| Four million people die of obesity and related diseases every year worldwide,of which cardiovascular-related diseases account for more than two-thirds.Obesity is a chronic progressive disease,which is closely related to the occurrence of hyperlipidemia,diabetes,coronary heart disease,hypertension and heart failure.Previous studies have confirmed that obesity is an independent risk factor for myocardial remodeling and heart failure.In addition to causing myocardial injury by inducing cardiovascular-related diseases,obesity-induced abnormal lipid metabolism in myocardial tissue can also directly cause myocardial injury.Intra-myocardial accumulation of lipids in obese patients is closely related to the damage of cardiac function.Lipids,free fatty acids and intermediate metabolites of fatty acids can disrupt the signal transmission of normal cardiomyocytes and further cause oxidative stress damage,damaging cardiomyocytes and cardiac function,which was defined as myocardial lipotoxicity.Therefore,improving fatty acid metabolism in myocardial tissue and avoiding the accumulation of excessive fatty acid in myocardial tissue may be a potential way to prevent myocardial injury caused by obesity.Cardiomyocytes mainly use fatty acids for energy production.70-90%of the energy supply of cardiomyocytes comes from fatty acids.Lipid droplets are organelles for lipid storage within cardiomyocytes.Mitochondria are the only place for fatty acid oxidation in cardiomyocytes.The interconnection between mitochondria and lipid droplets is an important structural basis ensuring lipid transport from lipid droplets to mitochondria for oxidative.Previous studies have also reported that the interconnection of lipid droplets and mitochondria can also prevent excess free fatty acids from entering other parts of the cell,thereby avoiding the occurrence of lipotoxicity.Both starvation and exercise treatments induce enhanced lipid droplet-mitochondrial interactions in cultured cells and skeletal muscle tissue.However,the structural basis of lipid droplet-mitochondrial interaction in myocardial tissue has not been studied,and the changes of this interaction in myocardial tissue of obese patients also need to be further studied.Mfn2 is mainly located in the mitochondrial outer membrane and is involved in the fusion of the mitochondrial outer membrane.In a previous study,we found that the expression of Mfn2 in myocardial tissue of obese mice with type 2 diabetes was significantly reduced,accompanied by a significant accumulation of lipids in myocardial tissue.Multiple studies have shown that Mfn2 is involved in the interaction between the membrane structures of various organelles in cells.However,whether Mfn2 is involved in myocardial mitochondria-lipid droplet membrane interaction has not been reported.This study intends to investigate the role of Mfn2 in myocardial lipid droplet-mitochondrial interaction and its role in the pathogenesis of obesity-induced myocardial injury by in vivo and in vitro experiments.MethodsPart Ⅰ:Changes in myocardial lipid accumulation and Mfn2 expression during the development of lipotoxic cardiomyopathyEight-week-old wild-type C57BL/6J mice were purchased and fed with chow-diet（CD）or high-fat diet（HFD）respectively.Body weight changes of the mice were monitored at the0th,5th,10th,and 20th weeks of feeding.Cardiac function was determined by echocardiography.Content of lipid droplets in mouse myocardial tissue and the interaction between lipid droplets and mitochondria were detected by electron microscope.The changes of Mfn2 protein levels were detected by Western-blot and RT-PCR.Part Ⅱ:The specific mechanism of Mfn2 promoting myocardial lipid metabolism and the structural basis of myocardial lipid droplet-mitochondrial interactionPrimary rat neonatal rat cardiomyocytes were isolated and extracted,and the cells were cultured in high-fat medium（containing 500μM palmitate）.Recombinant adenovirus was used to overexpress/knock down Mfn2 to observe the effect of Mfn2 on the content of lipid droplets in cardiomyocytes.Mitotracker-Red staining combined with Bodipy493/503 staining was used to observe the interaction between mitochondria and lipid droplets in cardiomyocytes.Fatty acid subcellular localization（in mitochondria or within lipid droplets）was further visualized using Bodipy 558/568 C12.Myocardial tissue of mice was extracted,and the target protein bound to Mfn2 was collected by IP experiment,and the lipid droplets of myocardial tissue were separated and purified.The target molecule was further knocked down by adenovirus,and the effect of the content change of the target molecule on the lipid droplet content and the interaction between mitochondria and lipid droplets was observed.Part Ⅲ:The relationship between Mfn2 and myocardial lipid metabolism,lipid droplet-mitochondria interaction was researched in cardiac-specific Mfn2 knockout mice.Conditional cardiac-specific Mfn2 knockout mice were constructed,induced by tamoxifen feeding.To investigate the in vivo causal relationship between the two,Mfn2 CKO mice and control mice were fed an HFD/CD for 5 weeks.Electron microscope was used to detect lipid droplet content and lipid droplet-mitochondrial relationship in mouse myocardial tissue.Oil red O staining was used to detect the lipid content of mouse myocardial tissue.Echocardiography was used to detect the cardiac systolic and diastolic function of mice.Part Ⅳ:The relationship between Mfn2 and myocardial lipid metabolism and lipid droplet-mitochondria interaction was studied using cardiac-specific Mfn2 transgenic mice.Conditional cardiac-specific Mfn2 transgenic mice was constructed.The effect of Mfn2overexpression in myocardial tissue was verified by Western-blot.To investigate the in vivo causal relationship between the two,Mfn2tg+mice and control mice were fed an HFD/CD for 10 weeks.Electron microscope was used to detect lipid droplet content and lipid droplet-mitochondrial relationship in mouse myocardial tissue.Oil red O staining was used to detect the lipid content of mouse myocardial tissue.Echocardiography was used to detect the cardiac systolic and diastolic function of mice.Part Ⅴ:of Hsc70 were myocardium-specific knockdown to study the relationship between Hsc70 and myocardial lipid metabolism and lipid droplet-mitochondria interactionsMfn2tg+mice and control mice were injected with adeno-associated virus to knock down Hsc70 by intramyocardial injection,and were fed an HFD/CD for 10 weeks.Electron microscope was used to detect lipid droplet content and lipid droplet-mitochondrial interaction in mouse myocardial tissue.Ultrasound was used to detect the cardiac systolic and diastolic function of mice.Part Ⅵ:Study the effect of post-translational modification of Mfn2 on its protein expression under lipid overload myocardiumPrimary cardiomyocytes were isolated and cultured in high-fat medium,and cells were collected at different time points to detect Mfn2 protein and mRNA levels.The acetylation and ubiquitination levels of Mfn2 protein were detected by acetylated antibodies and ubiquitinated antibodies.Point mutation was further applied to determine the acetylation modification site of Mfn2 protein,and the effect of acetylation modification on lipid droplet-mitochondria interaction was observed.Part Ⅶ:Further validation of in-vivo and in-vitro results in obese patientsClinical data and atrial muscle tissue of obese patients（BMI>30）and non-obese patients were collected,and statistical analysis was performed to observe the differences between the two groups.Further laboratory analysis was performed to observe the interaction of Mfn2-Hsc70 in the atrial muscle tissue of the patients,the difference in the expression of Mfn2 in the atrial muscle of the two groups of patients and the level of Mfn2post-translational modification.Results1.Compared with the chow diet（chow-diet,CD）fed mice,body weight of the mice in the high-fat diet（HFD）fed group increased significantly with the prolongation of feeding time.After 5 weeks’feeding,compared with the mice in the CD-fed group,cardiac function of HFD-fed mice was not significantly changed.After 10 weeks of high-fat feeding,the diastolic function of the mice in the HFD group began to be impaired,while the systolic function remained normal.After 20 weeks of high-fat diet feeding,the cardiac systolic and diastolic functions were significantly impaired in the HFD group compared with the CD group.Electron microscope results showed that there were very few lipid droplets in the myocardial tissue of CD-fed mice.After 5 weeks of feeding,little lipid droplets began to appear in the hearts of mice in the HFD group,and the connections between lipid droplets and mitochondria remained tight.After 10 weeks of high-fat diet feeding,the number and diameter of lipid droplets in myocardial tissue of HFD mice were significantly increased,and the interaction between lipid droplets and mitochondria was significantly weakened.After 20 weeks of high-fat diet feeding,the number and diameter of lipid droplets in the myocardium of HFD mice were further increased,and the connection between lipid droplets and mitochondria was looser.The expression of Mfn2 was detected by Western-blot.Compared with the mice in the CD group,the Mfn2 content in the myocardial tissue of the mice in the HFD group was significantly increased after 5 weeks of high-fat diet feeding;after 10 weeks of high-fat diet feeding,content of Mfn2 in myocardial tissue of HFD-fed mice decreased significantly,and the level of Mfn2 protein further decreased after 20 weeks of HFD feeding.2.Compared with the normal cultured primary cardiomyocytes,a large number of lipid droplets appeared in the primary cardiomyocytes after high-fat medium culturing.Overexpression of Mfn2 significantly reduced the lipid droplet content of the cardiomyocytes.Mito-tracker staining showed that Mfn2 overexpression significantly promoted mitochondrial fusion in high-fat cultured primary cardiomyocytes.Similarly,overexpression of Mfn1 and Opa1 in primary cardiomyocytes also significantly promoted mitochondrial fusion in primary cardiomyocytes under high-fat culture,however,fluorescent staining showed that overexpression of Mfn1 and Opa1 failed to reduce the number of lipid droplets in cardiomyocytes,indicating that Mfn2 does not promote cardiomyocyte lipid metabolism through mitochondrial fusion.3.Lipid droplet-mitochondria co-staining results showed that in normal cultured cardiomyocytes,lipid droplets were scattered and distributed closely with mitochondria,and the percentage of lipid droplet-mitochondria contact was about 80%.The percentage of lipid droplet-mitochondria contact in high-fat cultured cardiomyocytes decreased significantly,with lipid droplets distributing around the cell membrane in a ribbon-like manner.Overexpression of Mfn2 significantly promoted lipid droplet-mitochondrial interactions in cardiomyocytes,the percentage of lipid droplet-mitochondria contact was significantly increased.Knockdown of Mfn2 in normal cardiomyocytes resulted in a significant increase in lipid droplets in myocardial tissue and a significant decrease in the percentage of lipid droplet-mitochondria contact.4.In normal cultured cardiomyocytes,a small number of fatty acids are distributed in lipid droplets,and most of the fatty acids are co-localized with mitochondria,indicating that most fatty acids transferred to mitochondria for oxidative metabolism.In high-fat cultured cardiomyocytes,the co-localization ratio of fatty acids with mitochondria was significantly decreased,and the co-localization ratio with lipid droplets was significantly increased.Overexpression of Mfn2 significantly increased the co-localization rate of fatty acids and mitochondria in high-fat cultured cardiomyocytes,while the co-localization rate of fatty acids and lipid droplets decreased.Mfn2 knockdown in normal cultured cardiomyocytes induced a large number of fatty acids accumulated in lipid droplets,and the fatty acid-mitochondria colocalization rate was significantly reduced.Seahorse assay further confirmed that Mfn2 significantly promoted fatty acid-dependent mitochondrial oxygen consumption in primary cardiomyocytes.These data suggest that Mfn2 directly promote fatty acid transport from lipid droplets into mitochondria for oxidation.5.Mass spectrometry showed that Mfn2 directly binds to Hsc70 on the surface of lipid droplets.IP detection with different antibodies further confirmed the binding of Mfn2 to Hsc70.Immunofluorescence staining data showed that Mfn2 and Hsc70 formed a ring-like structure surrounding the lipid droplets.Fatty acid,mitochondria,and lipid droplet staining data showed that Hsc70 knockdown in normal cultured primary cardiomyocytes increased the content of lipid droplets significantly,and the contact between lipid droplets and mitochondria was significantly reduced,with large number of fatty acids deposited in lipid droplets.In primary cardiomyocytes overexpressing Mfn2,knockdown of Hsc70 also significantly increased the number of lipid droplets in cardiomyocytes,significantly decreased lipid droplet-mitochondrial interactions,and deposited fatty acids in lipid droplets.Seahorse data further showed that Hsc70 knockdown reduced fatty acid metabolism in Mfn2-overexpressing primary cardiomyocytes.These data suggest that Mfn2 binds to Hsc70 on the surface of lipid droplets,mediates mitochondrial-lipid droplet interactions,and promotes fatty acid oxidation from lipid droplets into mitochondria.6.Cardiac-specific Mfn2 knockout mice were successfully constructed.RT-PCR results showed that the expression of lipolysis-related genes（PPARa,CPT1b,ATGL）in myocardial tissue of Mfn2 CKO mice was significantly decreased.After 5 weeks of the CD-feeding,a small amount of lipid droplets was accumulated in the myocardial tissue in Mfn2CKO mice,and the interaction between lipid droplets and mitochondria was weakened,and cardiac function was not significantly changed.After fed with HFD for 5weeks,the number of lipid droplets in the myocardial tissue of the control mice increased slightly,the lipid droplet-mitochondria junctions were tight,and the cardiac function was not significantly impaired.A large number of lipid droplets appeared in myocardial tissue in 5-weeks’-HFD fed Mfn2 CKO mice,the connection between lipid droplets and mitochondria was weakened,and both cardiac systolic and diastolic functions were significantly impaired.Lipid metabolomics results showed that myocardial TAG content was significantly increased after Mfn2 knockout.High-fat diet further increased the content of diacylglycerol and ceramide in myocardial tissue,which may be an important cause of cardiac dysfunction.7.Cardiac specific Mfn2 transgenic mice were successfully constructed.RT-PCR results showed that the expression levels of lipolysis-related genes（PPARa,CPT1b）in myocardial tissue were significantly increased.After 10 weeks of CD-feeding,no significant lipid droplet formation was found in myocardial tissue of Mfn2tg mice,lipid droplets interacted closely with mitochondria,and no significant change in cardiac function was found.After 10 weeks of HFD feeding,the number of lipid droplets in myocardial tissue of control mice was significantly increased,lipid droplet-mitochondria junctions were weakened,and cardiac diastolic function was significantly impaired.Compared with control mice,the number of lipid droplets in Mfn2tg mice myocardial tissue was significantly reduced,the connection between lipid droplets and mitochondria was significantly enhanced,and there was no significant damage to cardiac systolic and diastolic functions.8.The content of Hsc70 was significantly decreased after adeno-associated virus injection.Electron microscopy results showed that knockdown of Hsc70 caused a large number of lipid droplets in myocardial tissue,and the interconnection between lipid droplets and mitochondria was weakened.Echocardiography results further showed that after Hsc70knockdown,the diastolic function of the control diet WT mice was significantly decreased.After feeding a high-fat diet for 10 weeks,there was no significant lipid droplet accumulation in myocardial tissue of Mfn2tg mice,tight lipid droplet-mitochondrial junctions,and no significant changes in cardiac function.After knockdown of Hsc70 in high-fat-fed Mfn2tg mice,a large number of lipid droplets appeared in myocardial tissue,and the interaction between lipid droplets and mitochondria was weakened,and the diastolic and systolic functions of the heart were significantly damaged,indicating that Mfn2 plays a role in high-fat diet-induced myocardial tissue.9.Western-blot combined with RT-PCR analyse showed that the changes of Mfn2 protein and mRNA levels in primary cardiomyocytes were not exactly the same with the prolongation of high-fat culture time.After 3-6 h of high-fat culture,the mRNA level further increased,while the protein level began to decrease significantly.Further treatment of cells with MG-132 combined with CHX found that high-fat culturing can significantly promote the ubiquitination and degradation of Mfn2.The acetylation and ubiquitination levels of Mfn2 were significantly increased with prolonged high-fat culture and high-fat diet feeding.High-fat-induced Mfn2 acetylation was significantly inhibited by NAM,and the decrease in Mfn2 protein level induced by high-fat culture could also be inhibited by NAM,indicating that high-fat can induce the acetylation of Mfn2 and further promote its ubiquitination degradation.11.Sequence comparison showed that the sequences near site 243 of Mfn2 were highly conserved among different species.The Mfn2KR243 mutation significantly increased the protein level of Mfn2,while the K243Q mutation significantly decreased the protein level of Mfn2.High-fat culture induced a decrease in the protein level of wild-type Mfn2,while the expression level of Mfn2KR243 mutant protein was not affected by high-fat.The reducing effect of the K243Q mutation on the protein level of Mfn2 was inhibited by MG-132.Fluorescence staining results showed that overexpression of wild-type and K243R mutant Mfn2 could promote lipid droplet-mitochondrial interactions and reduce lipid droplet numbers in primary cardiomyocytes under high-fat culture,while overexpression of K243Q mutant Mfn2 could not achieve the above effects.These evidences suggest that acetylation of Mfn2 at the K243 site further promotes its ubiquitination degradation,and the degradation of Mfn2 further induces a weakening of lipid droplet-mitochondrial interactions.12.Clinical data showed that compared with non-obese control patients,obese patients had significantly lower left ventricular ejection fraction,left ventricular short-axis shortening rate and E/A ratio than control non-obese patients.The ventricular septal thickness and left atrial diameter were significantly higher than those in the control non-obese patients.13.Clinical samples results showed that almost no lipid droplets were found in the myocardial tissue of control non-obese patients.In the myocardial tissue of obese patients,significant lipid droplet accumulation was observed,and lipid droplet-mitochondria were significantly separated,with almost no lipid droplet-mitochondria interaction.The Mfn2protein level in myocardial tissue of obese patients was significantly lower than that of non-obese controls,and the acetylation and ubiquitination levels of Mfn2 protein were significantly higher than those of non-obese controls.In human atrial myocardium,IP results confirmed that Mfn2 interacts with Hsc70,and the interaction between Mfn2 and Hsc70 was significantly attenuated in the cardiac tissue of obese patients.Conclusion1.With the development of obesity-induced cardiomyopathy,lipid droplet content in myocardial tissue was significantly increased,lipid droplet-mitochondria interactions were significantly weakened,and fatty acid oxidation capacity was reduced.2.Mfn2 interacts with Hsc70 on the surface of lipid droplets,mediates the interconnection between lipid droplets and mitochondria,and promotes fatty acids transferring from lipid droplets to mitochondria for oxidation.3.Mfn2/Hsc70 deficiency can directly induce cardiac lipid accumulation,weakened lipid droplet-mitochondrial junctions,and reduced lipid metabolism.Mfn2 overexpression promotes lipid droplet-mitochondrial interconnection,counteracts cardiac lipid accumulation,and protects cardiac function.4.Lipid overload induces acetylation of Mfn2 at the K243 site,and further promotes its ubiquitination and degradation,reduces the expression level of Mfn2,disrupts lipid droplet-mitochondrial interconnections,and leads to lipid accumulation in the heart.5.Obese patients have impaired cardiac function and significant myocardial remodeling.In atrial tissue from obese patients,lipid droplet content was significantly increased,with lipid droplet disconnecting to mitochondria.Compared with those of atrial tissue from non-obese patients,Mfn2 expression is significantly down-regulated,acetylation and ubiquitination levels are significantly increased,and Mfn2 interacts with Hsc70significantly weaken. |
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