The Role And Mechanism Of SIRT1/GCH1/Nrf2 In High Fatty Acid-induced Atrial Fibrillation And Fibrosis

Background As the most common clinical tachyarrhythmia,atrial fibrillation(AF)is a major study in the cardiovascular field because of its high disability rate and fatality rate.There are many factors related to the occurrence and development of atrial fibrillation,among which age and gender are unavoidable risky factors while heart failure,obesity,smoking,diabetes and metabolic syndrome are avoidable.According to some existing data,high-fat diet can cause left atrial enlargement through various mechanisms,lead to inflammatory infiltration,structural abnormalities,and conduction abnormalities,and increase the susceptibility to atrial fibrillation.Furthermore,experimental studies have also shown that triglyceride levels are positively correlated with the occurrence of atrial fibrillation,while High-density lipoprotein cholesterol was inversely associated with atrial fibrillation.At the same time,hyperlipidemia is one of the characteristic manifestations of obesity and metabolic syndrome,and it is also closely related to the occurrence of atrial fibrillation,but the exact triggering mechanism of hyperlipidemia is still unclear.Therefore,exploring the intrinsic link between high fat and atrial fibrillation is of great significance to clarify the pathogenesis of atrial fibrillation,and improve the prognosis of atrial fibrillation.Aims This study aims to explore how high fat affects myocardial fibrosis in atrial fibril lation.By constructing a high-fat rat model,a cardiomyocyte model under high-fat sti mulation,and a co-culture model of primary cardiomyocytes and primary fibroblasts,h e effects of high fat on cardiomyocyte apoptosis and atrial fibrosis will be clarified.F rom the perspective of abnormal lipid metabolism,the study will also clarify the mech anism by which SIRT1/GCH1/ROS/NRF2 pathway induces cardiomyocyte apoptosis lea ding to atrial fibrosis and facilitates the occurrence of atrial fibrillation under high-fat environment,in order to find out the new intervention strategies for the prevention an d treatment of high-fat mediated atrial fibrillations.Method 1.Construction of a high-fat rat modelTwenty healthy SD rats,male or female,were randomly selected and divided into control group(CON,n=10)and high-fat group(HFD,n=10).Electrophysiological me asurements were carried out before feeding,and then the control group was fed with normal feed,and the high-fat group with high-fatty acid feed for 2 months.Then,the corresponding biochemical indicators will be measured by electrophysiological measur ements,cardiac color doppler ultrasound measurements,and blood sampling from the a pex of the heart.2.Observation of myocardial tissue structure and detection of molecular biological indicatorsMitochondria were observed under transmission electron microscope,and the degree of fibrosis was observed by Masson staining.The protein expression levels of mitochondrial molecular indicators Mfn1,Mfn2,OPA1 and the protein expression level of antioxidant transcription factor Nrf2 were detected by Western Blot.3.Construction of a high-fat cultured cell model in vitro to verify the effect of SIRT1/GCH1/ROS/Nrf2 pathway on cardiomyocytesThe high-fat-stimulated cardiomyocyte model was constructed in vitro,the protein expression levels of Mfnl/Mfn2 and OPA1 were detected by Western Blot,the positiv e ratio of ROS and the ratio of apoptosis were detected by flow cytometry,and the p rotein expression of Nrf2 was determined by Western Blot.The protein expression lev el of Nrf2 after scavenging reactive oxygen species was measured after the scavenger NAC to clarify the effect of ROS on Nrf2.According to literature reports,the upstrea m protein GCH1 of Nrf2 was found,and the relationship between GCH1 and ROS w as determined by flow cytometry,and then the protein expression level of downstream Nrf2 was verified by interfering with the expression of GCH1,and the relationship b etween GCH1 and Nrf2 was clarified;Afterwards,the deacetylation level of GCH1 wa s verified by immunoprecipitation technology,the expression level of SIRT1 protein w as determined by Western Blot method,and the relationship between SIRT1 and ROS was verified by flow cytometry,SIRT1 was intervened,and the downstream GCH1 a nd Nrf2 proteins were detected by Western Blot method Change the level to clarify t he relationship of SIRT1-GCH1-ROS-Nrf2.Whether there is a correlation between SIR T1 and GCH under high fatty acid stimulation was verified by co-immunopritatio.4.Construction of the co-culture model in vitroPrimary cardiomyocytes and primary cardiac fibroblasts were extracted by enzymatic digestion and differential adhesion method,and primary cardiomyocytes were cultured in the upper layer and primary fibroblasts were cultured in the lower layer to establish a high-fatty acid-stimulated co-culture system.In the stimulated fibroblast group and the co-culture group of high-fat stimulated cardiomyocytes and fibroblasts,the proliferation of primary fibroblasts in the lower layer was verified by Edu staining,and the expressions of CollagenⅠ,CollagenⅢand Fibronectin in the lower layer of fibroblasts were detected by Western Blot method.,ROS scavenging and tBHQ activation of Nrf2 were performed on the primary cardiomyocytes in the upper chamber,the positive ratio of ROS in the lower primary fibroblasts was determined by flow cytometry,and the Collagen I and Collagen III in the lower primary fibroblasts were determined by Western Blot.,Fibronectin expression to clarify the effect of cardiomyocyte oxidative stress on fibroblasts in high-fat environment.5.Construction of an animal rescue modelThirty SD rats were randomly divided into CON(n=10),HFD(n=10),HFD+tBHQ(n=10).Electrophysiological indicators were measured before the experiment,and then the CON group was fed with common chow and HFD.The HFD+tBHQ group with high-fat diet,and then the HFD+tBHQ group with high-fat diet for 1 month,and then intraperitoneally injected with tBHQ every other day to activate Nrf2.After 2 months,electrophysiological measurement and blood sampling from the apex of the three groups were performed respectively.,Masson staining and Western Blot to determine the protein expression levels of CollagenⅠ,CollagenⅢ and Nrf2.Results Ⅰ.High-fat diet increases the incidence of atrial arrhythmias in ratsBy constructing a high-fat rat model in vitro,the CON group(n=10)and the HFD group(n=10)found that the body weight of the high-fat-fed rats increased significantly;the serum triglyceride,total cholesterol and low-density lipoprotein contents increased in rats;the atrial effective refractory period(AERP)shortened,the induction rate of atrial arrhythmia increased,the left atrial diameter(LAD)enlarged,and the content of triglycerides,total cholesterol and low-density lipoprotein increased.2.High-fat diet induced left atrial mitochondrial damage and increased atrial fibrosis in ratsHigh fat can induce left atrial mitochondrial damage and atrial fibrosis in rats,and the expression level of the antioxidant transcription factor Nrf2 in atrial tissue is up-regulated.Transmission electron microscopy of the tissue showed that the left atrium mitochondria of the HFD group were significantly damaged,vacuolized,and the mitochondrial cristae were broken.,and the results of electron microscopy were basically consistent;Masson results showed that there was obvious collagen deposition in the interstitial space of HFD group,and the expressions of collagen CollagenⅠ and CollagenⅢ increased.3.Cardiomyocyte mitochondrial damaged and increased apoptosis under high-fat stimulationHigh-fat stimulation can cause mitochondrial damage and increase apoptosis in H9C2 cells.In the in vitro cell model,H9C2 cardiomyocytes were stimulated by high fat,and the results of Western Blot showed that the ratio of mitochondrial protein S-OPA1/L-OPA1 increased,and the protein expressions of Mfnl and Mfn2 decreased.The results of flow cytometry showed that with the increase of fatty acid stimulation concentration,the positive ratio of reactive oxygen species(ROS)gradually increased,and at the same time,the ratio of apoptosis gradually increased,and the ratio of apoptosis-related protein Bax/Bcl2 increased.4.Downregulation of GCH1 leads to increasing ROS and activation of Nrf2 under high-fat stimulationHigh-fat stimulation caused a decrease in the expression of GCH1,an increase in the positive ratio of ROS,and an increase in the expression of Nrf2 in cardiomyocytes.After high-fat stimulation of H9C2 cardiomyocytes,the protein expression level of Nrf2 increased and the protein expression level of GCH1 decreased.After using the reactive oxygen species scavenger NAC,the positive ratio of ROS decreased and the expression level of Nrf2 decreased.After overexpression of GCH1,flow cytometry showed that the positive ratio of ROS decreased and the protein expression of Nrf2 decreased,while after GCH1 was silenced,the positive ratio of ROS and the protein expression of Nrf2 increased.5.Downregulation of SIRT1 affects the GCH1-ROS-Nrf2 axis under high-fat stimulationHigh fat caused the decrease of SIRT1 protein expression in cardiomyocytes,and it was found that SIRT1 could induce the changes of GCH1 and Nrf2.After H9C2 cells were stimulated with different concentrations of fatty acid,the protein expression level of SIRT1 gradually decreased,which was consistent with the protein expression level of GCH1.Intervention of SIRT1,after silencing SIRT1,the expression of downstream ROS increased,the protein expression level of GCH1 decreased,and the protein expression of Nrf2 increased;after overexpression of SIRT1,the expression of downstream ROS decreased,the protein expression level of GCH1 increased,and the protein expression of Nrf2 at the same time increased,indicating a correlation between SIRT1-GCH1-Nrf2 via ROS.6.GCH1 is deacetylated under high-fat stimulation,and there is a correlation between SIRT1 and GCH1It was found by immunoprecipitation that the expression of GCH1 pan-acetylated decreased under high-fat stimulation;the interaction between SIRT1 and GCH1 was verified by co-immunoprecipitation under high-fat stimulation,and the effect enhanced.7.Oxidative stress in cardiomyocytes promotes fibroblast proliferation and collagen formation under high-fat stimulationCompared with the high-fat-stimulated fibroblasts group,the lower primary fibrobl asts in the high-fat-stimulated cardiomyocytes and fibroblasts co-culture group proliferat ed significantly,and the expressions of fibrosis-related proteins Collagen Ⅰ,Collagen ⅡⅠ and Fibronectin increased.After NAC scavenged ROS from upper cardiomyocytes,t he proliferation of lower fibroblasts improved and the expression of fibrosis-related pro teins decreased.After activation of Nrf2 expression in upper cardiomyocytes with tBH Q,the proliferation rate of lower fibroblasts decreased and the expression of fibrosis-r elated proteins decreased.The above co-culture demonstrated that high-fat stimulation c an induce fibrosis and improve fibrosis by stimulating the expression of Nrf2.8.Activation of Nrf2 in vivo improves high-fat-induced atrial fibrosis and reduces the incidence of atrial fibrillationIntraperitoneal injection of tBHQ to stimulate Nrf2 in high-fat-fed rats ameliorated high-fat-induced atrial fibrosis and reduced the incidence of atrial fibrillation.Compared with the HFD group,the AERP of the HFD+tBHQ group significantly prolonged.The cardiac electrophysiological results showed that the LAD of the HFD+tBHQ group was shorter than that of the HFD group,and the induction rate of atrial fibrillation was lower;Compared with HFD group,lipoprotein decreased,the expression of antioxidant factor Nrf2 in HFD+tBHQ group increased compared with HFD group,and the expression of fibrosis-related protein decreased compared with HFD group.Conclusion 1.The high-fat-induced rat model showed left atrial myocardial fibrosis,and mitochondrial damage occurred in cardiomyocytes,which acted by activating the antioxidant transcription factor Nrf2.2.High-fat-stimulated SIRT1-GCH1-ROS-Nrf2 axis affects cardiomyocyte apoptosis and myocardial fibrosis,and promotes the aggravation of high-fat-stimulated fibrosis t hrough oxidative stress,thereby inducing atrial fibrillation.3.Activation of Nrf2 can improve high-fat-induced atrial fibrosis and reduce the i ncidence of atrial fibrillation.