Role And Mechanism Of Fragile X-Related Protein 1 In Right Ventricular Remodeling In Pulmonary Arterial Hypertension

Background: Pulmonary arterial hypertension(PAH)is a progressive pulmonary vascular disease with high morbidity and mortality.The sustained increase in pulmonary arterial pressure drives the remodeling of the right ventricle and ultimately leads to RV failure and death.Previous studies have shown that RV function is the major prognostic factor of patients with PAH.However,as the underlying mechanisms of RV remodeling remain poorly understood,no RV-specific therapies exist,which makes the prognosis of patients with PAH very poor.FXR1,an RNA binding protein,is the sole fragile X family member that is highly enriched in cardiac muscle and can modulate RNA stability and translation via binding with them.It has been confirmed that FXR1 played an important role in regulating myocardial remodeling and heart failure.However,the role of FXR1 in PAH right ventricular remodeling is uncertain.Objective: To explore whether FXR1 mediates the development of right ventricular remodeling in PAH,and analyze the specific regulatory mechanism,providing new ideas for prevention and treatment of right ventricular remodeling in pulmonary arterial hypertension.Methods:Part one: The PAH rats model was established by single intraperitoneal injection of monocrotaline at 60mg/kg.The rats were divided into dilated RV group,hypertrophic RV group and control group based on echocardiography.The RV remodeling of each group was evaluated via hemodynamic,RV structure and morphology using right cardiac catheterization,echocardiography and tissue staining.Expression of FXR1 in RV of each group was assessed by western blot and immunofluorescence staining.The neonatal rat cardiomyocytes(NRCM)were intervened with angiotensin Ⅱ for 48 hours to induce NRCM hypertrophy,and the cell size was measured by immunofluorescence staining and expression level of hypertrophy related genes was detected by RT-q PCR.Expression of FXR1 in hypertrophic NRCM was detected by western blot.NRCMs were infected with FXR1 adenovirus to overexpress FXR1 and transfected with small interfering RNA to knockdown FXR1.Observe the regulatory effect of overexpression or silence of FXR1 on cardiomyocyte hypertrophy based on the expression of hypertrophy related genes.Part two: RNA sequencing and bioinformatics analysis were used to screen the common differentially expressed genes of remodeling RV myocardium of PAH rats and FXR1 knockdown NRCM.GO analysis and KEGG pathway analysis were used to annotate the biological functions and pathways of these genes.The co-localization of FXR1 and mitochondria in remodeling RV of PAH rats was detected by confocal.Results:Part one: Right ventricular hypertrophy and dilation occurred in 21 th and 28 th after MCT injection.In both hypertrophic and dilated RV group,significant higher RV systolic pressure,RV/(LV+S)and RV/BW,thicker RV free wall and larger size of cardiomyocytes were detected compared with control group.The RV end diastolic dimension and myocardial fibrosis in dilated RV group was larger than hypertrophic group and control,while there was no difference between two groups.Compared with control group,FXR1 protein levels increased in dilated and hypertrophic RV group,and there was no difference during the remodeling process.In AngⅡinduced hypertrophic cardiomyocyte,the expression of FXR1 was also increased compared with control.Overexpression of FXR1 in NRCM can promote cardiomyocyte hypertrophy based on increased expression of Anp、Bnp、Myh7.Silence of FXR1 can inhibit the expression of Anp induced by 1u M AngⅡ.Part two: A total of 6673 differentially expressed genes in remodeling RV myocardium of PAH rats and 2199 in FXR1 knockdown NRCM were screened by RNA sequencing,and there were 1103 common differential genes in two groups.The top enriched KEGG pathway of these genes was metabolism.GO analysis showed that the top enriched biological process was “positive regulation of gene expression”,cellular component was“cytoplasm”,and molecular function was “protein binding”.Compared with control,the co-localization of FXR1 and mitochondria was stronger in remodeling RV of PAH rats detected by confocal.The genes related to mitochondria function,including Ndufv2、Ndufs2、Ndufa13、Hsp60,were identified in two groups which were involved in ATP synthesis and mitochondrial membrane integrity.Conclusion:1.The expression of FXR1 was increased in right ventricular myocardium in PAH and hypertrophic NRCM induced by AngⅡ;2.Increased expression of FXR1 promoted NRCM hypertrophy and silence of FXR1 inhibited AngⅡ induced NRCM hypertrophy;3.FXR1 appears to participant in the development of RV remodeling in PAH via regulating the function of mitochondrion.