| BackgroundAtrial fibrillation(AF)is the most common type of rapid heart rhythm disorder in clinical practice,with an increasing incidence and significant clinical impact.It poses a major challenge in the field of cardiovascular prevention and treatment.It is worth noting that a growing body of clinical and experimental studies suggest that cardiac autonomic remodeling plays a critical role in the initiation and maintenance of AF.Excessive regeneration and heterogeneous distribution of the cardiac autonomic nervous system can affect or alter the electrophysiological properties of the atria,thereby mediating the occurrence and development of AF.Dyslipidemia is characterized by elevated blood lipids due to high-fat diet and metabolic abnormalities.Epidemiological and clinical evidence indicates that lipid abnormalities are risk factors for cardiovascular disease(CVD),and reducing blood lipid levels can help reduce the risk of AF.However,the specific mechanisms underlying the relationship between dyslipidemia and AF remain unclear.High-fat diets can induce oxidative stress and pro-inflammatory status in cardiac tissue by increasing serum free fatty acids(FFAs)and damaging mitochondria.CRIF1 is a mitochondrial protein that regulates reactive oxygen species(ROS)production.ROS can modulate NO production through the SIRTl/eNOS axis,thereby affecting cardiovascular homeostasis.P21,as a widely acting cyclin-dependent kinase inhibitor,can suppress cell proliferation.Oxidative stress has been shown to be closely associated with autonomic remodeling,and reduced NO levels can alleviate the inhibitory effect on cell proliferation,thus promoting cell growth.We hypothesize that high-fat diet may promote AF by inducing cardiac autonomic remodeling via reduced NO production.AimsBy establishing a high-fat diet-fed rat model and a co-culture model of cardiomyocytes and nerve cells under conditions of high fatty acids,we elucidated the mechanism by which high-fat stimulation damages mitochondria and causes myocardial NO imbalance.The CRIF1/eNOS/p21 axis leads to atrial neural remodelling and thus induces the development of atrial fibrillation.These findings provide a new predictive index and intervention target for the prevention and treatment of atrial fibrillation in a high-fat diet-fed rat model and a co-culture model under high-fat conditions.Methods1.Cellular experimentsRat cardiomyocytes(H9C2)and rat adrenal pheochromocytoma cells(PC 12)came to be cultured in the DMEM medium containing 10%fetal bovine serum.Palmitic acid(PA)was added to construct a high-fat cell model.Cell viability was measured using CCK-8,ROS by fluorimetry and flow cytometry using DCFH-DA probe,mitochondrial membrane potential by JC-1,supernatant NO content by Griessy method,ATP by fluorimetry,and mitochondrial morphology by MitoTracker.The protein expression of SIRT1 and eNOS was detected using Western blotting(WB).The relationship between SIRT1/eNOS axis and NO was verified using an overexpression plasmid or si-RN A of SIRT1.The relationship between CRIF1/SIRT1/eNOS axis and NO was verified using the siRNA of CRIF1.Immunoprecipitation to detect the acetylation level of eNOS.A co-culture model was established using 0.4 μm Transwell cell culture plates to verify the interaction between cardiomyocytes and neuronal cells.The upper layer was inoculated with H9C2 cardiomyocytes and the lower layer with PC 12 neural cells.The protein expression levels of PGP9.5,TH and P21 in the lower layer of neuronal cells were measured by WB and the apoptosis level was measured by EdU after the corresponding stimulation according to the experimental plan.SNAP was added to PC 12 neuronal cells to simulate a high NO content environment,and the level of nitrosylation was detected using the iodoTMT reagent labelling method,and the protein expression level was detected by immunofluorescence.The relationship between Nrf2 and P21 was verified using Nrf2 small interference.Trimetazidine(TMZ)was added to the cells to verify its effect.2.Animal experimentsThirty 8-week male Sprague-Dawley(SD)rats 200g±20g rats were randomly divided into the control group(n=10),HFD group(n=10),and HFD group+TMZ group(n=10).Body weight was measured weekly.After anesthesia,electrodes were inserted through the right jugular vein into the high right atrium to detect the atrial effective expiration period(AERP),atrial fibrillation induction rate,and rapid irregular atrial rate lasting 2s was considered as successful induction of atrial fibrillation.Blood was collected from the apical part of the heart and serum was taken to measure high and low-density lipoproteins,total cholesterol,triglycerides,MDA,immunohistochemistry,and WB to detect relevant protein expression.Results1.PA stimulation increased ROS production and impaired mitochondrial morphological function in cardiomyocytes:After PA stimulation,ROS production increased,mitochondrial breakage increased,and fragmentation became more obvious.Expression of Mfn1 and Mfn2 proteins decreased.ATP production decreased,and mitochondrial respiratory chain-related protein expression decreased.2.PA can regulate the expression of CRIF1/SIRT1/eNOS through ROS and affect the production of NO:Fatty acid stimulation decreased CRIF1,SIRT1,and eNOS protein expression.Silencing CRIF1 and overexpressing SIRT1 using siRNA and plasmid respectively,we found that silencing CRIF1 could decrease NO production by decreasing SIRT1 protein expression and increasing eNOS acetylation.3.PA induces PC12 cell proliferation in a coculture model of H9C2 cardiomyocytes and PC 12 neuronal cells:Cardiomyocytes were cultured in the upper layer and neuronal cells in the lower layer.The addition of fatty acids to the co-culture model resulted in increased TH and PGP9.5 protein expression and decreased P21 protein expression in the lower layer of neuronal cells,and increased cell proliferation ratio.4.Overexpression of eNOS in cardiomyocytes increased the NO content and inhibited the proliferation of lower neuronal cells:In a co-culture model,after overexpression of eNOS in upper layer cardiomyocytes,the protein expression of TH and PGP9.5 in lower layer neuronal cells decreased,P21 expression increased,and cell proliferation ratio decreased.After NO scavenging with C-PTIO,TH and PGP9.5 protein expression increased,P21 protein expression decreased,and cell proliferation ratio increased in lower layer neuronal cells.5.NO inhibits proliferation by nitrosylating Keap1 to promote Nrf2 entry into the nucleus to agonize P21 transcription.6.Trimetazidine inhibits neural remodelling by promoting the protein expression of eNOS:Trimetazidine(TMZ)increased the protein expression of eNOS in cardiomyocytes in a concentration-dependent manner.The addition of trimetazidine in a co-culture model resulted in decreased TH and PGP9.5 protein expression,increased P21 protein expression,and decreased cell proliferation ratio in lower PC 12 cells.7.HFD induced atrial neural remodelling and increased the induction rate of AF,and TMZ improved neural remodelling and decreased the induction rate of AF:The high-fat diet reduced the atrial effective expiration period(AERP)and increased the induction rate of atrial fibrillation,and increased the expression of TH,CHAT,and PGP9.5 proteins in rats.The administration of trimetazidine reduced the AERP,decreased the induction rate of atrial fibrillation,decreased the expression of TH,CHAT,and PGP9.5 proteins,and improved the neural remodelling of atrial muscle.Conclusions1.High levels of fatty acids can damage mitochondria,affecting their morphology and function through the CRIF1/SIRT1/eNOS axis,which reduces the production of NO.2.NO promotes the entry of Nrf2 into the nucleus by nitrosoylation of Keap1 to activate the expression of P21,thus inhibiting the proliferation of nerve cells in the myocardial microenvironment stimulated by high fatty acids。3.Trimetazidine inhibits neural remodelling caused by HFD by increasing the protein expression of eNOS and decreasing the induction rate of atrial fibrillation. |
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