Study On The Molecuiar Mechanism Of Nicotine-induced Mitochondrial Dynamics Disorder And Cardiomyocyte Apoptosis

Background:Nicotine,as one of the main components of cigarettes,is an important risk factor for cardiovascular disease.Mitophagy,a selective autophagy different from autophagy,can remove aging,damaged and dysfunctional mitochondria in time,which plays an irreplaceable role in maintaining energy supply and realizing material recycling.The blockage of mitophagy will bring irreversible damage to cell growth,metabolism,signal transduction and other physiological processes.Our previous results show that nicotine impaired autophagy,causing cardiac dysfunction.However,it is not clear whether nicotine affects mitophagy in neonatal rat ventricular myocytes.Cathepsin L(CTSL)is a member of the papain family,which mainly exists in lysosomes and usually plays an important role in the later stage of autophagy.Whether cathepsin L is involved in mitophagy affected by nicotine remains to be studied.Torin1,an mTOR inhibitor,was used to pretreat cardiomyocytes damaged by nicotine.The activity of CTSL,mitophagy and mitochondrial dynamics were observed after Torin1 pre-treatment.There is a close relationship between mitophagy and mitochondrial dynamics.Mitochondrial fragmentation is accompanied by the decrease of mitochondrial membrane potential and precedes the formation of mitophagosomes.lt also has also been reported that when mitochondrial division-related factors are deleted,highly fused mitochondria can lead to mitophagy.Mitochondrial fusion can promote protective mitophagy.However,the relationship between the changes of mitophagy induced by nicotine and mitochondrial dynamics in cardiomyocytes is not clear.Therefore,more studies are needed to explore the crosstalk between mitophagy and mitochondrial dynamics.Our previous work showed that nicotine can induce abnormal accumulation of reactive oxygen species(ROS),damaging cardiac function through ROS/p38/JNK pathway.Therefore,we speculate that nicotine damages mitophagy through ROS/p38/JNK,which is closely related to mitochondrial dynamics.However,there are few reports about this and the specific mechanism needs to be further studied.According to the latest research progress,we speculate that nicotine may damage mitophagy through ROS/p38/JNK/CTSL and mTOR,resulting in mitochondrial dynamics disorder and increase of cardiomyocyte apoptosis rate.Torin1 can inhibit the above process.Objective:The purpose of this study is to explore the effects and mechanisms of nicotine on mitophagy,mitochondrial dynamics and apoptosis in cardiomyocytes,and to explore the relationship among them.Research contents:1.Nicotine damaged mitophagy through ROS/p38/JNK pathway.2.Nicotine impaired mitophagy through inducing abnormal accumulation of reactive oxygen species and inhibiting the activity of CTSL.3.Nicotine up-regulated the level of mitochondrial fission and inhibited mitochondrial fusion,causing mitochondrial dynamics disorder.4.Nicotine induced cardiomyocyte apoptosis,resulting in cardiomyocyte injury.Methods:1.Extraction of neonatal rat ventricular myocytes(NRVMs)2.Effect of nicotine on mitophagy3.Effect of nicotine on mitochondrial reactive oxygen species(mito-ROS)and CTSL activity4.Detect the change of Mitochondrial dynamics5.Detect cardiomyocyte apoptosis-related proteinsResults:1.Nicotine impairs Parkin/PINK1 mediated mitophagic flux in the late stage of mitophagy.2.Nicotine stimulation caused imbalance of mitochondrial fusion and fission.3.Nicotine induced mitophagy flux impairment through decreasing CTSL activity and ROS mediated p38/JNK pathway.4.Hindered mitophagic flux via declined CTSL activity and activation of ROS mediated p38/JNK pathway was contributed to nicotine caused mitochondrial fusion and fission imbalance.5.Nicotine induced cardiomyocytes apoptosis via ROS accumulation and mitochondrial fission in vitro.Conclusion:Our study showed that nicotine induced mitochondrial dynamics disorder through blocked mitophagic flux via repressing CTSL activity and activating ROS-mediated p38/JNK pathways,while Torin1 could rescue NRVMs by restoring CTSL activity and damaged mitophagic flux.These results may reveal a close link between mitophagy and mitochondria dynamics,which also provide new evidence of effects of nicotine on mitochondria dynamics in NRVMs.