GSNO-induced S-nitrosylation Of Drp1 Regulates The Directional Differentiation Of Mouse Embryonic Stem Cells Into Cardiomyocytes

Background Embryonic stem cells(ESCs)are derived from the inner cell mass of the preimplantation blastocyst,which could proliferate immortally and differentiate into all types of somatic cells in vitro.ESCs-derived cardiomyocytes have the structural and functional characteristics of early cardiomyocytes,which can be combined with animal experiments to evaluate the efficacy and toxicity of heart disease.S-nitrosoglutathione(GSNO)is an S-nitrosylated derivative of the antioxidant glutathione.Various biological processes were regulated by GSNO such as transcription,DNA repair damage,cell differentiation and apoptosis through proteins S-nitrosylation.Our group revealed that short-term exposure to GSNO could promoted the directed cardiomyocyte differentiation of mouse ESCs(mESCs).We also found that the S-nitrosylation of 104 proteins was upregulated by proteomic analysis,80% of which were located in organelles,especially mitochondria and ribosomes.Our further study showed that S-nitrosylation at C271 of mitochondrial connexin 43 could induce mitochondrial maturation,which was associated with myocardial differentiation.Mitochondria are continuously cyclically regulated by fission and fusion to form a stable network,which plays a key role in stem cell proliferation and initiation of differentiation.Mitochondrial dynamics-related protein 1(Drp1)is the key for mitochondrial fission.The activity and intracellular localization of Drp1 are regulated by its phosphorylation,ubiquitination,and S-nitrosylation.S-nitrosylation of Drp1 results in up-regulation of GTPase activity and conformational change,promoting mitochondrial fission and fusion.The mitochondria-associated endoplasmic reticulum membrane(MAMs)is a major intracellular region of mitochondrial fission,fusion,and calcium transport,where molecular events occur that contribute to the differentiation or pluripotency maintenance of stem cell.Based on the above research background,it has not been clarified whether GSNO promotes the directed myocardial differentiation of mESCs by influencing mitochondrial dynamics and calcium flow in the MAMs structure through S-nitrosylation of Drp1.Objective To reveal the role of S-nitrosylation of Drp1 in GSNO-induced differentiation of mESCs into cardiomyocytes.Methods and results Using mESCs myocardial differentiation model in vitro,embryonic bodies(EBs)were treated with 25 μM GSNO in the dark for 2 h on day 5.100 n M RA was treated as a positive control.Western blot and immunofluorescence staining on day 5+3 showed that the expression of myocardial specific protein α-actinin in EBs-derived cells of GSNO and RA group was significantly upregulated,and more mature light-dark sarcobar structure was formed than that of the negative group.Then,we investigated the effect of GSNO on EBs mitochondria.Mito Tracker? Green FM probe staining and Western blot showed that part of mitochondria in GSNO group were activated and divided,and the expressions of mitochondrial fusion protein Mfn2 and mitochondrial marker protein Tom20 were obviously increased.However,the total protein expression of Drp1 was not different from that of the negative control group.Furthermore,the S-nitrosylation protein in total protein was further extracted by Biotin-Switch,and it was found that the S-nitrosylation level of Drp1 was notably up-regulated after GSNO treatment.After the separation of cytoplasm and mitochondria on day 5 EBs,the expression of Drp1 in mitochondria was significantly increased after GSNO treatment,while the expression in cytoplasm was opposite.It is suggested that GSNO could facilitate mitochondrial fusion,fission and generation,promoting the transfer of Drp1 from cytoplasm to mitochondria.Next,we specifically overexpressed Drp1 and Drp1C650A(S-nitrosylation site mutant)in mESCs by transfection with lentiviruses.The results showed that in EBs on day 5,the S-nitrosylation level of Drp1 in the GSNO+Drp1C650A group was markedly down-regulated compared with the GSNO+Drp1 group.Correspondingly,on day 5+3,flow cytometry showed that the proportion of α-actinin positive cells and fluorescence intensity of α-actinin in EBsderived cells of GSNO+Drp1C650A group were significantly decreased,and the formation of mature sarcomore was reduced compared with the GSNO+Drp1 group.These results suggested that the mutation of Drp1 nitrosylation site was not conducive to GSNO-induced directional differentiation of mESCs into cardiomyocytes.In order to explore the effects of the Drp1 S-nitrosylation by GSNO on mitochondrial dynamics,mitochondrial morphology was observed by Mito Tracker probe staining.Compared with the GSNO+Drp1 group,the increase of elongated tubular mitochondria indicated that mitochondrial fission was weaken in GSNO+Drp1C650A group.Western blot showed that the expressions of p-Drp1(Ser616)in total proteins,Drp1 and p-Drp1(Ser616)in mitochondria were obviously down-regulated in GSNO+Drp1C650A group.It was suggested that GSNO-induced S-nitrosylation of Drp1 contributed to mitochondrial fusion and fission,maintaining the balance of mitochondrial network dynamically.Immunofluorescence co-staining showed that the co-localization of Drp1 and Tom20 as well as p-Drp1(Ser616)and Tom20 increased in EBs treated with GSNO on day 5,and the co-localization of GSNO+Drp1C650A group was significantly decreased compared with GSNO+Drp1 group.It was confirmed that GSNO-induced S-nitrosylation of Drp1 promoted the transfer of Drp1 and p-Drp1(Ser616)to the mitochondrial outer membrane.Next,we continued to explore the changes of downstream mitochondrial fission-related proteins.Western blot showed that the expression of Fis1 was obviously increased after GSNO treatment,and the co-localization of Fis1 and Drp1 was also notably enhanced,while that in the GSNO+Drp1C650A group was decreased compared with the GSNO+Drp1 group.The results suggested that the S-nitrosylation of Drp1 made it easier to bind to Fis1 and promoted Fis1 expression,which were conducive to mitochondrial fission.Then,Rhod-2AM calcium probe staining showed that the calcium concentration was increased notably in day 5 EBs,while the GSNO+Drp1C650A group was decreased compared with the GSNO+Drp1.Western blot showed that the expression of cell membrane calcium channel SLC8A1 had no difference between groups.And the mitochondrial calcium channel MICU1 was obviously increased after GSNO treatment,which was decreased in the GSNO+Drp1C650A group compared with the GSNO+Drp1.In day 5+3 EBs-derived cells,the expressions of IP3 R and Mfn2 were markly increased after GSNO treatment,while the GSNO+Drp1C650A group was down-regulated compared with the GSNO+Drp1.However,the expressions of Grp75 and VDAC1 had no changes.The morphology of MAMs was observed by transmission electron microscope,and it was found that the average length of MAMs increased significantly after GSNO treatment,while the GSNO+Drp1C650A group was decreased compared with the GSNO+Drp1.It was suggested that the mechanism of GSNO inducing myocardial differentiation was related to the increase of MAMs structure and the expression of IP3R-Grp75-VDAC1-MCU calcium axis molecule promoted by S-nitrosylation of Drp1.Conclusions 1.S-nitrosylation of Drp1 was induced by GSNO during the directional differentiation of mESCs into cardiomyocytes,accompanied by an increase in phosphorylation.It was beneficial for Drp1 to transfer to mitochondrial outer membraneand and bind with receptor protein Fis1,facilitating mitochondrial fission in early stage of myocardial differentiation.2.The length of MAMs and the expressions of IP3 R and Mfn2 were promoted by GSNO-induced S-nitrosylation of Drp1.The opening of mitochondrial calcium channel MICU1 was also activated by S-nitrosylation of Drp1,leading to change in mitochondrial calcium concentration.