P300/P53/Smad3 Pathway Mediates Atrial Fibroblasts Senescence And Aging-related Atrial Fibrosis

Objective: Atrial fibrillation(AF)is considered as a kind of "senile disease".Its prevalence increases with age.Fibrosis is a common pathological process of various cardiovascular diseases,including AF,and aging can participate in the occurrence of fibrosis.Previous studies have shown that the co-transcriptional activator and acetyltransferase p300 plays an important role in the pathological process of senescence and fibrosis.Therefore,this study intends to verify whether p300/p53/Smad3 pathway is involved in atrial fibroblasts senescence,and then causes aging-related atrial fibrosis,finally plays an important role in the occurrence and development of AF.To explore the potential mechanism of aging in AF pathological process and to provide a new target and experimental basis for AF prevention and treatment.Methods:(1)Atrial tissues of young and old patients with AF and SR were collected.Masson staining and Western Blot assay were used to detect the atrial fibrosis degree and protein expression of p300,tumor suppressor protein p53(p53),cyclin-dependent kinase inhibitor p21(p21),collagen type 3 alpha 1 chain(COL3A1),collagen type 1 alpha 1 chain(COL1A1),matrix metalloproteinase 2/9(MMP-2/9),transforming growth factor beta 1(TGF-β1),SMAD family member 3(Smad3)and phospho-Smad3(p-Smad3).(2)The animal model of natural aging mice was established.Then Masson staining and Western Blot was used to detect the atrial fibrosis degree and the expression of p300,p53/p21,COL3A1/COL1A1,MMP-2/9,TGF-β 1,Smad3 and p-Smad3 protein in each groups to verify the correlation between p300/p53/Smad3 and aging-related fibrosis.In order to evaluate the AF susceptibility,transvenous atrial stimulation in vivo was used to acquire the electrophysiological parameters and AF inducibility of C57BL/6 mice with different ages.(3)Mouse atrial fibroblasts were isolated and identified,and the replicative senescence model was established by passage.The model was identified by senescence associated β-galactosidase(SA-β-gal)staining and senescence associated protein p53/p21 expression level,and the protein expression of fibrosisrelated proteins COL3A1/COL1A1,MMP-2/9,TGF-β 1and p300/p53/Smad3 were detected in atrial fibroblasts with different passages.(4)p300 si RNA transfection was used to downregulate the expression of p300 protein in passage 11 atrial fibroblasts and Synergistic activation mediator(SAM)technique was applied to over-express p300 protein in passage 2or 3 atrial fibroblasts to verify the role of p300 in atrial fibroblasts senescence and fibrosis-related protein expression.(5)p53 si RNA transfection in passage 11 atrial fibroblasts,double fluorescence co-localization and Co-immunoprecipitation were performed to assessed p53/Smad3 co-localization and p53/Smad3 protein complex,further to investigate the regulatory mechanism of p53 on Smad3.Results:(1)Compared with SR patients,the older patients with AF had higher fibrosis degree and protein expression levels of p300,p53/p21 and COL3A1/COL1A1,MMP-2/9,TGF-β 1 and Smad3/p-Smad3,suggesting that p300/p53/Smad3 pathway is related to senescence and fibrosis.(2)In the natural aging mouse model,higher SA-β-gal activity,fibrosis degree and elevated levels of p53,p21 and fibrosis-related proteins in 13-month or18-month mice,accompanied by the up-regulation of p300 protein expression.In addition,the P-wave duration(PDW),PR interval,sinus node recovery time(SNRT)and corrected SNRT(CSNRT)were prolonged,the atrial effective refractory period(AERP)60ms was shortened,and the inducibility of AF was increased in the aged group.(3)The protein expression levels of p300,COL1A1,MMP2,TGF-β1,p53 and p21 was significantly increased in passage 11 than in passage 3 atrial fibroblasts.Passage 11 atrial fibroblasts were transfected with p300 si RNA successfully,then p53/p21,Smad3/p Smad3 protein and fibrosisrelated proteins were down-regulated,while overexpression of p300 in passage 3 atrial fibroblasts could up-regulate the expression of p53,Smad3/p-Smad3 and fibrosis-related proteins.(4)After transfected p53 si RNA,the expression of p21 in passage 11 atrial fibroblasts was down-regulated,with decreased fibrosis-related proteins,but the decrease of Smad3/p Smad3 was not statistic significantly.Moreover the co-localization of p53 and Smad3 was found by cell double fluorescence co-localization,and the p53/Smad3 protein complex was confirmed in atrial tissues by co-immunoprecipitation.Conclusion: With the increase of age,the expression of p300 protein in atrial fibroblasts increases,which can up-regulate p53,activate p21 and Smad3,and form p53/Smad3 protein complexes,then up-regulate the expression of senescence and fibrosis associated proteins,to participate in atrial senescence and aging-related fibrosis,and finally lead to the occurrence of AF.