histone H3 methylation in Gata6 promoter.Results In sham group,the knockout of lnc RNA Fendrr did not affect cardiac function,and there were no any significant differences in IVSd,LVEDd,LVPWd,IVSs,LVEDs,LVPWs,LVEF,and FS between Fendrr-KO mice and Fendrr-Flox mice(P>0.05).8 weeks after AB,knockout of lnc RNA Fendrr obviously alleviated the deterioration of cardiac function under pressure overload,characterized by the lower levels of LVEDd,LVPWd,IVSs,LVEDs and LVPWs in Fendrr-KO mice than that in Fendrr-Flox mice(P<0.05),but a higher levels of LVEF and FS in Fendrr-KO mice(P<0.05).8 weeks after AB,knockout of lnc RNA Fendrr apparently attenuated the AB induced cardiac remodeling.The HW,HW/BW and HW/TL in Fendrr-KO mice were significantly lower than that in Fendrr-flox mice(P<0.05).The results of HE staining confirmed that myocardial CSA of Fendrr-KO mice was significantly lower than that of Fendrr-Flox mice under pressure overload(P<0.05).The results of PSR staining indicated that the degree of collagen deposition in the myocardial interstitium of Fendrr-KO mice was lower than that of Fendrr-Flox mice under pressure overload challenge(P < 0.05).q PCR results suggested that the m RNA levels of Anp,Bnp,β-MHC,Tgf-β,Collagen Ⅰ,Collagen Ⅲ in Fendrr-KO mice were lower than that in Fendrr-Flox mice(P < 0.05),but the m RNA levels of α-MHC and SERCA2α in Fendrr-KO mice were significantly higher than that in Fendrr-Flox mice(P<0.05).In the sham groups,there were no any significantly differences between Fendrr-TG and NTG mice(P>0.05).8 weeks after AB,the specific overexpression of Fenddr in cardiomyocytes further aggravated cardiac dysfunction induced by AB.The IVSd,LVEDd,and LVEDs of Fendrr-TG mice were significantly higher than those of NTG mice(P<0.05),but LVEF and FS were significantly lower in NTG mice(P<0.05).The specific overexpression of Fendrr in cardiomyocytes exacerbated AB induced cardiac remodeling.8 weeks after AB,the levels of HW,LW,HW/BW,LW/BW and HW/TL in Fendrr-TG mice were significantly higher than those in NTG mice(P<0.05).In addition,the CSA of cardiomyocytes,content of myocardial interstitium collagen,cardiac hypertrophy markers and fibrosis markers in Fendrr-TG mice were significantly higher than those in NTG mice(P<0.05).The knockdown of Fendrr in NRCMs clearly mitigated the PE induced myocardial hypertrophy(P<0.05),also notably reduced the m RNA levels of Anp and Bnp(P<0.05).By contrast,the overexpression of Fendrr in NRCMs overtly aggravated the PE induced myocardial hypertrophy(P<0.05),and obviously increased the m RNA levels of Anp and Bnp(P<0.05).The potential downstream targets of Fendrr were quantified by q PCR and Western Blot,which showed that knockout of Fendrr rescued the AB induced downregulation of Gata6 m RNA and protein to the normal level.After Gata6 was silenced by small interfering RNA,the protective effect of myocardial hypertrophy caused by Fendrr knockdown disappeared.The result of CHIP-q PCR indicated that the knockout of Fendrr increased tri-methylation of histone H3K4 and inhibited the tri-methylation of H3K27 in Gata6 promoter,thereby promoting Gata6 gene transcription.Conclusions The specific knockout or knockdown of Fendrr in myocardial cells attenuated pressure overload induced cardiac remodeling,while the specific overexpression of Fendrr in myocardial cells exacerbated pressure overload induced cardiac remodeling.Fendrr regulated cardiac remodeling by regulating histone H3 methylation level in Gata6 promoter,thus affecting transcription of Gata6.Fendrr effectively restored the abnormal expression of Gata6 induced by pressure overload to the normal level,thus Fendrr is a potential target for the treatment of pressure overload induced cardiac remodeling.