Using Mitochondrial Dynamics-related Proteins To Explore The Mechanism Of Cardiac Dysfunction In Sirt3 Knockout Mice

Background:Mitochondria are one of the most critical organelles to maintain the function of cardiomyocytes,they have been widely concerned in the study of cardiac dysfunction by regulating cell metabolism,apoptosis,and oxidative stress.Sirt3 is the most important deacetylating protease in the mitochondrial matrix,and its metabolic function is closely related to its protective effect against oxidative stress damage.Current research suggests that Sirt3 is involved in different cardiac pathophysiological processes,but its mechanism is not very clear.At present,existing experiments have found that Sirt3,which is a mitochondrial sirt family,has attracted the attention of researchers as an antioxidant molecule by regulating the functions of antioxidant enzymes such as SOD2.Mitochondrial fission and fusion are critical for maintaining the health and normal function of cardiomyocytes.At present,studies have confirmed that excessive fusion or fission of cardiac mitochondria will change the shape of mitochondria,which will lead to pathological changes in cardiomyocytes,and ultimately affect the overall function of the heart,suggesting that mitochondria may change their shape when responding to oxidative stress,and even cause changes in their dynamics.In addition,our early laboratory studies found that micro RNA-210(mi R-210)is closely related to oxidative injury and regulates energy metabolism by influencing mitochondrial function,but whether it is involved in the regulation of heart function and mitochondrial dynamics by Sirt3 is still unclear.Therefore,this study took Sirt3 knockout mice as the research object,focusing on the perspective of mitochondrial dynamics,combined with basic mitochondrial functions such as energy metabolism,to explore the mechanism by which Sirt3 affects mitochondrial dynamics,thereby leading to the decline of cardiac function.At last,we hope our study could provide a new experimental and theoretical basis for the prevention and treatment of ischemia heart disease.Objective:By exploring the changes of energy metabolism and mitochondrial dynamics in cardiomyocytes under oxidative stress,and further analyzing the changing trends of mitochondrial dynamics related proteins,to explore the mechanism of the decline of Sirt3 function leading to the cardiac dysfunction.Methods:1.The Sirt3 knockout mice models were constructed to evaluate the effect of Sirt-3 deletion on mice cardiac function;q PCR technology was used to detect the expression level of mi R-210;Western Blot technology was used to detect the changes of mitochondrial dynamic-related proteins.2.The oxidative stress model of H9C2 cells was constructed by using H₂O₂.CCK8was used to evaluate cell viability,ATP and lactate kit were used to compare intracellular ATP and lactate contents,and Mito-Tracker and Hoechst 33342colocalization staining was used to evaluate mitochondrial dynamics and cell survival state.The levels of mitochondrial dynamic-related proteins were detected by Western Blot.3.The H9C2 cell model with high expression of mi R-210 was constructed by adenovirus transfection,and the relative content of ATP and lactate after the pretreatment of mi R-210 was detected again.Mito-Tracker and Hoechst 33342colocalization staining was applied to assess mitochondrial network level and cell survival status.The levels of mitochondrial motility-related proteins were detected by Western Blot.4.The H9C2 cell model with low expression of mi R-210 was constructed by adenovirus transfection,the relative content of ATP and lactate content after mi R-210pretreatment were detected.Mito-Tracker and Hoechst 33342 colocalization staining was applied to assess mitochondrial dynamics and cell survival status.The levels of mitochondrial dynamic-related proteins were detected by Western Blot.Results:1.In the animal experiments,it was found that the cardiac ventricular ejection fraction of the Sirt3 knockout mice was decreased,and the short-axis shortening rate was further decreased,indicating that the cardiac function of the mice was damaged.At the same time,it was found that the expression level decline of mi R-210 was consistent with that of Sirt3;in Sirt3 knockout mice,the protein expressions of Sirt3,DRP1,OPA1,and Fis1 decreased.2.Under the state of oxidative stress,the cell viability decreased significantly with the passage of time,the ATP production of H9C2 cells decreased significantly,and the lactate was the highest at 1 hour,and then the lactate content gradually decreased with the passage of time.Inhibitors of glycolysis further reduced ATP production,as well as lactate production.The content of Sirt3,DRP1 Ser637 were further reduced under oxidative stress,in the H9C2 cells which were dealed with the glycolysis inhibitor(2-DG).However,the total amount of DRP1 protein showed an increasing trend under oxidative stress,while the phosphorylated DRP1 Ser637 protein showed a decreasing trend.The level of OPA1,which promotes fusion,decreased under acute oxidative stress.Under oxidative stress,mitochondrial fission was further aggravated,and the expression of related proteins was further reduced.3.After mi R-210 intervened in H9C2 cells,it was found that the high expression of mi R-210 increased the energy supply of H9C2 cells to a certain extent under oxidative stress.By increasing anaerobic metabolism,H9C2 cells were reversed in the case of increased lactate production(due to glycolysis)and decreased ATP production under oxidative stress.Meanwhile,mi R-210 reversed the deterioration of mitochondrial dynamics under oxidative stress.In H9C2 cells with high expression of mi R-210,the level of Sirt3 did not change significantly compared with the control group under oxidative stress,but the high expression of mi R-210 reversed the decrease in the level of DRP1 Ser637 phosphorylated to a certain extent and maintains mitochondrial dynamic,while the overall trend of DRP1 is still rising.However,the level of fusion-promoting OPA1 decreased with oxidative stress under acute oxidative stress,and mi R-210 failed to reverse the decreasing trend of OPA1.4.The high expression of mi R-210 under oxidative stress can promote cellular energy metabolism,while its low expression aggravates the damage of oxidative stress to cellular energy metabolism.High expression of mi R-210 under oxidative stress has a protective effect on the maintenance of mitochondrial dynamics,while its low expression accelerates the collapse of mitochondrial dynamics.Under oxidative stress,low expression of mi R-210 further increased the total amount of DRP1 protein,but the phosphorylation of DRP1 Ser637 decreased more significantly,while high expression of mi R-210 reversed the reduction phosphorylation DRP1 Ser637 to a certain extent.Conclusion:1.The cardiac ejection fraction,short-axis shortening rate and cardiac function of the Sirt3 knockout mice were decreased,and the functional changes may be related to the decrease of mitochondrial dynamics-related proteins DRP1,Fis1,and OPA1.The mi R-210 expression of gene knockout mice was reduced,suggesting that mi R-210 may be involved in the induction of cardiac dysfunction due to the low expression of Sirt3.2.Using H₂O₂ to replicate the oxidative stress model of cardiomyocytes,it was found that the changes in cardiomyocyte function may be related to changes in mitochondrial dynamics-related proteins(DRP1,DRP1 Ser637,OPA1).Glycolysis played a role in antagonizing H₂O₂-induced H9C2 cell injury through Sirt3 and changes in mitochondrial dynamics,suggesting that oxidative stress regulates ATP generation through glycolysis and induces changes in mitochondrial dynamics.3.There was no significant change in Sirt3 expression when mi R-210 was highly expressed,so we speculated that Sirt3 may act as the upstream of mi R-210 to regulate intracellular energy metabolism by regulating mi R-210,further regulating the phosphorylation level of DRP1 Ser637,and finally regulating the changes of mitochondrial dynamics.