Time-Restricted Feeding Protects Against Pressure Overload-Induced Heart Failure By Improving Myocardial Arginine Metabolism

Background:Heart failure（HF）is the advanced stage of various heart diseases.It is characterized by a poor prognosis and brings a heavy burden to society and families.The heart is a high energy-consuming organ.A large and continuous supply of ATP maintains the activity of myocardial ion channels and normal contraction function.Myocardial energy substrates are mainly fatty acids,followed by glucose,ketone bodies and amino acids.In cardiac hypertrophy and HF caused by pressure and volume overload,the switch of energy substrates from fatty acids to glucose is one of the signs of disease progression.With the progress of heart failure,the oxidation rate of fatty acids and glucose gradually decreases.Although the glycolysis process in the myocardium is gradually enhanced,overall the production of ATP gradually decreases,which is insufficient to meet the myocardial energy demand.In the heart of HF patients,the myocardial energy metabolism disorder causes insufficient energy supply and oxidative stress,which in turn leads to the occurrence and development of cardiac systolic dysfunction.Metabolic failure plays a central role in the pathogenesis of heart failure.Cardiometabolic therapy is showing its positive effect on improving the prognosis of HF patients,which suggests that improving myocardial metabolism can be used as a therapeutic target for HF.Evidences showed that HF patients exhibit metabolic disorders of energy substrates,leading to the progression and deterioration of HF.Cardiometabolic therapy shows its positive effect on improving the prognosis of HF patients,and improving myocardial lipid metabolism can be used as a therapeutic strategy for HF.Time-restricted feeding（TRF）refers to restricting the eating time within 24 hours to a certain time,usually 4-8 hours.TRF is essentially different from calorie restriction（CR）.CR is a dietary pattern that reduces calorie intake by 20%-40%per day,while TRF emphasizes restricting eating time,not the amount of food intake.Animal studies have shown that TRF with a feeding period of 8-12 hours per day prevents fatty liver,dyslipidemia and glucose intolerance,and improves blood glucose control,insulin levels,inflammation,weight regulation,energy expenditure,exercise coordination,heart contraction,sleep and exercise endurance levels.Animal studies have shown that TRF with a feeding period of 8-12 hours a day can prevent fatty liver,reduce blood lipid levels,increase energy consumption and exercise coordination,reduce body weight and inflammation,improve insulin sensitivity and exercise endurance,and improve glucose tolerance,cardiac function and sleep.In addition,human clinical studies have also proved that TRF reduces body weight,improves glucose intolerance,and reduces cardiovascular disease risk factors（reducing cholesterol,triglycerides and low-density lipoprotein levels,and improving insulin sensitivity）.The results of animal experiments and clinical studies have shown that the caloric intake in TRF is equal to or slightly less than the normal caloric intake,so the impact of TRF is not due to the restriction of caloric intake,but mainly from its impact on metabolism.Whether TRF can prevent and correct metabolic disorders and prevent cardiovascular diseases,especially the occurrence and development of HF through its metabolic remodeling effect,is not clear.Objective:To test whether TRF resists pressure overload-induced heart failure and explore its underlying mechanism.Methods:C57BL/6（male,8-10 weeks old）mice were subjected to transverse aortic constriction（TAC）surgery to establish a pressure overload-induced heart failure.The TRF was conducted as follows:during the feeding period（from 21:00 daily to 5:00 the next day,8 hours in total）,mice were exposed to sufficient food and water;during the fasting period（5:00-21:00 daily,16 hours in total）,the mice were exposed to sufficient drinking water,but not food.The control group was the ad libitum（AL）group.Targeted or non-targeted metabonomics were used to detect the metabolic changes in heart tissue and plasma.Six kinds of amino acids（L-Pro,L-Arg,L-Met,L-Ser,L-Thr and L-Cit）were formulated into an amino acid mixed solution,which was orally supplemented to mice in AAs group.The cardiac function was detected by animal ultrasound,pathological staining and various conventional molecular biology methods.Myocardial lipid metabolism was detected by quantitation of stable isotope-labeled fatty acid metabolites.Cardiac-specifically knock-out argininosuccinate lyase（ASL）mice was used to test the role of ASL in TRF’s cardiac protection.Cardiac systolic function was evaluated by left ventricular ejection fractions（LVEF）.Cardiac hypertrophy indicators included left ventricular left ventricular posterior wall;diastolic（LVPW;d）,diastolic ventricle interventricular septum;diastolic（IVS;d）,heart weight（HW）,heart weight/body weight（HW/BW）and average cross-sectional area of cardiomyocytes.Evaluation of heart failure included lung weight/body weight（LW/BW）and plasma brain natriuretic peptide（BNP）concentration.Masson staining was used to detect cardiac fibrosis by quantifing left ventricular fiber volume.Results:1.TRF was conducted immediately after TAC.After 8 weeks,compared to the TAC+AL group,the cardic function of the TAC+TRF group was significantly improved.LW/BW was reduced,and the BNP concentration in plasma was reduced.HW and HW/BW were reduced.IVS;d and LVPW;d were decreased.The fibrosis of mice heart was reduced,and the cross-sectional area of myocardial cells was reduced.The transcription levels of molecules related to heart failure and fibrosis were reduced.2.HF was established through TAC surgery（AL for 8 weeks after TAC）.TRF intervention was conducted after HF establishment,and related indicators were tested after TRF intervention for 8 weeks.Compared with the AL group,TRF inhibited the further reduction of cardiac function in HF mice,reducing LW/BW and plasma BNP content;inhibiting the further hypertrophy of myocardium（HW,HW/BW,IVS;d,LVPW;d and the cross-sectional area of myocardial cells were reduced after TRF intervention）.Pathological staining also showed that the degree of fibrosis was reduced.The transcription levels of molecules related to heart failure and fibrosis were reduced.3.The results of plasma non-targeted metabonomics showed that,after TRF intervention,the changes of metabolites in plasma were mainly amino acids,followed by lipids and cofactors.Further amino acid targeting metabolomics results showed that after TRF intervention,the levels of amino acids such as asparagine,citrulline,methionine,ornithine,proline,threonine,tyrosine,valine,glycine,alanine,arginine,serine,and lysine were increased.Myocardial tissue non-targeted metabolomics results showed that after TRF intervention,the differential metabolites in myocardial tissue were mainly lipids,followed by carbohydrates,amino acids and cofactors.Cell experiments have preliminarily confirmed that six amino acids such as L-arginine,L-methionine,L-proline,L-serine,L-threonine and L-citrulline promoted myocardial lipid metabolism.4.After the TAC surgery,mice were given oral supplemental amino acid solution immediately.After 8 weeks,compared with the TAC group,the cardic function of the TAC+AAs group was improved;LW/BW,LVPW;d and IVS were reduced.Pathological staining also showed that the degree of myocardial interstitial and vascular fibrosis was reduced.The cross-sectional area of myocardial cells was reduced.The transcription level of the molecules related to heart failure and fibrosis-related molecules was reduced.5.The urea cycle of cardiomyocytes was inhibited in heart failure.After TRF intervention,ASL enzyme activity in cardiomyocytes was increased.Studies of intracellular amino acids have confirmed that amino acids were converted into endogenous arginine through the urea cycle.Endogenous arginine increased the production of NO and promotes myocardial lipid metabolism.6.ASL^{c KO} mice were given TRF intervention after TAC operation.After 8 weeks,the results showed that TRF failed to improve the cardic function and decrease LW/BW,plasma BNP,HW,HW/BW,IVS;d,LVPW;d,myocardial interstitial fibrosis and cross-sectional area of myocardial cells in failure heart.Conclusions:1.TRF improves heart function in TAC mice,reduces myocardial hypertrophy and fibrosis,and resists pressure overload-induced heart failure.2.TRF increases the level of amino acids in the circulation of TAC mice and improves myocardial lipid metabolism.3.Partial amino acids（L-Pro,L-Arg,L-Met,L-Ser,L-Thr and L-Cit）elevated by TRF improve the cardiac function,reduce myocardial hypertrophy and fibrosis,resist heart failure indued by pressure overload in mice.4.TRF increases the level of amino acids which increase the urea cycle flux incardiomyocytes.Then the metabolism of arginine which is improved in cardiomyocytes play a key role in TRF resisting pressure overload-induced heart failure.