Research On The Molecular Mechanism Of Nicotine-induced Cardiac Hypertrophy Through Impairing Autophagy Flux

Background and purpose:Cardiomyocytes hypertrophy is characterized by cardiomyocyte hypertrophy and is an important pathological process in the development of heart failure.Heart failure is a clinical disease with high morbidity and high mortality.In recent years,it has shown a younger trend and brought huge economic and mental pressure to society.The molecular mechanism related to heart failure has always been the focus of research,but its specific underlying molecular mechanism is not yet clear.Studies have confirmed that nicotine is an important risk factor for myocardial hypertrophy.How nicotine induces myocardial hypertrophy is not cler.Autophagy is the formation of autophagosomes by cells that form double-layer membrane coating,which coat damaged organelles or misfolded abnormal proteins to form autophagosomes,which are fused with lysosomes to form autophagolysosomes.It degrades the coating to remove harmful substances,and at the same time realizes the recycling of substances and maintains the process of normal metabolism of cells,which plays an important role in the occurrence and development of myocardial hypertrophy.Complete autophagy flow is a necessary condition for cells to maintain normal function.Cathepsin B(cathepsin B,CTSB)is the most important enzyme that degrades myocardial lysosomes in vivo,and plays a key role in the late stage of autophagic flow.Cilostazol(Cilostazol),by inhibiting the activity of PDEs,has the effect of dilating blood vessels and anti-platelets,clinically mainly used to treat peripheral vascular diseases.Studies have shown that cilostazol can treat Parkinson’s disease by regulating autophagy.Therefore,in this study,cilostazol was used to pre-treat cardiomyocytes to observe their effects on CTSB enzyme activity,nicotine-induced autophagic flow injury,and myocardial hypertrophy.So does nicotine block autophagic flow and CTSB enzyme activity?A number of studies have shown that oxidative stress is involved in the occurrence and development of myocardial hypertrophy,and reactive oxygen species(ROS)as the main oxidative intermediate products play a key role in this process.While p38MAPK/JNK is the downstream molecule of ROS,ROS accumulation will activate p38MAPK/JNK.It has been confirmed that the activation of JNK is related to the blockage of autophagic flow.From this we speculate:Does nicotine block autophagic flow through the ROS/p38MAPK/JNK signaling pathway,thereby inducing cardiac hypertrophy?Based on the above background,this paper aims to explore the role of nicotine in regulating myocardial hypertrophy by regulating autophagy and the underlying molecular mechanism to provide new ideas and potential targets for clinical prevention and treatment of heart failure and cardiac hypertrophy.Research content:1.Nicotine impaired autophagy flux and induced cardiac hypertrophy.2.Nicotine blocked autophagy flux through inhibiting CTSB enzyme activity.3.Nicotine-induced autophagic impairment was related to ROS/p38MAPK/JNK signaling.4.Nicotine regulated autophagy flux and cardiac hypertrophy through CTSB/ROS/p38MAP/JNK feedback loop.Objective:Cardiomyocytes hypertrophy is an important pathological process of heart failure.Studies have confirmed that nicotine is an important risk factor for cardiomyocyte hypertrophy.Autophagy is a process that maintains the normal metabolism of cells by degrading waste proteins and damaged organelles in cells,and plays an important role in the development of cardiomyocyte hypertrophy.Cathepsin B is the most important enzyme that degrades substrates in lysosomes of cardiomyocytes,and plays a key role in the late stage of autophagic flux.Cilostazol(Cilostazol),by inhibiting the activity of PDEs,has the effect of dilating blood vessels and anti-platelets,which mainly used to treat peripheral vascular diseases clinically.Studies shown that cilostazol can treat Parkinson’s disease by regulating autophagy.However,the role and relative mechanisms of cilostazol in nicotine-induced autophagy impairment and cardiomyocyte hypertrophy have not been reported.Therefore,in this study,cilostazol was used to pre-treat cardiomyocytes to observe their effects on CTSB enzyme activity,nicotine-induced autophagic flow injury,and myocardial hypertrophy.This subject aims to explore how nicotine can induce the potential molecular mechanism of myocardial hypertrophy by blocking autophagic flow,and provide new ideas and targets for clinical treatment of myocardial hypertrophy..Method:The cells used in this project are neonatal rat ventricular myocytes(NRVMs).1.Extraction of primary neonatal rat ventricular myocytes(NRVMs)from Wistar rats[1].2.Effect of nicotine on cardiac hypertrophy.(1)Hematoxylin-eosin staining was used to detect the surface area of cardiomyocytes;(2)Quantitative polymerase chain reaction(qRT-PCR)was used to test the mRNA level of myocardial hypertrophy marker:ANP,BNP and β-MHC after nicotine treatment.3.Detection of autophagyAutophagy flux impairment is an important part of cardiac hypertrophy.Therefore,on the basis of clarifying that nicotine can induce cardiac hypertrophy,we studied the role of autophagy in this process.(1)Transmission electron microscope(TEM)detected the effect of nicotine on autophagosomes and autophagolysosomes in cardiomyocytes;(2)Western blot was used to detect the effect of nicotine on autophagy-related proteins and autophagosome marker proteins;(3)Transfected cardiomyocytes with ADV-RFP-GFP-LC3 to observe the changes of autophagy flux in cardiomyocytes under nicotine treatment.4.Effect of nicotine on lysosomal function.In order to investigate whether nicotine-induced autophagy flux impairment affects the function of lysosomes,we next examined the changes of cathepsin B(CTSB)enzyme activity in lysosomes of cardiomyocytes.(1)Magic Red cathepsin activity kit was used to detect the CTSB enzyme activity in cardiomyocytes after nicotine treatment,which reflected the effect of nicotine on lysosomal function,bafilomycin(Baf Al)or rapamycin and PP242 as negative or positive controls.After pretreatment with CTSB activator cilostazol,the activity of CTSB was also detected;(2)qRT-PCR was used to detect whether nicotine affects its enzyme activity by interfering with CTSB mRNA levels,cilostazol and bafAl treated groups were set at the same time;(3)Western blot was used to detect the changes of CTSB and its precursor protein pro-CTSB protein level.5.The relationship between CTSB enzyme activity and autophagic impairment and cardiac hypertrophy.(1)Western blot technique was used to detect the change of autophagy marker LC3 II and substrate p62 after cilostazol pretreatment;(2)Transfection of ADV-RFP-GFP-LC3 was used to observe the effect of cilostazol on autophagy flux;(3)qRT-PCR technology was used to detect the effect of cilostazol on the mRNA level of cardiac hypertrophy marker protein.6.The homeostasis of intracellular oxidative stress is related to autophagy.(1)Applied 2,7-dichlorofuorescin diacetate(DCFH-DA)probe labeled cardiomyocytes to detect the effect of nicotine on the level of reactive oxygen species in cardiomyocytes,and NAC treatment group was set as positive control;(2)In order to further clarify the role of oxidative stress in nicotine-induced myocardial hypertrophy,we applied NAC,an inhibitor of oxidative stress,and used western blot to detect the effect of NAC on autophagy marker protein;(3)qRT-PCR was used to detect the effect of NAC on cardiac hypertrophy marker protein;(4)ADV-RFP-GFP-LC3 transfection was used to detect the effect of cilostazol on autophagy flux.7.Detection of the relationship between intracellular reactive oxygen species and MAPKs signaling pathway.(1)Western blot was applied to detect the effects of nicotine treatment alone and co-treatment of nicotine with SB203580(p38MAPK inhibitor)and SP600125(JNK inhibitor)on corresponding signal molecules and autophagy marker proteins;(2)Transfection of ADV-RFP-GFP-LC3 was used to observe the changes of autophagy flux after treatment with nicotine and pathway inhibitors;(3)qRT-PCR was used to detect the effect of inhibitors on of cardiac hypertrophy markers under nicotine treatment;(4)CTSB enzyme activity kit to detect changes in CTSB enzyme activity after inhibitor treatment;(5)Reactive oxygen detection kit was applied to detect the effect of inhibitors on the level of reactive oxygen in cardiomyocytes.8.The relationship between CTSB enzyme activity and ROS/p38MAPK/JNK pathway.In order to clarify the role of CTSB enzyme activity in nicotine-induced myocardial hypertrophy,we applied cilostazol to treat cardiomyocytes to activate CTSB enzyme activity.(1)ROS detection kit was used to detect ROS level of cardiomyocytes;(2)Western blot was applied to detect the changes of p38MAPK and JNK phosphorylation levels.Results:1.Different concentrations(0,1,10,100,500μM)of nicotine treatment significantly induced cardiac hypertrophy.(1)HE staining results showed that the surface area of cardiomyocytes increased significantly after treatment of nicotine compared with the control group(Figure 1 A).(2)qRT-PCR results showed that myocardial hypertrophy markers ANP,BNP andβ-MHC mRNA levels were also increased significantly.(Figure 1B-D)2.Nicotine induced autophagy impairment in cardiomyocytes.(1)Transmission electron microscopy results showed that,the number of autophagosomes in cardiomyocytes increased significantly after nicotine treatment(Figure 2A).After treatment with different concentrations of nicotine,the levels of autophagy-related protein LC3Ⅱ and substrate p62 were significantly increased,similar to those in the bafilomycin-treated group,LC3Ⅱ level increased in the rapamycin-treated group,while the substrate p62 expression level was down-regulated(Figure 2B).(2)After co-treatment with nicotine and bafilomycin,the blockage of autophagy flux was significantly worse than that treated with nicotine alone,but there was no significant difference compared with bafilomycin alone(Figure 2C).(3)After ADV-RFP-GFP-LC3 transfection,red and green fluorescence in cardiomyocytes can be seen under the microscope,which are increased compared with the blank control group,similar to bafilomycin,red fluorescence increased and green fluorescence weakened after rapamycin treatment(Figure 3A).(4)The level of autophagy lysosomal marker protein LAMP2 protein decreased,while there was no significant change in lysosomal marker protein LAMP1(Figure 3B).3.Nicotine inhibited cathepsin B(CTSB)activity in cardiomyocytes.PP242 as a mTORC1 inhibitor and rapamycin were set as positive controls,and bafilomycin was set as negative control.(1)The results of CTSB enzyme activity test showed that after treatment with different concentrations of nicotine,CTSB enzyme activity decreased in a concentration-dependent manner,which was consistent with the change after treatment with bafilomycin;rapamycin and PP242 treatment could significantly increase enzyme activity(Figure 4A).Cilostazol as an activator of CTSB enzyme activity can significantly improve the nicotine-induced decrease in CTSB enzyme activity(Figure 4B).(2)qRT-PCR results showed that nicotine did not affect the CTSB mRNA level,and there was no significant change after treatment with cilostazol(Figure 4C).(3)Western blot results show that nicotine can increase CTSB protein levels.At the same time,higher concentrations of nicotine can also increase CTSB precursor protein levels(Figure 4D-E).4.Cilostazol regulated autophagic flux by activating CTSB enzyme activity and relieves cardiac hypertrophy.(1)Cilostazol can improve nicotine-induced autophagy flux blockage:LC3Ⅱ and substrate p62 were both down-regulated(Figure 5A).(2)The results of ADV-RFP-GFP-LC3 transfection showed that the green fluorescence in the cells was significantly reduced after cilostazol pretreatment(Figure 5B).(3)Cilostazol relieves nicotine-induced cardiac hypertrophy.qRT-PCR results showed that cilostazol reduce the expression level of myocardial hypertrophy marker protein significantly(Figure 6A-C),but only increased CTSB mRNA levels at higher concentrations(500μM),and had no effect at 100μM(Figure 6D).5.Nicotine induced the accumulation of reactive oxygen species in cardiomyocytes.(1)After treatment with different concentrations of nicotine,the level of reactive oxygen in cardiomyocytes increased significantly,and NAC(reactive oxygen scavenger)group was set as positive control(Figure 7A).(2)NAC treatment reduced the expression level of LC3 Ⅱ and p62 in cardiomyocytes(Figure 7B-D).(3)The results of qRT-PCR showed that the markers of myocardial hypertrophy were significantly downregulated in the NAC and nicotine co-treated group compared with the nicotine-treated group alone(Figure 7E).6.Reactive oxygen accumulation was related with activation of MAPKs signaling pathway.(1)Nicotine activated p38MAPK/JNK signaling pathway in cardiomyocytes(Figure 8A-C),NAC,SB203580(p38MAPK inhibitor)and SP600125(JNK inhibitor)\significantly reduced the phosphorylation level of the corresponding signal molecules,SB203580 and SP600125 significantly decreased the levels of LC3 Ⅱ and p62(Figure 8D-F,9A-B).(2)ADV-RFP-GFP-LC3 transfection was used to detect,the green fluorescence in cardiomyocytes decreased after inhibitors treatment(Figure 10A).(3)SB203580 and SP600125 significantly reduced ANP,BNP,β-MHC mRNA levels(Figure 10B-C).(4)After treatment with NAC,SB203580 and SP600125,the intensity of ROS fluorescence in cardiomyocytes decreased significantly(Figure 11).7.CTSB enzyme activity and ROS/p38MAPK/JNK signaling pathway jointly regulate autophagy flux and cardiac hypertrophy(1)NAC,SP600125 and SB203580 could improve the nicotine induced reduction of CTSB enzyme activity.(2)Cilostazol significantly reduced the accumulation of ROS in cardiomyocytes,(3)Western blot results showed that cilostazol can reduce the phosphorylation level of p38MAPK/JNK.That is:CTSB and ROS/p38MAPK/JNK jointly regulate autophagic flow and participate in cardiac hypertrophy.Conclusion:Collectively,these findings firstly demonstrated that nicotine impaired autophagy flux through inhibiting CTSB activity,Cilostazol,as an activator of CTSB enzyme,could alleviate nicotine induced cardiomyocytes hypertrophy through restoration of autophagy flux by activation of CTSB and inhibiting ROS/p38/JNK pathway,which exhibiting a feedback loop on regulation of autophagy and cardiomyocytes hypertrophy.These results will further improve the theoretical system of the molecular mechanism of nicotine-induced cardiac hypertrophy,and provide new standpoint for clinical treatment of cardiac hypertrophy.