Enhancing Fatty Acid Utilization Ameliorates Mitochondrial Fragmentation And Cardiac Dysfunction Via Rebalancing OPA1 Processing In The Failing Heart

BackgroundHeart failure(HF)is regarded as the "last battle" of cardiovascular diseases.Elucidation of its pathophysiological mechanisms bears great theoretical and clinical significance and contributes to the development of potential therapeutic approaches.The heart has sustained high energy demands and primarily relies on the oxidation of fatty acids(FA)(~60-90 %).During the transition process towards heart failure(HF),the cardiac substrate preference shifts to a greater reliance on glucose as substrate with a concomitant suppression of FA utilization.The metabolic shift towards glucose utilization has long been interpreted as a protective mechanism against oxygen deficiency and lipotoxicity,and hence inhibition of FA utilization has been suggested as an approach to treat patients with HF.However,recent studies suggest a novel concept that a diet high in fat may prevent the progression of HF.Studies have shown that enhancement of FA utilization through feeding animals a high fat diet(HFD)attenuated cardiac dysfunction and inhibition of FA utilization through genetic manipulation aggravated cardiac dysfunction in response to pressure overload.These advances suggest that targeting cardiac FA utilization may be a potential strategy for the treatment of HF.FA oxidation occurs primarily in the mitochondria that continually alter their morphology by undergoing fission and fusion.Evidence has shown that the balance of mitochondrial fission and fusion is strictly associated with their bioenergetics.Unbalanced mitochondrial fusion/fission,such as mitochondrial fragmentation,occurs in response to stress and in diseases.It has been shown that mitochondrial fragmentation is a cause of HF and mitochondrial fragmentation-induced cardiac dysfunction can be reversed by HFD feeding,suggesting that the metabolic switch may be associated with the imbalanced mitochondrial fission/fusion in failing hearts.However,the underlying mechanism is poorly understood.Therefore,this study aims to investigate the mechanism of mitochondrial fragmentation and FA utilization in the failing heart using a model of post-load-induced heart failure.Purposes1)To examine the effect of the increased myocardial FA utilizaiton on cardiac function during heart failure and investigate the underlying mechanisms.Methods1)C57 mice(male,8 weeks)were used for induction of HF through pressure overload.Pressure overload was produced by transverse aortic constriction(TAC)as described previously.Briefly,the aorta was approached via minimal sternal incision and a 7-0 ligature placed around the vessel using a 27-gauge needle to ensure consistent occlusion.Sham-operated mice underwent the same surgery without constriction.After surgery,the mice were fed with either control diet or HFD for 2 months.All the diets were obtained from Research Diets Inc.(NJ,USA).Control diet consists of 10% of calories derived from FA,and HFD consists of 45% of calories derived from FA.The cardiac and mitochondrial functions were determined 2 months post-surgery.2)C57 mice(male,4 weeks)were randomly chosen to receive a single-bolus tail vein injection of either AAV9 encoding CD36 or AAV9 encoding RFP at 1*1011 viral genomes per mouse.After 4 weeks,mice were subjected to TAC or sham surgery.2)Cardiac tissues were paraffin embedded and sectioned at a thickness of 5 μm for staining of Hematoxylin and Eosin.Hematoxylin and Eosin staining was performed according to the manufacturer’s protocols.Echocardiography to assess cardiac function was performed in mice 2 months post-surgery.Transmission electron microscopy were used for measuring mitochondrial size and number.3)We examined the effects of palmitic acid(PA)and oleic acid(OA)on the expression of OPA1 and the ratio of L-OPA1 to S-OPA1,and confocal was used to observe the morphology of mitochondria.H2O2 was used to induce mitochondrial division and then the effects of FA were detected again.4)The expression of YME1 L and OMA1 which modulate the cleavage of OPA1 was detected on neonatal rat cardiomyocytes.The effect of YME1 L on FA-regulated OPA1 expression was investigated using si-RNA to reduce the expression of YME1 L.5)Bioinformatic prediction suggested that YME1 L contains a PPARγ response element.Therefore,si-RNA was used to konckdown the expression of PPAR-γ to identify the mechanism of FA regulating YME1 L expression.6)To further confirm the role of YME1 L in the cardioprotection against HF,we specifically overexpressed YME1 L in hearts of TAC mice using AAV9.After 8 weeks,the morphology and function of mitochondria and the heart were detected.We also measured the expression level of moleculars associated with FA metabolism and mitochondrial dynamics.Results1)Feeding TAC mice a HFD increased cardiac FA utilization and improved cardiac function and survival.Compared with age-matched sham mice,TAC mice showed a 6.5% decrease in body weight,a 97.6% increase in heart weight,and a 114.0% increase in heart weight-to-tibia length ratio 2 months post-surgery.In addition,pressure overload impaired cardiac function as evidenced by the decreased left ventricular(LV)ejection fraction and the increased LV chamber diameter.HFD feeding restored body weight,and decreased heart weight by 26.8% and heart weight-to-tibia length ratio by 24.7% in TAC mice.It also increased LV ejection fraction and normalized LV chamber diameter in TAC mice.Feeding TAC mice HFD increased the circulating levels of FFAs and TG,cardiac CD36 and CPT-1b expressions,and intramyocardial lipid droplets and TG contents.CD36 overexpression yielded the similar effects to HFD feeding.2)Enhancement of FA utilization attenuated mitochondrial fragmentation and improved mitochondrial function in the failing heart.Pressure overload induced cardiac mitochondrial fragmentation as evidenced by the increased mitochondrial number and the decreased mitochondrial size in TAC mice.It also decreased mitochondrial respiratory control ratio and ATP content,while increasing mitochondrial ROS.Notably,the cardiac mitochondrial fragmentation and dysfunction were attenuated by both HFD and cardiac-specific overexpression of CD36 in TAC mice.3)FA inhibited OPA1 cleavage.No differences in the contents of mfn1,mfn2,Drp1,fis1 and Drp1 phosphorylation were detected in TAC mice.Neither HFD nor cardiacspecific overexpression of CD36 had effects on these proteins.Specifically,OPA1 showed profound changes in TAC mice,and such changes could be reversed by both HFD and CD36 overexpression.Pressure overload decreased the contents of total OPA1,L-OPA1,and L-OPA1/S-OPA1,while it had no effects on the contents of S-OPA1.Imbalanced OPA1 processing was attenuated by either HFD or cardiac-specific overexpression of CD36 in TAC mice.4)FA inhibited OPA1 cleavage through upregulation of YME1 L.Treatment with oleic acid or palmitic acid had no effects on OMA1 expression,while it increased YME1 L expression in a dose-dependent manner in isolated cardiomyocytes.In addition,both oleic acid and palmitic acid attenuated the loss of YME1 L contents in a dose-dependent manner in cardiomyocytes under oxidative stress(10 μM H2O2 for 2 h).Knockdown of YME1 L inhibited FA-induced upregulation of YME1 L and L-OPA1/S-OPA1 in neonatal myocytes under oxidative stress.In addition,both dietary fat and cardiacspecific overexpression of CD36 restored the pressure overload-induced decrease of YME1 L content in TAC hearts.Taken together,the results suggest that upregulated YME1 L expression was responsible for FA-induced inhibition of OPA1 cleavage and mitochondrial fragmentation.5)Upregulation of YME1 L expression exerted cardioprotection against pressure overload.Cardiac-specific overexpression of YME1 L increased the contents of YME1 L and LOPA1/S-OPA1 and attenuated mitochondrial fragmentation in TAC mice,accompanied with increased mitochondrial respiratory control ratio and cardiac ATP level.In addition,cardiac-specific overexpression of YME1 L decreased heart weight by 24.6% and heart weight-to-tibia ratio by 21.7% in TAC mice.It also increased LV ejection fraction and decreased chamber diameter in TAC mice.Restoration of mitochondrial morphology with cardiac-specific overexpression of YME1 L increased the contents of CD36 and CPT-1b,as well as intramyocardial storage of lipids as evidenced by the increased intramyocardial lipid droplets and TG in TAC mice,suggesting that improvement of mitochondrial dynamics increased FA utilization in the TAC heart.Conclusions1)HFD or CD36 overexpression in the myocardium increases myocardial FA utilization and ameliorates cardiac dysfunction in the failing heart.2)FA inhibts OPA1 cleavage by up-regulating the expression of PPAR-γ,which increases YME1 L expression,and thus improves mitochondrial dynamics.Taken together,we found that the suppressed FA utilization by the heart contributes to cardiac dysfunction and enhancement of FA utilization exerts protective effects against pressure overload-induced HF.Mechanically,FA plays an important role in regulation of mitochondrial dynamics through activation of nuclear receptor PPARγ which increases the expression of YME1 L.YME1L inhibits OPA1 cleavage and mitochondrial fragmentation in the heart.These findings suggested that FA utilization is essential in the maintenance of mitochondrial dynamics and enhancing cardiac FA utilization may be a promising approach for intervention of HF.