Effects Of Regulation Of Mitochondrial Autophagy And Biogenesis On Isoproterenol-induced Cardiomyocyte Injury

Background:Mitochondrial quality control is critical for the development of myocardial hypertrophy,in which insufficient energy supply of mitochondria plays an important role.Studies have shown that PGC-1α is closely related to energy metabolism,but previous studies on PGC-1α have different conclusions on myocardial hypertrophy.Previous studies have found that simply increasing PGC-1αto promote mitochondrial biogenesis can promote the occurrence of dilated cardiomyopathy in mice,which may ignore the relationship between mitochondrial biogenesis and autophagy.Recent studies have proved that PINK1 is involved in mitochondrial quality control.PINK1 degrades rapidly in normal mitochondria,but accumulates in damaged mitochondria and initiates mitochondrial autophagy to clear damaged mitochondria.However,it is not clear whether PINK1 can mediate isoproterenol(Iso)-induced mitochondrial autophagy and whether the comprehensive strategy of coordinating mitochondrial autophagy and biogenesis is useful for Iso-induced cardiomyocyte injury.Objective:The changes of PINK1 and autophagy activity were measured in the model of cardiomyocyte injury induced by Iso.The effects of regulation mitochondrial autophagy and biogenesis on myocardial mitochondrial structure and function and its mechanism were explored in a cardiac hypertrophy model.Methods:Primary rat cardiomyocytes were extracted and stimulated with Iso(10uM)for 48 hours to construct a model of cardiomyocytes injury.The role of PINK1 in cardiomyocyte injury model was studied by adenovirus-mediated PINK1 overexpression.Then PGC-1α was activated by Metformin,and the damage indexes of reactive oxygen species(ROS),mitochondrial membrane potential(MMP)and cardiomyocytes apoptosis were detected in each group.Mitochondrial morphology and mitochondrial autophagy were measured by transmission electron microscopy.The autophagy-related protein LC3B,Beclinl and P62 was detected by immunoblotting.Colocalization of lysosomes and mitochondria was observed by confocal microscopy.Protein levels of PINK1,PGC-1α,TF AM and NRF1 were detected by immunoblotting.Cell activity was detected by CCK-8;ATP level was detected by luciferase method;Mitochondrial respiratory function was measured by oxygen consumption rate.Results:1.The changes of PINK1 and MFN2 in the model of cardiomyocyte injury induced by Iso stimulation:the protein level of PINK1 increased gradually with the stimulation time of Iso,reached the highest at 12 hours,and then decreased gradually after 48 hours,while the protein level of MFN2 decreased gradually with the stimulation time of Iso and maintained balance from 12 hours to 48 hours.MMP decreased,ROS level and apoptosis rate increased,and autophagy related protein expression increased.2.The effects of overexpression of PINK1 on cardiomyocyte function and expression of downstream gene:overexpression of PINK1 promoted the increase of Parkin in the model of cardiomyocyte injury induced by Iso.Compared with AD-Control+Iso group,The level of MMP increased,autophagy markers were up-regulated,ROS level and apoptosis rate decreased.Meanwhile,cell viability,ATP synthesis and mitochondrial respiratory function were improved.3.The effects of increasing the expression of PGC-1α on the function of cardiomyocytes and downstream genes expression while overexpression autophagy:Metformin can increase the expression of PGC-1α,which increased gradually with the stimulation concentration of Metformin to a certain extent.In the model of cardiomyocyte injury induced by Iso,metformin could also increase PGC-1α after overexpression of PINK1.Compared with Iso+PINK1 group,although overexpression of PGC-1α,NRF1,TFAM and mitochondrial biogenesis,the synthesis of ATP and respiratory function of mitochondria were not significantly enhanced.4.The effects of overexpression of MFN2 on cardiomyocyte function while overexpression the level of PINK1 and PGC-1α protein:Overexpression of MFN2 could increased mitochondrial fusion while increasing mitochondrial autophagy and regeneration in the model of cardiomyocyte injury induced by Iso.Compared with Iso+PINK1 group,ROS level and apoptosis rate decreased,cell viability,ATP synthesis and mitochondrial respiratory function were further improved.Conclusion:1.In the model of Iso-induced cardiomyocyte injury,the expression level of PINK1 increased in the early stage and decreased in the middle and late stages.2.The expression level of MFN2 gradually decreased in the model of cardiomyocyte injury induced by Iso.3.In the model of cardiomyocyte injury induced by Iso,the level of mitochondrial autophagy was up-regulated and the cardiomyocyte injury was aggravated.4.Overexpression of PINK 1 can inhibit the decrease of mitochondrial membrane potential induced by Iso,reduce the level of reactive oxygen species and the rate of apoptosis,and alleviate cardiomyocyte hypertrophy.5.Overexpression of PINK1 can promote the expression of autophagy related proteins and up-regulate the activity of mitochondrial autophagy.6.Increasing the expression of PGC-1α can increase the biogenesis of mitochondria.7.Metformin promotes mitochondrial regeneration in cardiomyocytes by increasing the expression of PGC-1α.8.In the model of Iso-induced cardiomyocyte injury,increasing mitochondrial autophagy and regeneration,promoting mitochondrial fusion and improving mitochondrial quality control can reduce cardiomyocyte injury and improve capacity supply.9.In the model of cardiomyocyte injury induced by Iso,MFN2 can promote mitochondrial regeneration in cooperation with PGC-1α.