Inhibition Of ALCAT1 Improves Myocardial Mitochondrial Dysfunction In Myocardial Infarction And Its Molecular Mechanism

Backgrounds:Myocardial infarction（MI）is an extremely high mortality and disability rate of CHD.MI is characterized by myocardial cell death attributable to prolonged myocardial ischemia.Despite advances in clinical and pharmacological interventions,MI represents the leading cause of morbidity and mortality worldwide.Although a variety of adjunctive agents to reduce MI,such as β-blockers,have been tested in clinical settings,currently there is no effective treatment for MI.Consequently,despite the improvements in time －to-reperfusion and the use of evidence-based therapies in the past decade,in-hospital mortality for MI has not declined.Moreover,the precise molecular mechanisms leading to cardiac dysfunction and adverse remodeling in MI remain poorly understood,which has hindered ongoing efforts to develop more effective treatments for this debilitating disease.Therefore,the identification of novel therapeutic targets that improve cardiac function in patients with Mi-induced heart failure remains a major priority.Cardiolipin（CL）is a signature phospholipid of mitochondria and is required for the maintenance of normal mitochondrial function,including membrane structure,dynamics;,oxidative phosphorylation,and mitophagy.The biosynthesis disorder and pathological remodeling of CL is invoveled in severe dieases,such as obesity,type 2 diabetes,fatty liver disease,and Parkinson’s disease.ALCAT1 is an acyltransferase that catalyzes the pathological remodeling of CL with aberrant acyl compositions,including enrichment of docosahexaenoic acid（DHA）in CL,leading to tetralinoleoyl cardiolipin（TLCL）depletion and mitochondrial dysfunction.Moreover,up-regulated ALCAT1 protein expression by oxidative stress is implicated in the mitochondrial etiology of various aging-related diseases.Furthermore,ALCAT1 is most abundantly expressed in the heart,and therefore is poised to modulate cardiac function through pathological CL remodeling.The underlying connection remains elusive.Objectives:（1）To investigate the ablation of ALCAT1 or inhibition by Dafa improves the survival rate and the onset of MI-induced dilated cardiomyopathy and cardiac dysfunction.（2）To investigate the ablation or pharmacology inhibition of ALCAT1 by Dafa prevents the onset of MI-induced oxidative strss,inflammation and apoptosis.（3）To evaluate the ablation or pharmacology inhibition of ALCAT1 by Dafa restores the mitochondrial dynamics in response to MI.Methods:（1）In vivo experiments:8-week old male C57BL/6 wild-type（WT）and ALCAT1 gene deletion（KO）mice were subjected to the left anterior descending coronary artery（LAD）sugery to induced Ml.Sham-operated mice which underwent the operation without ligation served as control.C57BL/6 mice were orally gavaged with vehicle（5%CM-cellulose,Sigma）or ALCAT1 inhibitor（Dafa,10 mg/kg）twice daily after MI surgery for 4 consecutive weeks.（2）In votro experiments:H9c2 cells were obtained from ATCC（CRL-1446）.H9c2 vector（Vec）,H9c2 ALCAT1 overexpression（AL）and ALCAT1 gene knockout（KO）cells mediated by the CRISPR/Cas9 system were cultured in DMEM with 10%heat-inactivated fetal bovine serum,1%penicillin,and streptomycin and maintained in 95%air plus 5%CO2 at 37℃.For hypoxia treatment,cells were transferred to a hypoxia incubator（Thermo Electron Corporation,3130）with 5%CO2 and 1%O2 for indicated time（0,0.5,1,2 hrs）.Mito Q（0,1,5 μM）and ALCAT1 inhibitor（Dafa,3 μM）were used for the treatment H9c2 cells in hypoxia experiments for 3 hrs.The neonatal cardiomyocytes were then diluted to 1x106 cells/ml and plated in a 24well Seahorse plate for analysis.After 5 days,the primary neonatal cardiomyocytes were treated with 1%O2 in a hypoxia incubator（5%CO2 and 1%O2）or in the presence or absence of Dafa（3 μM）for 6 hrs.（3）Analysis methods:Cardiac function were analyzed by echocardiography.The size of cardiomyocytes were evaluated by WGA starining.Hematoxylin and Eosin（H＆E）,and Masson’s trichrome staining was performed.TUNEL staining was used to analysis apoptosis.Mitochondrial OCR level was evalusated by Seahorse.The protein expressions of ALCAT1,HIF-1α,HIF-1β,VHL,TXNIP,NLRP3,Bax,C-casp3,DRP1,OPA1,MFN1 and MFN2 were detected by Western Blot.The mRNA levels of Anf,Bnp,β-Mhc,Col1a1,Col3a1,Tnf,Nfkb,Il-1b and Il-6 were detected by RT-PCR.Results:（1）The LAD surgery led to a high mortality rate in WT control mice,likely as a consequence of heart failure caused by MI.In support of this notion,ablation and pharmacological inhibitor of ALCAT1 not only improved the survival rate,but also mitigated MI-induced dilated cardiomyopathy,as evidenced by changes in heart-weight to bodyweight ratio（HW/BW）and heart morphology which was analyzed by hematoxylin and eosin（H＆E）staining of LV sections.（2）We further demonstrated that MI-induced dilated cardiomyopathy also led to bradycardia and LV dysfunction in WT mice,as shown by a representative M-model echocardiogram and by decreased levels of heart rate（HR）,LV ejection fraction（LVEF）,and LV fractional shortening（LVFS）.ALCAT1 deficiency and pharmacological inhibition not only normalized the heart morphology and HR,but also prevented the loss of LVEF and LVFS induced by MI.ALCAT1 deficiency and pharmacological inhibition also restored levels of LV posterior wall end-diastole and end-systole（LVPWd and LVPWs）,interventricular septal end-diastole and end-systole（IVSd and IVSs）,LV internal diameter end-diastole and end-systole（LVIDd and LVIDs）,and LV volume end-systole and end-diastole（LV volume s and LV volume d）in MI mice.（3）ALCAT1 deficiency and pharmacological inhibition also significantly decreased mRNA expression levels of key biomarkers associated with cardiac hypertrophy in the heart of MI mice,including atrial natriuretic factor（Anf）,brain natriuretic peptide（Bnp）,and β-myosin heavy chain（β-Mhc）as demonstrated by results from RT-qPCR analysis.（4）Moreover,ablation of ALCAT1 and pharmacological inhibition also significantly decreased the mRNA expression levels of both Col1al and Col3a1 in response to MI.ALCAT1 deficiency and pharmacological inhibition significantly attenuated the excessive accumulation of collagen fibers in the heart,as demonstrated by decreased collagen volume fraction（CVF%）and by results from Masson’s trichrome staining of LV samples of MI mice.（5）Ablation of the ALCAT1 or pharmacological inhibition by Dafa significantly attenuated the expression of both HIF-1α,HIF-1β,and key biomarkers for inflammation and apoptosis.（6）Ablation of the ALCAT1 or pharmacological inhibition by Dafa significantly attenuated the levels of both reactive oxygen species（ROS）and malondialdehyde（MDA）.（7）ALCAT1 deficiency or inhibition by Dafa also attenuated mRNA expression levels of several pro-inflammatory cytokines,including Tnf,Nfkb、Il-1b and Il-6 in the heart of MI mice.（8）MI also upregulated the expression of TXNIP,NRLP3,Bax,and cleaved-caspase 3（C-cas 3）,as shown by results from western blot analysis,implicating a role of hypoxia in myocardial inflammation and apoptosis.Ablation or pharmacological inhibition of ALCAT1 by Dafa also mitigated myocardial apoptosis,as demonstrated by the results from TUNEL staining of apoptotic cells in the LV of the heart samples from MI mice.（9）MI caused remarkable disarray of mitochondrial morphology,including mitochondrial fragmentation,swelling,and loss of cisterna structure,as evidenced by the results from EM analysis.ALCAT1 deficiency and pharmacological inhibition not only restored mitochondrial dynamics,but also significantly increased mtDNA copy number.（10）The onset of MI-induced cardiac hypertrophy led to a profound loss in mitochondrial respiration,as evidenced by decreased mitochondrial citrate synthase（CS）activity and oxygen consumption rate（OCR）.Again,ALCAT1 deficiency or inhibition by Dafa not only restored CS activity,but also OCR level in the LV of MI mice or in primary neonatal cardiomyocytes.（11）MI also significantly increased DRP1 protein expression in the heart of MI mice.Surprisingly,MI also significantly upregulated the expression of OPA1,MFN1,and MFN2,likely as a consequence of excessive mitochondrial remodeling in response to mitochondrial damage.In support of this notion,ablation or inhibition of ALCAT1 not only attenuated DRP1 expression,but also normalized expression levels of OPA1,MFN1,and MFN2 to those of the sham controls.（12）In response to mitochondrial fission,DRP1 translocates from cytosol to mitochondria,a key process required for mitochondrial fission.Indeed,MI significantly stimulated mitochondrial DRP1 translocation,as evidenced by results from subcellular fractionation analysis.In further support of the role of ALCAT1 in promoting mitochondrial fission,ablation or inhibition of ALCAT1 by Dafa significantly attenuated these defects.（13）The results showed that the onset of MI induced heart failure not only caused depletion of TLCL,but also significantly increased DHA content in the heart of MI mice.In final support of ALCAT1 in mitochondrial dysfunction in MI,ablation or pharmacological inhibition of ALCAT1 by Dafa not only restored TLCL level,but also significantly decreased DHA content in CL,common defects associated with heart failure and various aging related diseases.（14）ALCAT1 overexpression significantly upregulated HIF-1α expressions in H9c2 cells with stable overexpression of the enzyme,whereas ALCAT1 deficiency significantly attenuated these expressions in response to hypoxia in H9c2 cells with ablation of ALCAT1 gene mediated by the CRISPR/Cas9 system.Additionally,treatment of cardiomyocyte cell line H9c2 with mitoquinone（Mito Q）,a mitochondrial targeted antioxidant,significantly inhibited hypoxia-induced HIF-1α expression in H9c2 cells with stable overexpression of ALCAT1 or the empty vector.Moreover,pharmacological inhibition of ALCAT1 by Dafa also inhibited HIF-1α expression in response to hypoxia,suggesting that ALCAT1 mediates hypoxia-induced damage to the heart in part by promoting oxidative stress.（15）Mito Q and ALCAT1 inhibitor Dafa signidicantly inhibited hypoxia-induced HIF-1α expression in neonatal cardiomyocytes.Conclusions:（1）Ablation or pharmacological inhibition of ALCAT1 by Dafa not only prevents high mortality,but also effectively mitigates MI-induced LV hypertrophy,fibrosis,and contractile dysfunction.（2）Ablation or pharmacological inhibition of ALCAT1 by Dafa significantly decreases the HIF-1α expressions,also effectively mitigated MI-induced oxidative stress and lipid peroxidation,and prevents inflammation and apoptosis.（3）Ablation or pharmacological inhibition of ALCAT1 not only prevented mitochondrial fragmentation and dysfunction,but also mitochondrial translocation of DRP1.Ablation or pharmacological inhibition of ALCAT1 not only restored TLCL levels to that of the sham control mice,but also significantly decreased DHA content in CL.