The Mechanism Of Trans-cinnamaldehyde Regulating Microtubule Detyrosinization To Improve Cardiac Hypertrophy

Purpose:Epidemiological investigations show that cardiovascular disease has become the main cause of death in the world,so its prevention and treatment are imperative.Chinese herb Guizhi has excellent efficacy in treating cardiovascular diseases,but the research on its mechanism is rarely reported.Trans-cinnamaldehyde（TCA）is the main active ingredients of Guizhi,this study uses phenylephrine（PE）to induce cardiac hypertrophy model in vivo and in vitro,in order to identify therapeutic effects by which Trans-cinnamaldehyde inhibits phenylephrine-induced cardiac hypertrophy,microtubule detyrosination,STIM1/Orai1 and downstream Ca N/NFAT signaling pathway and elucidate the mechanisms.Method:In vivo,animal model of cardiac hypertrophy was established by which8-week-old C57BL/6J mice were continuously infused with PE（75 mg/kg per day）through a micro-osmotic pump for 2 weeks with or without TCA treatment（50 or 100mg/kg/d）.After 2 weeks of treatment in each group,cardiac mass index,echocardiography analysis and morphological indicators were used to assess myocardial hypertrophy.The expression of myocardial tissue microtubule detyrosination and STIM1/Orai1 was observed with Western blotting and immunofluorescence.In vitro,neonatal cardiac myocytes（NRCMs）and adult mice ventricular myocytes（AMVMs）were stimulated with PE 50μΜfor 48h with or without TCA treatment（5μΜ）and changes inα-tubulin detyrosination,microtubule densification,store-operated Ca²⁺entry（SOCE）,STIM1/Orai1 translocation,and calcineurin/NFAT pathway were also analyzed by Western blotting,immunofluorescence analysis,and transcriptome analysis.Ca²⁺handling,sarcomere shortening,junctophilin-2 and T-tubule distribution pattern in the AMVMs were observed by confocal microscope.After VASH1 overexpression adenovirus transfection,the protein changes of detyrosinated microtubules were detected by Western Blot.The endoplasmic reticulum staining was used to observe the distribution of endoplasmic reticulum in neonatal rat cardiomyocytes and immunofluorescence and RT-PCR methods were used to detect the surface area of cardiomyocytes and hypertrophy-related genes.Results:In vivo experiments show,trans-cinnamaldehyde inhibited PE-induced the increase in the thickness of the ventricular septum and posterior wall during diastole and systole,reduced PE-induced heart weight/body weight and heart weight/tibia length,significantly down-regulated the expression of Nppa,Nppb and Mhy7 m RNA,and inhibited the detyrosination of microtubules,VASHs and SVBP in mice.In vitro,trans-cinnamaldehyde could down-regulate Tuba4a,Tubb2a,Tubb4a and TTL genes caused by PE and inhibit the protein levels of de Tyr-tubulin,VASH1,SVBP and TTL.TCA had demonstrated activity against enhanced SOCE by reducing the massive translocation of STIM1 and Orai1.TCA could inhibit PE-induced Ca N activity and nuclear translocation of NFAT,as well as increased surface area of cardiomyocytes in neonatal cardiac myocytes.In adult mice ventricular myocytes,TCA had been revealed to restore the Ca²⁺handling and sarcomere shortening,and to rebuild the highly ordered junctophilin-2 and T-tubule distribution pattern.After VASH1 overexpression adenovirus transfection,trans-cinnamaldehyde could inhibit the increase of detyrosinated microtubules,reduce the accumulation of endoplasmic reticulum near the cell membrane,decrease the surface area of cardiomyocytes and hypertrophic genes.Conclusion:TCA can improve PE-induced cardiac hypertrophy.The effect may be involved in regulating of the STIM1/Orai1 pathway and the downstream Ca N/NFAT pathway by modulating the detyrosination of microtubules.