The Mechanism Of CARM1/CLOCK/NAMPT On Myocardial Energy Metabolism Remodeling Via Regulating NAD~+ In Aging Related Atrial Fibrillation

BackgroundAtrial fibrillation is the most common persistent cardiac arrhythmias and is closely related to aging.Energy metabolism remodeling is an important mechanism for the occurrence and development of atrial fibrillation,however,the mechanism of age-related atrial fibrillation is still unclear.During the aging process,the levels of nicotinamide adenine dinucleotide(NAD_total,NAD⁺as oxidized form,NADH as reduced form)and its limiting enzyme nicotinamide phosphoribosyltransferase（NAMPT）are down-regulated,leading to impaired energy metabolism.The mechanism by which histone arginine methyltransferase 1（CARM1）interacts with the circadian rhythm output cycle protein kaput（CLOCK）to regulate the expression of NAMPT,thereby modulating the levels of silent information regulator proteins（Sirtuins,SIRTs）and protein acetylation,leading to age-related atrial fibrillation myocardial energy metabolism remodeling,is still unclear.Objective1.Observing the changes of NAD⁺and energy metabolism in the samples of patients with clinical atrial fibrillation,the atrial tissue of dogs with age-related atrial fibrillation,the atrial tissue of mice with age-related atrial fibrillation and the atrial fibrillation model of aging cardiomyocytes.2.Clarify the effect of NAMPT on NAD⁺metabolism in aging atrial fibrillation.3.Investigate the specific mechanism by which CARM1/CLOCK/NAMPT regulates NAD⁺and leads to energy metabolism remodeling,in order to find new intervention targets for preventing and treating aging atrial fibrillation.Methods1.In order to detect the changes of NAD⁺and energy metabolism during the occurrence and development of atrial fibrillation,the left atrial appendage and whole blood samples of patients with atrial fibrillation were collected.NAD_totaland NADH/NAD⁺were detected by WST-8method,ATP content was detected by ATP detection kit,SIRT1 and protein acetylation levels were detected by western blot,and NAMPT in peripheral blood was detected by enzyme-linked immunosorbent assay.2.Exploration of clinical issues in animal and cellular atrial fibrillation models:An aged canine atrial fibrillation model was constructed through rapid atrial pacing for six weeks,while an aged mouse atrial fibrillation model was established by intravenous injection of a mixture of calcium chloride and acetylcholine for six weeks.The successful modeling of animal atrial fibrillation models was validated by echocardiography,electrophysiological,and pathological examination.The senescence of HL-1 atrial myocytes was induced by hydrogen peroxide,and the aging HL-1 atrial myocytes were stimulated by a cell culture electrical stimulation system to construct an atrial fibrillation model of aged atrial myocytes.The detection of ion channel proteins verified the success of cell modeling.The atrial tissue of dogs and mice with aging atrial fibrillation,and the aging cell model of atrial fibrillation were collected and detected NAD_total,NADH/NAD⁺,ATP content,SIRT1level and protein acetylation level.Comprehensive observation of NAD⁺levels and energy metabolism changes in the occurrence and development of age-related atrial fibrillation from mammalian,rodent models and cell model levels.3.Non-targeted metabolomic profiling was performed on the cardiac tissue of aged mice with atrial fibrillation,and changes in metabolites during atrial fibrillation were identified by chromatography-mass spectrometry analysis.4.Changes in NAMPT were detected in the animal and cellular atrial fibrillation models using western blot,and the positive and negative effects of NAMPT on NAD⁺metabolism were verified at the cellular level by using NAMPT activators and inhibitors.5.Changes in CARM1 and CLOCK were detected in the animal and cellular atrial fibrillation models using western blot,and the co-localization of CARM1,CLOCK,and NAMPT in cells was detected using immunofluorescence.6.The mechanisms by which CARM1,CLOCK and NAMPT cause NAD⁺metabolism changes leading to energy metabolism remodeling in atrial fibrillation were investigated at the cellular level.First,the protein-protein interaction between CARM1 and CLOCK was verified using co-immunoprecipitation in 293T cells.Second,the mechanisms were explored using rescue experiments with the simultaneous use of a CARM1 inhibitor and NAMPT inhibitor in the atrial fibrillation cell model.Results1.Clinical samples showed a decrease in atrial NAD⁺levels,accompanied by a decrease in SIRT1 levels and ATP content,and an increase in protein acetylation levels.2.The changes of the above indicators were validated in canine,murine,and aging cell models of atrial fibrillation.A non-targeted metabolomics approach was used to detect myocardial metabolites in an aging murine model of atrial fibrillation,identifying a total of 420metabolites.KEGG metabolic pathway analysis revealed significant metabolic differences between atrial fibrillation and control mice in the tricarboxylic acid cycle.3.NAD⁺,SIRT1 and ATP levels in atrial tissue were up-regulated while protein acetylation levels were decreased after the intervention of nicotinamide mononucleotide（NMN）in the mouse model of age-realetd atrial fibrillation,indicating that NMN can partially reverse the energy metabolism disorder in atrial fibrillation.4.Down-regulation of NAMPT was detected in clinical specimens,different aging animal models and cellular atrial fibrillation models.Cell-level verification revealed that the NAMPT activator up-regulated the NAD⁺level in the atrial fibrillation group,whereas the NAMPT inhibitor down-regulated the NAD⁺level.5.CARM1 and CLOCK were found to be co-localized in the nuclei of atrial myocytes in mice with atrial fibrillation,and cell Co-IP experiments suggested that there was an interaction between them.CARM1 expression was up-regulated in atrial fibrillation,and rescue experiments showed that NAD⁺levels decreased when CARM1 inhibitor and NAMPT inhibitor were used simultaneously.Taken together,these results suggest that CARM1 regulates the CLOCK/NAMPT pathway to affect NAD⁺metabolism.Conclusion1.Age-related atrial fibrillation is associated with changes in NAD⁺metabolism and impaired energy production.2.Down-regulation of NAMPT expression in age-related atrial fibrillation results in decreased levels of NAD⁺and SIRT1,leading to energy production impairment.3.The expression of CARM1 is up-regulated in the age-related atrial fibrillation,and there is a protein interaction between CARM1 and CLOCK.CARM1 regulates CLOCK/NAMPT,leading to reduced NAD⁺production and mediating energy metabolism impairment.Figures:45,Tables:22,References:90.