Modulatory Effect Of Drp1 On VDAC1 In Cardiac Hypertrophy

Background:Pathlogical cardiac hypertrophy is cardiac adaptive response to cardiomyocyte damage caused by a series of pathological factors including left ventricular afterload,mechanical strain and activation of neurohumoral system.This pathological process is characterized by the increase in cardiomyocyte size.It’s common in multiple heart diseases and is an independent risk factor for the death of cardiovascular diseases.Although various signal pathways are involved in the process of myocardial hypertrophy,the underlying pathogenesis has not yet been full illustrated.Therefore,further elucidation of the mechanism of cardiomyocyte hypertrophy even apoptosis is of great significance for clinical practice.Mitochondria are bilayer organelles that are the “powerhouse” of cells,generating ATP through a series of biological process(such as oxidative phosphorylation).Mitochondrial dysfunction is an important feature of many diseases.Previously,it was considered that mitochondria were static and isolated organelles.However,it is now clear that mitochondria exist in dynamic networks,in which they balance between dividing(fission)and joining together(fusion)and move about the cell(trafficking),this process is termed mitochondrial dynamics.It has been clear that mitochondrial dynamics is regulated by a number of highly conserved guanosine triphosphatases(GTPases).Mitochondrial fission is controlled by dynamic-related protein 1(Drp 1)interaction with other factors,such as Mid51,Mid49.Mitochondria in mature cardiomyocytes are mainly distributed in the interfibrillar,subsarcomembrane and perikaryonic areas,restricting of mitochondrial movement.Palisade arrangement is less influenced in Drp1 or Mfn2 knock out adult cardiomocytes,indicating that mitochondria dynamics is rare in adult cardiomyocytes.However,the abundance of Drp1 is high in heart,brain and muscle,besides,Drp1 knock out is lethal in adult heart.Therfore,we hypothesis that Drp1 may carry out novel fission-independent role in heart.Drp1 consists of four different functional domains,including the GTPase domain,the mediate domain,the variable domain and the effector domain.As a kind of cytoplasmic protein,Drp1 could transfer to mitochondria after receiving the signal,forming oligomers around the outer membranes.Current evidence indicated that Drp1 mediated mitochondria fission is a main feature of cardiac hypertrophy.However,other studies indicated that in the early stage of cardiac hypertrophy,the expression of Drp1 increased to maintain the autophagy flux,alleviating the Deterioration of cardiac function.All these evidences illustrated that it is not yet very clear about the role of Drp1 in cardiac hypertrophy.It’s reported that mitochondria fission mainly occurs at mitochondria-associated membranes mitochondria-associated membranes(MAMs),a specific domain between endoplasmic reticulum and mitochondria outter membrane.MAMs play key roles in calcium transformation,lipid transformation etc.There are some evidences indicated that Drp1 regulated FUNDC1 at MAMs,regulating mitophagy.In addition,mitochondria fission could mediate VDAC1 oligomerization at MAMs in ischemia reperfusion injury model.Although many evidences implied that Drp1 plays a role in MAMs,there is no data of the characteristics of Drp1 in cardiac hypertrophy.Taken together,in this study we aim to explore the role of Drp1 in myocardial hypertrophy and its possible regulation mechanism.Objective:(1)To explore the expression of Drp1 in different tissues and MAMs of cardiomyocytes.(2)To observe the expression changes of Drp1 in the pathological processes of cardiac hypertrophy.(3)To determine the molecules interacting with Drp1 on MAMs and the underlying sites.Explore the possible mechanisms in this process.Materials and Methods:1.Detect the Drp1 levels in different tissues and its impact on mitochondria morphology.(1)Measurement of Drp1: Western-blot and Real-time PCR were employed to detect the gene and protein levels of Drp1 in different tissue.(2)The impact of Drp1 decrease on mitochondria: Treat WT or Drp1 KO cardiomyocytes with Drp1 K38 A,sh Drp1 adenovirus or treat with Mdivi-1(10umol/L)for 24 hours.Observe the mitochondria morphology with Confocal.(3)Detect the mitochondria morphology in MEF after Drp1 restoration: Culture Drp1 KO MEFs and transfection with wild type Drp1 adenovirus with GFP tag,observe the mitochondria morphology with mitotracker.2.Detect Drp1 on MAMs(1)Detect the relationship between Drp1 and MAMs with immunofluorescence:Cardiomyocytes from SD rats were isolated and cultured,treating with immunofluorescent staining and observing the Drp1 on MAMs with Confocal.(2)Detect the relationship between Drp1 and MAMs with immunogold transmission electron microscope(TEM): Cardiomyocytes from SD rats were isolated and cultured,observe the Drp1 on MAMs by immunogold TEM.(3)Detect the relationship between Drp1 and MAMs with western blot: Isolate MAMs from hearts of SD rats.Identify the purification of MAMs and detect Drp1 by western-blot.3.To establish myocardial hypertrophy with ISO and detect Drp1 on MAMs.(1)Establish cardiac hypertrophy model in vivo: Mice models of cardiac hypertrophy were induced by ISO injection.After 7 days,14 days and 28 days injection,echocardiography was carried out to measure the cardiac structure and function,and hematoxylin-eosin(HE)staining was followed to observe the hypertrophic pattern.LV mass,Heart/body ratio and heart/tibia ratio were applied.Caspase 3was detected by western blot and the apoptosis kit.m PTP opening time was measured by Confocal.(2)Evaluate Drp1 expression on MAMs in heart: Western-blot was applied to detect the Drp1 level on MAMs and whole heart tissue after ISO injection.(3)Detect Drp1 characteristics on MAMs incardiac hypertrophy: Blue-native gel was employed to measure Drp1 oligomers and GTPase activity was detected by the kit.(4)Evaluate the expression of Drp1 on MAMs in vitro: Cardiomyocytes from mice were isolated and cultured,then stimulated with ISO in different time and dose gradient.Drp1 levels were detected by western-blot.4.Screen the interaction target of Drp1 on MAMs.(1)Screen the underlying protein interacting with Drp1 in vitro: Cardiomyocytes from SD rats were isolated and cultured;CO-IP was included to prepare the sample.MS analysis was used to detect the underlying target interaction with Drp1.(2)Screen the underlying protein interacting with Drp1 in vivo: MAMs was isolated from SD rats and repeated the work in last step.(3)Identify the interaction between Drp1 and VDAC1: Cardiomyocytes from mice were isolated and cultured,transfection with dominant negative VDAC1 adenovirus and then stimulated with ISO,m PTP opening time and caspase3/7activity were used to detect the apoptosis,in VDAC1 KO MEF,transfection with Drp1 K38 A adenovirus,masure the mtichondria flash.(4)Screen the site of Drp1: a: establish different Drp1 truncation transfected into 293 cell and CO-IP to identify which domain interacted with VDAC1;b: different site mutations of Drp1,all these plasmids were transfected into 293 cell,CO-IP was applied to detect the interaction site between Drp1 and VDAC1.c: establish different VDAC1 truncation transfected into 293 cell and CO-IP to identify which domain interacted with Drp1.5.Detect mitochondria fission,Drp1 oligomers and GTPase activity of Drp1K582 Q.(1)Evaluate the characteristics of Drp1 K582 Q mutation a)Transfect Drp1 K582 Q adenovirus into Drp1 KO MEF and observe the mitochondria morphology with Confocal.a)Transfect Drp1 K582 Q adenovirus into Drp1 KO MEF and observe the Drp1 puncta with Confocal.b)Transfect Drp1 K582 Q adenovirus into H9c2,and blue-native gel was used to detect Drp1 oligomers.c)Purify Drp1 wild type and K582 Q protein and detect GTPase activity with kit.6.Detect the impact of Drp1 K582 Q on cardiac apoptosis.(1)Cardiomyocytes from mice were isolated and cultured,transfection with Drp1K582 Q adenovirus and then stimulated with ISO,m PTP opening time.(2)Cardiomyocytes from mice were isolated and cultured,transfection with Drp1K582 Q adenovirus and then stimulated with ISO,western-blot was used to detect the change of caspase 3,and caspase 3/7 activity were used to detect the apoptosis.(3)Cardiomyocytes from Drp1 KO mice were isolated and cultured,transfection with Drp1 K582 Q adenovirus and then stimulated with ISO,caspase 3/7 activity were used to detect the apoptosis.(4)Cardiomyocytes from Drp1 KO mice were isolated and cultured,transfection with Drp1 K582 Q adenovirus and then stimulated with ISO,m PTP opening time was measured with Confocal.Results:1.Drp1 is abundant in heart and comparing with MEF,Drp1 mildly influenced mitochondria morphology.(1)Compared with kidney,liver and lung tissue,the m RNA and protein levels of Drp1 in heart,brain and muscle are relatively high.(2)Compared with control group,the mitochondria morphology in Drp1 overexpression group,K38 A group and sh RNA group is mildly changed.Similar results could also be observed in Mdivi-1 group.(3)Mitochondria are fragmented after Drp1 transfection in Drp1 KO MEF.2.Drp1 is mainly located on MAMs.(1)Quantitative analysis shows that 83.76 ± 2.60 of the DRP1,punctate particles overlapped with TOM20,and 93.95 ± 2.06% were within 1 pixel of distance,the same analysis strategy revealed that 71.02 ± 3.40% of DRP1 aggregates were located within 196 nm radius of Ry R2,suggesting that these DRP1 oligomers,accumulated on MAMs.(2)The quantitative analysis of DRP1-IG particles shows 74 ± 4.05% of the particles on the MAMs.When normalized to the length,the number of particles per μm in MAMs was 8.09 ± 1.68-fold higher.(3)The Drp1 level in cyto is 2.25±0.23 folds than SR and 8.05±1.72 folds than c Mito.The Drp1 in SR is 3.64±0.26 fold higher than c Mito.While the Drp1 on MAMs is 2.52±0.08 folds higher than c Mito.3.The expression of Drp1 on MAMs in myocardial hypertrophy model increased significantly.(1)Compared with mice received ISO 7 days or 14 days,the cardiomyocyte apoptosis level increased significantly in mice receiving ISO injection for 28 days.The Drp1 level on MAMs also increased,while its level in whole heart did not change.(2)In myocardial hypertrophy,Drp1 on MAMs mainly exhibits as tetramers,in addition,the tetramers increased after ISO injection.The GTPase activity also increased.(3)Cardiomyocyte hypertrophy occurred after ISO stimulation at 0.1 umol/L after 24 hours.We applied this condition in the following experiments.(4)Compared with control group,Drp1 increased significantly in cardiomyocyte hypertrophy.4.Drp1 interacted with VDAC1 on MAMs,and Drp1 K582 regulated the interaction.(1)MS analysis indicated that Drp1 interacted with VDAC1 and this interaction increased after ISO stimulation.(2)MS analysis in MAMs sample identified this interaction.(3)In cardiomyocytes which transfected with dominant negative VDAC1 adenovirus,the apoptosis level decreased after Drp1 adenovirus transfection in the condition of ISO stimulation,VDAC1 KO could attenuate the mitochondria flash caused by Drp1.(4)CO-IP of all the Drp1 truncations indicated that Drp1 and VDAC1 interacted with each other via the variable domain and Drp1 K582 lysine regulated this interaction.(5)CO-IP of all VDAC1 truncations indicated that Drp1 and VDAC1 interacted with each other via the 25-amino acid peptide in N terminal.5.Drp1 K582 Q did not change the mitochondria morphology,Drp1 oligomers and GTPase activity.(1)Compared with Drp1 WT,there is no obvious change of mitochondria morphology in MEF cells after Drp1 K582 Q overexpression.(2)Compared with Drp1 WT,there is no obvious change in the number of GFP puncta in cardiomyocytes after Drp1 K582 Q overexpression.(3)Compared with Drp1 WT,Drp1 mainly exerts in tetramers after Drp1 K582 Q overexpression.(4)Compared with Drp1 WT,the GTPase activity of Drp1 K582 Q did not change.6.Drp1 K582 Q could inhibit cardiomyocyte apoptosis.(1)Compared with blank group,the m PTP opening time is longer after Drp1 K582 Q overexpression.(2)Compared with blank group,the caspase 3/7 activity decreased significantly after Drp1 K582 Q overexpression.(3)Compared with control group,there is no obvious inhibition of apoptosis in cardiomyocytes from Drp1 KO mice after Drp1 K582 Q overexpression.Conclusions:1.Drp1 is abundant in heart and it’s mainly located on MAMs.2.Drp1 interacts with VDAC1 on MAMs3.Drp1 K582 site modulates the interaction of Drp1 and VDAC1.4.Drp1 K582 Q could inhibit cardiomyocyte apoptosis.