Role And Mechanism Of Abnormal Mitochondrial Fusion And Fission In Alcoholic Cardiomyopathy

Background:The effect of alcohol on the heart has always been a topic of interest in the field of cardiology.According to statistics in the World Health Organization,an estimated 237 million men and46 million women worldwide suffer from alcohol-related diseases.The annual per capita consumption of alcohol in Europe States(especially in Russia)is the highest in the world,and the mortality of alcoholic cardiomyopathy(ACM)is also the highest.ACM is defined as acquired dilated cardiomyopathy associated with long-term alcoholism(> 80g/ day,lasting for more than 5 years).Exploring the pathogenesis of alcoholic cardiomyopathy and finding its potential therapeutic targets remain an important issue in the field of cardiology.As an important organelle in the cardiomyocytes,mitochondria provide more than 90% of the energy supply to cardiomyocytes and maintain the its basic function.Studies have pointed out that mitochondria are one of the main organelles attacked by alcohol,long-term alcohol intake leads to abnormal structure and function of cardiac mitochondria,resulting in mitochondrial fragmentation.It is suggested that abnormal mitochondrial dynamics may be involved in the development of alcoholic cardiomyopathy,but the specific mechanism has not been elucidated.The purpose of this study is to explore the role and underlying mechanism of abnormal mitochondrial fusion and fission in alcoholic cardiomyopathy through performing in vivo and in vitro experiment.Objectives:1.To elucidate the changes of mitochondrial fusion-fission in alcoholic cardiomyopathy;2.To observe the effect of regulating mitochondrial fusion-fission on alcoholic cardiomyopathy and the possible mechanism.Methods:Part Ⅰ: Changes of mitochondrial morphology and related proteins during the initiation and development of alcoholic cardiomyopathy.8-week-old male C57 mice were randomly divided into two groups after two weeks of adaptation: normal fluid diet group and alcohol fluid diet group.After 8 weeks of feeding,cardiac function was monitored by echocardiography.Plasma was collected to measure LDH level,and the oxidative stress level of myocardial tissue was detected by DHE staining.WGA staining and Masson staining were used to assess myocardial hypertrophy and fibrosis in the mice.TUNEL staining was used to detect cardiomyocyte apoptosis.Transmission electron microscope was used to observe the morphological changes of myocardial mitochondria.Western Blotting was used to detect the expression of mitochondrial fusion-fission related proteins(Mfn1,Mfn2,Opa1,Drp1,Fis1)and apoptosis-related proteins(Casepase 3,Bcl2,Bax).Part Ⅱ: In vitro cell model mimics the changes of myocardium and mitochondrial in alcoholic cardiomyopathy in vivo.The primary cardiomyocytes of neonatal rats were isolated and cultured with ethanol or/and acetaldehyde.The apoptosis of cardiomyocytes was detected by flow cytometry.The oxidative stress levels of cells and mitochondria were detected by Cellular ROS and Mito-SOX staining respectively.The changes of mitochondrial membrane potential were detected by JC-1 staining.The morphological changes of mitochondria were observed by Mito-Tracker Red staining.The changes of mitochondrial fusion-fission proteins and apoptosis-related proteins were detected by Western Blotting.Part Ⅲ: Upregulation of Mfn2 alleviates cell damage induced by ethanol and acetaldehyde mixture.Primary cardiomyocytes were isolated and cultured with ethanol-acetaldehyde mixed treatment.Adenovirus overexpressed Mfn2 was transfected into cardiomyocytes.Apoptosis of cardiomyocytes was detected by flow cytometry.Oxidative stress levels of cells and mitochondria were detected by Cellular ROS and Mito-SOX staining respectively.Mitochondrial membrane potential changes were detected by JC-1 staining,and mitochondrial morphological changes were observed by Mito-Tracker Red staining.Results:1.Compared with the normal fluid diet group,after 8 weeks of alcohol fluid diet,the left ventricular ejection fraction and left ventricular fraction shortening were significantly decreased,and the plasma LDH level was significantly increased.Myocardial oxidative stress and apoptosis were significantly increased,and myocardial hypertrophy and fibrosis were induced in the mice receiving alcohol diet.Alcohol diet increased myocardial mitochondrial fragmentation and inhibited the expression of mitochondrial fusion protein Mfn2.The expressions of apoptosis-related proteins Caspase3 and Bax were increased,and the expression of anti-apoptotic protein Bcl2 was decreased in the mice receiving alcohol diet.These results suggest that alcohol intake causes myocardial and mitochondrial damage.2.At the cellular level in vitro,the apoptosis of cardiomyocytes treated with ethanol alone had no significant change,and the damage of mitochondria was not significant.In the cardiomyocytes treated with acetaldehyde alone or ethanol plus acetaldehyde,the apoptosis was significantly increased,and the oxidative stress levels of cells and mitochondria were significantly increased.Acetaldehyde or ethanol plus acetaldehyde increased mitochondrial fission and reduced mitochondrial membrane potential.Moreover,the m RNA and protein levels of mitochondrial fusion protein Mfn2 were significantly decreased.Compared with acetaldehyde alone treatment,the injury effect of ethanol plus acetaldehyde treatment was more significant.Thus,the mixed treatment of ethanol and acetaldehyde in vitro was used tomimic the myocardial injury of alcohol in vivo.3.Overexpression of Mfn2 significantly reduced cardiomyocyte apoptosis,inhibited cellular and mitochondrial oxidative stress,improve mitochondrial membrane potential,prevented mitochondrial fragmentation and promoted mitochondrial fusion in the cardiomyocytes treated with ethanol plus acetaldehyde.Conclusions:1.The mitochondrial fission is abnormally increased and the expression of mitochondrial fusion protein Mfn2 is down-regulated in alcoholic cardiomyopathy.2.Up-regulation of Mfn2 inhibits mitochondrial fission,reduces myocardial apoptosis and oxidative stress,improves mitochondrial function and prevents the development of alcoholic cardiomyopathy.