Experimental Study Of NMN Improves Cardiac Function In SIRT3 Knockout Mice Via SIRT1/PGC-1α Pathway

Background:Cardiac dysfunction is a common clinical syndrome.Due to mitochondrial dysfunction,cardiac pumping function cannot meet the needs of the body,and the quality of life of patients is often severely affected.The mechanism of mitochondrial dysfunction during cardiac insufficiency is not entirely clear,but existing studies have suggested that Sirtuin 3(SIRT3)plays a key role in it.SIRT3 is a deacetylase that uses Nicotinamide adenine dinucleotide(NAD+)as a substrate and plays an important role in maintaining mitochondrial function.SIRT3 can regulate the expression of Peroxisome proliferative activated receptor,gamma,coactivator 1α(PGC-1α),a cofactor in mitochondrial biosynthesis,and the synthesis of respiratory chain complexes.Studies have found that SIRT3 knockout mice have severe impairment of mitochondrial biosynthesis and oxidative respiration,and they also exhibit cardiac dysfunction.There is an interactive compensatory mechanism between the effects of Sirtuins.Some studies have found that the expression of Sirtuin 1(SIRT1)can compensate for the loss of SIRT3,and the cofactor PGC-1α in mitochondrial biosynthesis is the main molecule that interacts between the two.In the absence of SIRT3,changes in intracellular NAD+levels can be detected through SIRT1/PGC-1α pathways to affect mitochondrial function,but the role of this pathway in myocardium is unclear.NAD+is the substrate of various enzymes in cells,and its content is also closely related to mitochondrial function.Administration of Nicotinamide mononucleotide(NMN)can upregulate the ratio of NAD+/NADH to improve mitochondrial function.Therefore,we speculate that although SIRT3 knock out mice will impaire mitochondrial function after the loss of SIRT3,which affects cardiac function,we can also improve cardiac dysfunction by using other pathways such as SIRT1 to improve mitochondrial function through compensatory mechanisms involving the cross-talk between sirtuins.Objective:To investigate the mechanism of NMN improving mitochondrial function and cardiac dysfunction through the compensatory effect of SIRT1 in SIRT3 knock out mice based on the model of cardiac dysfunction in 8-week-old SIRT3 knockout mice,and provide new ideas for the treatment of cardiac dysfunction in clinical practice.Methods:Preliminary experiment:Take 6 wild type(WT)and SIRT3 knockout(Sirt3-/-)type with similar body weight,with half male and half female,for relevant experimental operations.Subsequent experiments:Six-week-old wild type(WT)and SIRT3 knockout(Sirt3-/-)type with similar weight were divided into physiological saline(Control)group and NMN group,with 6 in each group,half males and half females.Saline or NMN solution was intraperitoneally injected into each animal at 500 mg/kg/d for 14 days,and the relevant experimental procedures were started on day 15.1.Measure the body length and weight of WT mice and Sirt3-/-mice at 0,2,4,6,and 8 weeks of age;2.Using an animal treadmill to detect the total running time and maximum allowable running speed of WT mice and Sirt3-/-mice aged 4 and 8 weeks;3.Examining the left ventricular ej ection fraction and left ventricular fractional shortening of mice by echocardiography;4.Used HE staining to observe the myocardial cell structure and tissue morphology of mice;5.Used electron microscopy to observe the morphology of mouse cardiomyocytes and mitochondria;6.ATP content in mouse myocardial tissue was determined by ATP detection kit;7.Used Western blot and immunohistochemical staining to detect respiratory chain complex protein expression in mouse myocardial tissue;8.Used NAD detection kit(WST-8 method)to detect the NAD+,NADH content and NAD+/NADH ratio in mouse myocardial tissue;9.Used Western blot to detect the expression of SIRT1 protein in mouse myocardial tissue.10.Used Western blot to detect the expression of PGC-1α and its downstream proteins NRF1,NRF2 and TFAM in mouse myocardial tissue.Results:1.There was no significant difference in body length between the two types of mice,but the weight of Sirt3-/-mice was lower than that of WT mice from the age of 2 weeks;2.Compared with WT mice,the total running time and the maximum running speed that Sirt3-/-mice can withstand did not significantly change at the age of 4 weeks,but both decreased at the age of 8 weeks;3.Echocardiographic results showed that the left ventricular ejection fraction and left ventricular fractional shortening of Sirt3-/-mice were significantly lower than those of WT group from the age of 8 weeks.After administration of NMN,the left ventricular ejection fraction and left ventricular fractional shortening of Sirt3-/-mice were significantly increased;4.Compared with WT mice,the myocardial cells of 8-week-old Sirt3-/-mice were more disorderly arranged,with deeply stained mast cells visible.After NMN administration,the myocardial cells of Sirt3-/-mice were more orderly arranged,with more uniform staining,and fewer mast cells in vision;5.Compared with WT mice,the structure of the sarcomere in the myocardium of 8-week-old Sirt3-/-mice is not clear,the structure of the mitochondrial bilayer membrane and ridge is not clear,and there are fused and enlarged mitochondria.After administration of NMN,the structure of the sarcomere in the myocardium of Sirt3-/-mice is clearer,the mitochondrial size is more uniform,and the structure of the bilayer membrane and ridge is clearer;6.ATP content in myocardial tissue of Sirt3-/-+NMN mice decreased compared with Control mice,but it was significantly improved compared with Sirt3-/mice;7.Western blot results showed that the expression of MTND1,SDHB,MTCO2,and ATP5 A in myocardial tissue of Sirt3-/-mice decreased compared with WT mice,and increased after NMN administration.Immunohistochemical staining results showed that the expression of SDHB and MTCO2 in Sirt3-/+NMN group were higher than those of Sirt3-/-group;8.The results of NAD kit showed that NAD+and NAD+/NADH ratio in myocardial tissue of WT mice and Sirt3-/-mice were increased after NMN supplementation;9.Western blot results showed that SIRT1 protein expression in myocardial tissue of WT mice and Sirt3-/-mice was increased after NMN administration;10.Western blot results showed that the expression of PGC-1α,NRF1,NRF2 and TFAM were decreased in myocardial tissue of Sirt3-/-mice compared with WT mice,and were increased after NMN administration.Conclusion:1.SIRT3 knockout mice develop cardiac dysfunction by 8 weeks of age;2.The decreased protein expression levels of mitochondrial biogenesis related molecules PGC-1α,NRF1,NRF2 and TFAM and respiratory chain complex subunits MTND1,SDHB,MTCO2 and ATP5A in SIRT3 knockout mice indicate that the decreased mitochondrial biogenesis capacity is associated with the development of cardiac dysfunction in SIRT3 knockout mice;3.NMN supplementation can be achieved through SIRT1/PGC-1α pathway via compensatory effect of SIRT1 improves mitochondrial and cardiac function in SIRT3 knockout mice.