Research On The Mechanism Of Piezo1 Regulating TET1-mediated DNA Demethylation In Myocardial Fibrosis Induced By High Hydrostatic Pressure

Objective:Increased ventricular pressure is an important pathogenic factor of myocardial fibrosis.The baroreceptor Piezo1 has been found to be able to convert mechanical stress into intracellular Ca²⁺signals,and Ca²⁺regulation abnormalities are closely related to myocardial fibrosis.However,whether Piezo1 mediates high hydrostatic pressure to induce myocardial fibrosis remains to be further explored.In addition,DNA demethylation also plays an important role in the pathogenesis of myocardial fibrosis,in which DNA demethylase（TET）is an important factor regulating DNA demethylation,but the relationship between TET and myocardial fibrosis remains to be further clarified.Therefore,this study aims to investigate whether Piezo1 can regulate TET mediated DNA demmethylation to participate in myocardial fibrosis induced by high hydrostatic pressure.Methods:（1）Selecting spontaneously hypertensive rat（SHR）model,Wistar rats were as control,and detecting the blood pressure and cardiac function of SHR and Wistar rats.（2）The neonatal rat cardiac fibroblasts（NRCFs）were treated with 0,120 and 180mm Hg hydrostatic pressure for 24 hours to simulate normal human left ventricular pressure and increased left ventricular pressure,respectively.The cell fibrosis model induced by high hydrostatic pressure（HHP）was established.（3）The expression levels of collagen 1（COL1-A1）,collagen3（COL3-A1）,transforming growth factorβ1（TGF-β1）,β-catenin,DNA demmethylation enzyme TET and baroreceptor Piezo1 in the ventricular tissue of SHR and Wistar rats and in NRCFs were detected by Western blot.（4）The pathological changes of myocardium in SHR and Wistar rats were detected by HE and Masson staining.The levels of 5-hm C and the intracellular localization ofβ-catenin in NRCFs were determined by cellular immunofluorescence.NRCFs loaded by Ca²⁺indicator Fluo4/AM was used to measure intracellular Ca²⁺concentration.（5）CCK8 was used to detect the cell proliferation rate of NRCFs.（6）The levels of 5-hm C and 5-m C in the promoter region of TGF-β1 in NRCFs were detected by q RT-PCR.Results:（1）In the myocardium tissue of SHR,the myocardial fibrous collagen increased,the myocardial interstitial arrangement was disordered,and the myocardial hypertrophy was accompanied by the increased expression of TGF-β1,collagens COL1-A1 and COL3-A1.Besides,the demetylation related proteins TET1 and TET2,baroreceptor Piezo1 andβ-catenin were increased.（2）TGF-β1,COL1-A1 and COL3-A1 expressions were increased in NRCFS model induced by HHP,and the proliferation rate of NRCFS was increased.This was accompanied by increased expression of Piezo1,β-catenin and TET1,increased levels of5-hm C,and increased Ca²⁺influx.（3）Based on the success of NRCFs myocardial fibrosis induced by HHP,Piezo1 inhibitor（Gs MTx4）,si-Piezo1,β-catenin inhibitor（XAV939）and sh-TET1 could reverse the expression of TGF-β1,COL1-A1 and COL3-A1.Inhibition or knockdown of Piezo1 could reverse HHP-induced Ca²⁺influx increase,elevated TET1expression andβ-catenin nuclear translocation increase.Inhibition ofβ-catenin reversed the HHP-induced increase in TET1 protein and 5-hm C levels,but did not affect Piezo1expression.TET1 knockdown could reverse the HHP-induced increase in 5-hm C level,increase the level of 5-hm C in the TGF-β1 promoter region,and decrease the total levels of5-m C and 5-hm C.Conclusion:This study reveals a new mechanism of myocardial fibrosis induced by HHP.HHP can induce the upregulation of Piezo1 and mediate the increase of Ca²⁺influx,thereby increasing the protein expression of TET1 by promoting the nuclear translocation ofβ-catenin,and further promoting the demmethylation of the DNA promoter region of TGF-β1to activate the expression of TGF-β1,ultimately leading to myocardial fibrosis.