The Role And Intervention Mechanism Of Peroxisome Proliferator–activated Receptor γ Coactivator 1-α(PGC-1α) In Septic Cardiomyopathy Rat Model

Objective: Mitochondrial damage is considered one of the main pathogenetic mechanisms in septic cardiomyopathy.The heart is a continuously powered organ in need of a lot of ATP to maintain normal systolic and diastolic functions.Mitochondria are the main ATP-producing organelles,accounting for about one third of the myocardial volume;if damaged,this would be harmful on the myocardial energy supply and cardiac function.Moreover,injured mitochondria can produce increased reactive oxygen species(ROS)and other toxicants,causing further damage to cells and tissues.Peroxisome proliferator–activated receptor γ coactivator 1-α(PGC-1α),a powerful transcriptional co-activator,is critical for maintaining energy homeostasis in different organs and in various physiological and pathological states.Moreover,PGC-1α is a key regulatory gene of mitochondrial biogenesis that can also affect lysosome synthesis through transcription factor EB,and further affect autophagy.Although,PGC-1α has attracted wide attention as a potential therapeutic target in disease,few studies exist on its use in cardiovascular diseases,especially septic cardiomyopathy.In our study,we developed a rat model of septic cardiomyopathy in which PGC-1α was measured and disrupted in myocardiocytes treated with lipopolysaccharide(LPS).We observed an improvement in myocardial cell injury and explored the possible mechanisms involved.This research may provide novel ideas for the treatment of myocardial depression in sepsis.Methods: A rat model of septic cardiac dysfunction was established by intravenous administration of LPS.Using random fraction method,SPF-grade male Wistar rats were randomly divided into groups of 3h,6h,and 12 h after LPS administration,with corresponding control group set at each time point,and 6 rats in each group.Femoral artery and common carotid artery were exposed for cannulation.Biopac multi-conducting physiological recorder pressure transducer was connected to monitor the mean arterial pressure(MAP),the left ventricular end-diastolic pressure(LVEDP),the maximum rate of left ventricular pressure drop during diastole(-dp/dt max),the maximum rate of increase in left ventricular pressure during systole(+dp/dt max),and other indicators.After reaching the corresponding time points,rats in each group were anesthetized with the left ventricular muscle collected.HE staining was used to observe the damage of myocardial tissue,and the ultrastructural changes of myocardial tissue were observed under transmission electron microscopy.Flow cytometry was used to detect mitochondrial membrane potential changes.Immunohistochemistry and immunofluorescence were used to observe the expression of PGC-1α,mt TFA,LC3 B and P62.RT-PCR was used to detect the m RNA expression of PGC-1α,mt TFA,LC3 B,P62,PINK1 and Parkin,and western blot was used to observe their protein expression.H9c2 rat cardiomyocytes were cultured,and the optimal intervention concentration of LPS was determined to be 10μg/m L and the intervention time was 24 h by CCK8 method,to construct the model of septic cardiomyopathy at the cell level.The cells were intervented with ZLN005,the intervention cells were divided into control group,LPS group and LPS+ZLN005 group.Apoptosis of H9c2 cells in each group was detected by flow cytometry,protein changes of PGC-1α were detected by Western blot,and mitochondrial damage was observed by electron microscopy.Intraperitoneal injection of 15 mg/kg ZLN005 intervention septic cardiomyopathy rats model,rats were divided into control group,ZLN005 group,LPS group and LPS + ZLN005 group,Western blot detected PGC-1α protein expression,after intervention 12 h,observed myocardial pathologic structure and microstructure change under electron microscopy.Western blot and immunofluorescence tested Bcl2 and Bax protein expression in myocardial tissue,flow detection of mitochondrial membrane potential changes between groups.We used lentivirus transfection method to overexpress H9c2 cells’ PGC-1α gene,and screened by purinomycin to found stable transfection cell lines,and the transfection was verified by Western blot and immunofluorescence.After successful transfection,cells were divided into control group,LPS group and PGC-1α++LPS group.The apoptosis level of each group was detected by flow cytometry,and mitochondrial damage was monitored by detecting ATP level,ROS level,cytochrome C level and mitochondrial membrane potential.Western blot and immunofluorescence were used to detect the expression changes of PGC-1α and mt TFA proteins,and RT-PCR was used to detect the m RNA expression level of PGC-1α and mt TFA genes,as well as the changes of mt DNA in each group,so as to evaluate the activation of mitochondrial biogenesis;western blot and immunofluorescence were used to detect the protein expression change of LC3 B,P62,PINK1 and Parkin,and RT-PCR was used to detect m RNA expression level of LC3 B,P62,PINK1 and Parkin,so as to evaluate autophagy and mitophagy activation.Results: 1.Myocardial structural disorder occurred at 6 hours after LPS administration,demonstrating widened muscle fiber spacing,broken muscle fibers,with a small amount of inflammatory cell infiltration observed.The myocardial structural disorder and muscle fiber breakage were more obvious at 12 hours after LPS administration.The myocardial fiber structure was basically normal in the 3h after LPS administration group,but some mitochondria had already showed vacuolar degeneration and sputum rupture.The myofilament was partially expanded in LPS 6h group,with visible Z line,but unclear band I and band A.Mitochondria were swelling with adventitial destruction,vacuolar degeneration and tendon rupture,and number of mitochondria was decreased.The muscle fibers were abnormally disordered,mitochondrial damage was serious and the number of mitochondria decreased significantly in the 12 h LPS administration group.In the 3h experimental group,the myocardial mitochondrial membrane potential decreased.As time progressed,the number of cells with decreased membrane potential increased gradually,with statistical difference compared with the corresponding control group.The 12 h group was the peak.2.Western blot and immunofluorescence results showed that PGC-1α and downstream protein mt TFA increased significantly in the LPS 3h group,but decreased activation in the LPS 6h and LPS 12 h groups over time.Autophagy related protein LC3 B in myocardial tissue was always activated,but P62 and the key protein of mitophagy: PINK1 and Parkin,were not significantly changed.3.The expression level of PGC-1α decreased after LPS intervention in H9c2 cardiomyocytes.After ZLN005 drug treatment,PGC-1α gene expression level increased,accompanied with the improvement of apoptosis level.After ZLN005 intervention 12 h in the rat model of septic myocardiopathy,the expression level of PGC-1α in the myocardial tissue of the intervention group was significantly higher than that of the LPS group,but there was no significant difference in the general and microstructure change of the myocardial tissue between the two groups,and the level of apoptosis and the decrease of mitochondrial membrane potential were not improved.4.Lentivirus transfection with overexpressed of PGC-1α gene in H9c2 cardiomyocytes significantly improved the level of apoptosis after LPS intervention.5.After lentivirus transfection with overexpressed PGC-1α gene in H9c2 cardiomyocytes,the PGC-1α++LPS group’s apoptosis level was significantly improved after LPS intervention.Compared with the LPS group,the mitochondrial ATP level in the PGC-1α++LPS group increased,but there was no statistical significance.The ROS level and cytochrome C level decreased significantly,and mitochondrial membrane potential improved significantly.6.Compared with the LPS group,PGC-1α and mt TFA increased significantly in PGC-1α++LPS group,and mt DNA number also significantly increased.Autophagy related protein LC3 B was not significantly increased,but P62 was significantly decreased.The expression of PINK1 increased,and the m RNA of PINK1 gene increased,but there was no significant statistical difference.There were no significant changes in Parkin.Conclusions: 1.The mitochondrial damage was significantly observed in the myocardium of the model of septic cardiomyopathy,accompanied with the decrease in the number of mitochondria,and the change was getting worse over time.PGC-1α,a key protein in mitochondrial biogenesis,was significantly activated in the myocardial tissue of the model group,but the activation decreased with disease progression.2.The small molecule agonist ZLN005 can significantly increase the expression level of PGC-1α protein in H9c2 cells.Meanwhile,the apoptosis level of H9c2 cells improved significantly after the intervention of ZLN005,but it did not work in vivo.3.After the lentiviral transfection method specifically increased the expression of PGC-1α in H9c2 cells,the cells’ apoptosis level was improved compared with the LPS group.PGC-1α++LPS group also showed improved mitochondrial function,reduced mitochondrial damage and increased ATP synthesis.4.Mitochondrial biogenesis and autophagy were more effectively activated in PGC-1α++LPS group than in LPS group,and the number of mitochondrial DNA copies increased.This may be the main underlying mechanism that the reduction of LPS-induced mitochondrial damage in cardiomyocytes and the improvement of apoptosis.