Trimethylamine-N-Oxide(TMAO) Promotes Myocardial Hypertrophy Through Autophagy-Induced Cardiomyocyte Calcium Perturbation

Background:Myocardial hypertrophy is an adaptive compensatory disease in the condition of the pathological stimulation.Continuous progress can lead to heart failure and even death.However,its pathogenesis has not been fully elucidated,which restricts the effectiveness and innovation of clinical treatment.Trimethylamine N-oxide（TMAO）is an intestinal flora metabolite discovered in 2011.In recent years,many clinical studies have reported that TMAO could be a new specific biomarker due to its closely association with the occurrence and development of cardiovascular diseases,such as hypertension,atherosclerosis and heart failure.TMAO has been used in clinical diagnosis as a disease warning molecule in some countries.Although the relationship between TMAO and myocardial hypertrophy has been realized,its specific mechanism is still unclear.The imbalance of intracellular calcium homeostasis is known to be one of the important mechanisms leading to myocardial hypertrophy,and the calcium channels on the cell membrane and sarco/endoplasmic reticulum calcium ATPase 2a（SERCA2a）are important proteins on calcium homeostasis regulation.Therefore,this study intends to further explore the role of TMAO in myocardial hypertrophy and the specific mechanism of regulating calcium homeostasis,which will provide a new experimental basis for revealing the relationship between intestinal flora and cardiovascular diseases,and benefit the future prevention and control.Objective:This study intends to confirm the effect of TMAO on calcium homeostasis in cardiomyocytes and its role in myocardial hypertrophy,and further explores the specific mechanism of TMAO promoting the autophagy degradation of SERCA2a,leading to calcium homeostasis imbalance and thus inducing myocardial hypertrophy.Methods:1.Animal experiments:6-week-old C57BL/6J mice were divided into eight different treatments:Normal control group;TMAO treatment group（0.12%TMAO drinking water for 8 weeks）;Isoproterenol（ISO）group(（ISO（7.5 mg/kg/day,s.c.）was administered two weeks before the mice were killed）;TMAO+ISO group（0.12%TMAO drinking water for 8weeks,combined with ISO（7.5 mg/kg/day,s.c.）during weeks 7-8）;TMAO+3-Methyladenine（3-MA）group（0.12%TMAO drinking water for 8 weeks,combined with 3-MA（10 mg/kg/day,i.p.）during weeks 7-8）;choline treatment group（1%choline diet for 8 weeks）;Choline+DMB group（choline diet based mice were given 1%DMB water for 8 weeks）.UPLC-MS/MS was used to detect the plasma TMAO level and ultrasound instrument was used to detect the changes in cardiac function;The heart tissue was isolated,and the general changes of the heart were detected,heart weight/body weight（HW/BW）and heart weight/tibia length（HW/TL）were measured;HE,WGA and Masson staining were used to observe the changes of myocardial histopathology;The expressions of hypertrophy marker protein,SERCA2a protein and autophagy related protein in myocardial tissue were detected by Western Blot.The m RNA level of SERCA2a was detected by RT-PCR;Autophagosomes of myocardial tissue were detected by transmission electron microscopy.2.Cell experiment:Using calcium chelators,lentivirus-mediated overexpression of SERCA2a and other intervention methods,the intracellular Ca²⁺levels in H9c2 cells,SERCA2a protein levels,cell surface area and expression of hypertrophy markers were measured through techniques such as calcium ion fluorescence probe Fura-4/AM,Western Blot,and rhodamine-phalloidin staining.These methods confirmed the regulatory role of SERCA2a in TMAO-induced myocardial hypertrophy.In a TMAO-stimulated cardiac hypertrophy cell model,interventions such as silencing,overexpression,and inhibitors were used to analyze the expression of SERCA2a,autophagy-related markers,hypertrophy marker proteins,cell surface area,and using techniques like Western Blot,q PCR,rhodamine-phalloidin staining,immunofluorescence,and Co-IP experiments.This study aimed to investigate the mechanism by which TMAO induces SERCA2a autophagic degradation to promote myocardial hypertrophy.Results:The results in animal level were as follows:TMAO drinking water group showed elevated levels of TMAO in mouse plasma,decreased heart function,significant pathological changes such as myocardial hypertrophy,increased cardiomyocyte volume,increased expression of hypertrophy markers and decreased protein expression of SERCA2a.The autophagy inhibitor 3-MA alleviated the above changes induced by TMAO drinking water.Choline diet group showed elevated levels of TMAO in mouse plasma,decreased heart function,significant pathological changes such as myocardial hypertrophy,increased cardiomyocyte volume and increased expression of hypertrophy markers.DMB alleviated the above changes induced by the choline diet.ISO group showed elevated levels of TMAO in mouse plasma,decreased heart function,significant pathological changes such as myocardial hypertrophy,increased cardiomyocyte volume,increased expression of hypertrophy markers,and TMAO drinking water significantly exacerbated the above changes induced by ISO.The results in cell level were as follows:TMAO incubation promoted hypertrophy of H9c2 cells,increased cytoplasmic Ca²⁺levels,induced autophagy,and reduced protein expression of SERCA2a.Chelation of calcium ions alleviated TMAO-induced hypertrophy of H9c2 cells.Overexpression of SERCA2a via lentivirus alleviated the elevated cytoplasmic Ca²⁺levels and cell hypertrophy induced by TMAO.Inhibition of autophagosome-lysosome fusion alleviated the decrease in SERCA2a protein levels caused by TMAO,inhibited colocalization of SERCA2a with LAMP1,and promotes colocalization of SERCA2a with LC3.Co-immunoprecipitation experiments showed that TMAO-incubated H9c2 cells and SD rat cardiomyocytes exhibited significantly increased interaction between SERCA2a and ATG5.Immunofluorescence experiments demonstrated a significant increase in fluorescence colocalization between SERCA2a and ATG5.Silencing of ATG5 alleviated TMAO-induced decrease in SERCA2a protein levels,increase in cell surface area,and elevation of hypertrophy marker protein levelsConclusion:1.TMAO,a metabolite of intestinal flora,can significantly induce and aggravate myocardial hypertrophy,and its effect is related to inducing calcium homeostasis imbalance.2.TMAO can induce calcium homeostasis imbalance through autophagy degradation of SERCA2a,thus promoting myocardial hypertrophy.61 figures,15 tables,134 references...