The Roles Of N-terminal Truncated Peroxisome Proliferator Activated Receptor Gamma Coactivator-1 Alpha (NT-PGC-1α) In Cardiac Fatty Acid Oxidation

Background:It appears obvious that heart failure is a symptom of cardiac energy depletion,and myocardial metabolic reconstruction are involved in its onset and progress which might be a potential target for heart failure therapy.Given to the crucial role of mitochondria in the heart,peroxisome proliferator activated receptor gamma coactivator-la(PGC-la)which plays key roles in energy metabolic is a versatile transcription factors coactivator that combined with various nuclear receptors to satisfy the physiological demand of the heart.However,studies on PGC-la are controversial and these findings might suggest that the therapeutic window of PGC-la are narrow and it is difficult to regulate its physiological effects precisely.N-terminal truncated PGC-la(NT-PGC-la)is a kind of alternative splice variants of PGC-1α which predominantly distribute in cytoplasm and only contains 270 amino acid from N terminal.Although NT-PGC-1α is shorter than(Full-length)FL-PGC-la,parts of the binding domains of transcription factors were preserved which made it reserved most of its physiological function.Together,we hypothesis that NT-PGC-la can compensate energy deficiency by shuttling into nucleus and co-activating nuclear receptor,and further enhancing downstream target genes transcription.We explored our hypothesis in 4 parts.Firstly,we aimed to detect the expression of PGC-1α and NT-PGC-1α in MI-induced heart failure mice by using western blotting and real time PCR.Secondly,we examined the influence of NT-PGC-la on neonatal rat cardiomyocytes(NRCMs),and further investigated the signal pathways and possible mechanism involved in NT-PGC-la.Thirdly,we explored the role of CRM1 in regulating the distribution of NT-PGC-la.Finally,we explored the influence of CRM1 inhibitor on MI-induced heart failure mice.Objective:To detect the expression of PGC-1α,NT-PGC-la and the enzymes involved in fatty acid oxidation(FAO)in myocardial infarction induced heart failure mice.In addition,we would explore the influence of NT-PGC-1αoverexpression on energy metabolism and the pathway involved in these process.Besides,we would also investigate the mechanism of CRM1 inhibitor on the regulation of NT-PGC-1α.Method:The present study was performed in 4 parts including:(1)the alteration of NT-PGC-1α in mice post MI;(2)the influence of NT-PGC-1α on mitochondrial energy metabolism;(3)the regulation of CRM1 on the shuttling of NT-PGC-1α;(4)the application of CRM1 inhibitor in vivo.The detail projects are as follows:1.Heart failure mice model was established by permanent ligation of left coronary artery under the monitoring of electrocardiograph(ECG),then cardiac function,the expression of PGC-1α,NT-PGC-1α and FAO related enzymes were detected by echocardiography,qPCR,Western blotting and immuohistochemical staining,respectively.2.By using nuclear and cytoplasm protein separation assay,we separated nuclear and cytoplasm protein from heart tissue of C57BL/6j mice.Then we detected PGC-1α and NT-PGC-la by western blotting.Besides,immunofluorescence was also performed in NRCMs with or without the expression of mCherry-NT-PGC-1α.3.NRCMs overexpress NT-PGC-1α were exposed to angiotensinll or phenylephrine,then ATP generation,ROS generation and mitochondrial membrane potential were detected.By using Co-immunoprecipitation technology,we explored the interaction of NT-PGC-la.Furthermore,we explored the role of NT-PGC-1α in PE treated cells and detected the lipid droplets and extracellular oxygen consumption when clutured in hpid medium.4.NRCMs transfected with AdV-NT-PGC-1α were performed by RNA extraction and qPCR process.CCK8 assay was performed to detect the viability of cells exposed to CRM1 inhibitor Selinexor.Then cells over express NT-PGC-1α and NLS-NT-PGC-1α were handled by Selinexor in an appropriate concentration and observed by laser confocal microscopy.Followed by NLS-NT-PGC-1α overexpression and treated with Selinexor,the levels of related mRNA were detected by qPCR.5.Theanti-hypertrophy effeect of CRM 1 inhibitor Selinexor were confirmed by phalloidin staining and western blotting.After that,wild type mice and FL-PGC-1α(-/-)mice were subjected to coronary ligation and Selinexor treatment.One and four weeks after the operation,echocardiography and survival analysis were carried on.Finally,we employed Histone H3 acetylation detection kit to analysis the effect of Selinexor and TSA.Results:1.Heart failure mice induced by permanent myocardial infarction presented a drop in PGC-1α,NT-PGC-la and FAO related enzymes.2.(1)Immunofluorescence and western blotting showed that PGC-1α and NT-PGC-1α are predominantly distribute in nucleus and cytoplasm,respectively.(2)NRCMs exposed to AngⅡ or PE displayed a decrease in ATP generation,and NT-PGC-1α can ameliorate the declining of ATP.(3)NT-PGC-la overexpression abolished PE induced mitochondrial membrane potential reduction and ROS generation.(4)NT-PGC-1α overexpression active PPAR-α pathway to increase Acadm and CPT2,which alleviate the inhibition PPAR-a by PE.(5)NT-PGC-1α overexpression contribute to the decrease of lipid droplets and the increase of oxygen consumption.3.(1)Nuclear localization sequence(NLS)and Selinexor led to redistribution of NT-PGC-1α.(2)Selinexor manifested the inhibition of P-MHC expression and cell hypertrophy,and further actived downstream target genes in NLS-NT-PGC-1αoverexpression cells.4.(1)Selinexor did not affect the survival rate and cardiac function of WT mice and FL-PGC-1α(-/-)mice post MI in one month.(2)Selinexor failed to active downstream target genes in wild type NRCMs.Conclusion:(1)C57/BL6 mice which underwent coronary artery ligation were accompanied with a declining in PGC-la,NT-PGC-1α,SOD2 as well as enzymes of FAO,which suggest the potential roles of NT-PGC-1α in FAO and anti-oxidative stress.(2)NT-PGC-1α and PGC-la distribute in cytoplasm and nuclear in cardiomyocyte,respectively.(3)NT-PGC-la overexpression ameliorates AngⅡ or PE induced ATP reducing,mitochondrial membrane potential declining and ROS generation.(4)NT-PGC-1α overexpression can enhance fatty acid oxidation via PPAR-α pathway.(5)NT-PGC-1α overexpression improves extracellular oxygen consumption and weakens lipid droplet accumulation in lipid medium.(6)Admission of Selinexor and NLS fusion expression boost nuclear localization of NT-PGC-1α,then further active downstream target.(7)Selinexor shows anti-hypertrophy effects but do not improve ejection function in MI mice.(8)Selinexor activates downstream target of PGC-1α only under the condition of NT-PGC-1α overexpression.