Regulation Of Rictor/mTORC2 On Mitochondrial Function Of Mouse Embryonic Stem Cell-derived Cardiomyocvtes Via Mitochondrial Cx43

The mammalian target of rapamycin is an evolutionarily conserved serine/threonine protein kinase.It is one of the phosphatidyl inositol kinase(PI3K)related protein kinase family.mTOR plays a central role in regulating cellular processes including cell growth,metabolism and survival.mTOR forms two independent complexes,as mTORC1 and mTORC2.mTORCl contains Raptor,mTOR,G?L,DEPTOR and PRAS40,whereas the mTORC2 composed of mTOR,Rictor,G?L and SIN1.Raptor and Rictor are the core components of mTORC1 and mTORC2,respectively.mTORCl is involved in protein and lipid synthesis,cell growth and metabolism.mTORC2 could regulate embryonic development,tumor genesis and cardioprotection in myocardial ischemia-reperfusion injury by phosphorylating downstream signaling such as Akt and PKC.The functions and underlying mechanisms of Rictor/mTORC2 in cardiac development remains poorly understood.Herein,it is urgent to start the research.Embryonic stem(ES)cell is derived from the inner cell mass of blastocysts.They have the ability of self-renewal and differentiating into diverse types of somatic cells in vivo or in vitro.ES cells could differentiate into functional cardiomyocytes,which is applied to the regenerative therapy of heart disease and drug discovery.However,the process of cardiac differentiation is complicated and the regulatory mechanisms are not yet clear.Our previous studies have found that knockdown of Rictor inhibited the cardiomyocytes differentiation from mouse ES cells.The cardiomyocytes showed abnormal electrophysiology and unorganized sarcomeres in shRNA-Rictor group.The mechanisms by which Rictor/mTORC2 may regulate cardiac differentiation remains poorly understood.The heart is one of the earliest formed organs during embryonic development.Cardiomyocytes possess the largest density of mitochondria and have high demand for energy.Currently,researchers pay more attention to investigate the mechanisms of cardiac development from the perspective of mitochondria.Mitochondrial function plays pivotal roles in preserving pluripotency and regulating stem cells differentiation.Mitochondrial energy metabolism switches from glycolysis to oxidative phosphorylation to ensure the normal cardiomyocyte differentiation and cardiomyocyte excitation-contraction coupling.Mitochondria-associated endoplasmic reticulum(MAM)is physical contacts formed between the endoplasmic reticulum(ER)and mitochondria.ER and mitochondria are tethered to each other by mitofusion 2(Mfn2)and glucose regulated protein 75(Grp75).Grp75 forms trimeric complex with 1,4,5-phosphoinositide receptor(IP3R)in ER and voltage-dependent anion channel(VDAC)in mitochondria.Calcium could transfer from ER to the mitochondria through the IP3R-Grp75-VDAC1 complex.Ca2+ in mitochondria promotes the synthesis of ATP in a certain concentration.Studies have reported that mTORC2 is localized in MAM structure in MEF cell.The coupling structure of MAM and mitochondrial function were destroyed by knockdown of Rictor.In our study,we investigated the localization of Rictor/mTORC2 in cardiomyocytes and the effect of Rictor/mTORC2 on structure and function of MAM.Furthermore,the relationships between Rictor/mTORC2 and mitochondrial function in regulating cardiomyocyte differentiation would be studied.Cx43 is a major component of gap junction in cardiomyocytes,which is involved in cardioprotection of ischemia preconditioning and electrophysiological activity.Cx43 is also confirmed to localize at mitochondria and mainly phosphorylated.Mitochondrial Cx43 is proposed to be associated with mitochondrial energy metabolism,reactive oxygen species(ROS)generation,mitochondrial potassium flow and so on.Cx43 is transported to mitochondrial inner membrane mainly by heat shock 90(Hsp90)and translocase of outer membrane 20(TOM20).Our previous study found that knockdown of Rictor affected the expression and distribution of Cx43 in the mouse ES cell-derived cardiomyocytes(ESC-CMs),which may finally lead to the disordered electrophysiological activity.Our research is aimed to investigate whether mitochondrial Cx43 is involved in the Rictor/mTORC2 regulated mitochondrial function during cardiomyocyte differentiation,to provide a basis for further study the mechanisms of Rictor/mTORC2 in cardiac development.Mitochondrial permeability transition pore(mPTP)is a non-specific muti-protein pore on mitochondria,which plays an important role in information exchange within and outside the mitochondria.mPTP is consisted of VDAC in mitochondrial outer membrane,adenine nucleotide translocase(ANT)in inner membrane and cyclophilin D(CypD)in matrix.Opening of mPTP could result in losing shielding function of inner membrane,decreasing mitochondrial membrane potential,destroying mitochondrial respiratory chain and dcreasing ATP production.It has been reported that Akt and GSK3? are associated with the opening of mPTP.The activity of GSK3? is inhibited after phosphorylation of GSK3? at Ser9 by Akt,which further prevents the opening of mPTP.GSK3? could combine with ANT and increase the threshold of mPTP opening.Mitochondrial Cx43 and p-Cx43Ser368 are proved to be the downstream moleculars of GSK3?.Activation of mitochondrial Cx43 could reverse the calcium overload-induced opening of mPTP.Little is known about the direct relationship of mitochondrial Cx43 and mPTP.We hypothesize that mitochondrial Cx43 is associated with the components of mPTP.Rictor/mTORC2 might influence mitochondria Cx43 by Akt-GSK3p signaling,thereby affecting the opening of mPTP and further influencing mitochondrial funtion.Therefore,our research is hope to investigate the the mitochondrial damage and the related mechanisms by knockdown of Rictor,to reveal the relationship of the localization of Rictor/mTORC2 in cardiomyocytes and the structure of MAM,to clarify whether Rictor/mTORC2 regulates mitochondrial function via mitochondrial Cx43 through influencing the respiratory chain enzyme activity and mPTP opening,to further explore the physiological function of Rictor/mTORC2 in cardiac development.The study also provides new experimental basis to explore targets for regulating cardiomyocyte differentiation and a useful reference for application of the induced pluripotent stem cells.Objective:To investigate the effects and related mechanisms of Rictor/mTORC2 on mitochondrial function in cardiac differentiation from mouse ES cells,to indicate the relevance of the localization of Rictor/mTORC2 and the structure of MAM,to clarify the mechanisms of regulation of Rictor/mTORC2 on mitochondrial function by mitochondrial Cx43.Methods:In this study,we suppressed the expression of Rictor in mouse ES cells by using shRNA lentivirus.Classical ES cell cardiogenesis model was used to differentiation of mouse ES cells into cardiomyocytes.Western Blot was used to investigate the expression of Rictor during mouse ES cells differentiation into cardiomyocytes.The efficiency of cardiomyocyte differentiation from mouse ES cells was evaluated by flow cytometry and western blot.The methods of immunofluorescence,western blot and immunoprecipitation were used to detect the localization of Rictor in ESC-CMs.The viability of mouse ES cells after transfection was assessed by MTT assay.The expression levels of components of mTORC2(SIN1,G?L,mTOR,p-mTORSser2481)and p-AktSer473 were detected by western blot.Intracellular ATP production were measured after knockdown of Rictor.JC-1 and flow cytometry were used to assess the mitochondrial membrane potential of cardiomyocytes.The level of cytochrome c was detected by Western Blot.Transmission electron microscope was used to observe the ultrastructure of mitochondria and MAM in ESC-CMs.Western blot was used to detect the expression of Mfn2 in ESC-CMs.The immunoprecipitation was used to detect the effect of shRNA-Rictor on IP3R-Grp75-VDACI complex.Mitochondrial Ca2+ transient of ESC-CMs was determined by the living cell workstation.Then we used western blot and immunofluorescence to investigate the influence of shRNA-Rictor on the expression and distribution of Cx43 and HDAC6 in ESC-CMs.Immunoprecipitation was used to evaluate the influence of knockdown of Rictor on the interation of Cx43 with Hsp90 and TOM20,Hsp90 and acetyl lysine.shRNA-mitoCx43 was used to overexpression the mitochondrial Cx43 of mouse ES cells.The ATP and mitochondrial membrane potential were detected in shRNA-Rictor+mitoCx43 treated cells.At the same time,flow cytometry analysis was used to determine the effect of shRNA-Rictor+mitoCx43 on the cardiac differentiation efficiency.The activities of mitochondrial respiratory chain complex ?,?,? and ? were detected in ESC-CMs.Fluorescence staining and immunoprecipitation were used to detect the opening of mPTP in ESC-CMs.The expression levels of Akt,p-AktSer473,GSK3? and p-GSK3?Ser9 were assessed by western blot analysis.Then we used immunoprecipitation to evaluate the combination of Cx43,p-Cx43Ser368,GSK3?,p-GSK3?Ser9,ANT and CypD in ESC-CMs.Results:1.The expression level of Rictor was up-regulated during cardiomyocyte differentiation from mouse ES cells.Knockdown of Rictor significantly decreased cardiomyocyte differentiation from mouse ES cells detected by flow cytometry and western blot analysis.Immunofluorescence and immunoprecipitation analysis showed that Rictor was highly overlapped with ER and mitochondria and associated with IP3R,Grp75 and VDAC1.The Rictor could be detected in ER and MAM except for pure mitochondria.Western blot analysis found that the expression level of mTOR and G?L have no significant change whereas SIN1,p-mTORSer2481 and p-AktSer473 were markedly decreased after knockdown of Rictor.Knockdown of Rictor significantly reduced the production of ATP in EBs and ESC-CMs.The mitochondrial membrane potential was significantly reduced in shRNA-Rictor group by JC-1 staining and flow cytometry.However,the expression of cytochrome c was not changed between the two groups.Transmission electron microscope showed that the mitochondria appeared to be swollen with vacuolar structure in shRNA-Rictor treated ESC-CMs.The amplitude of mitochondrial Ca2+ transient was significantly decreased after knockdown of Rictor.At the same time,the MAM structure was reduced,the level of Mfn2 decreased and the interaction between IP3R,Grp75 and VDAC1 weakend in shRNA-Rictor treated cells.These results demonstrated that knockdown of Rictor decreased ATP production,reducing mitochondrial membrane potential,resulting in mitochondrial swollen,while the level of cytochrome c did not change,and did not induce mitochondria-dependent apoptosis.Loss of Rictor destroyed MAM structure,which lead to the reduced Ca2+transferring from ER to mitochondria.2.Immunofluorescence and western blot analysis showed that Cx43 in mitochondria was significantly reduced in shRNA-Rictor cells.And the expression level of the total Cx43 in ESC-CMs was decreased whereas Cx43 in cytoplasm increased in comparison to the control group.The results indicated that knockdown of Rictor induced reduced expression of mitochondrial Cx43,which may associated with the inhibition of mitochondrial translocation of Cx43 from cytoplasm.Knockdown of Rictor decreased the binding of Cx43 with Hsp90 and TOM20.The protein level of HDAC6 was significantly down-regulated and the acetylation of Hsp90 was increased in Rictor knockdown ESC-CMs.It demonstrated that the reduced translocation of Cx43 into mitochondria was attributed to the HDAC6 induced acetylation of Hsp90,which resulted in weaken association of Cx43 with Hsp90 and TOM20.3.The up-regulation of ATP production and mitochondrial membrane potential were detected in shRNA-Rictor+mitoCx43 cells compared to the shRNA-Rictor group.Flow cytometry analysis found that the cardiac differentiation was enhanced after transfected with shRNA-mitoCx43(13.57±1.02%in shRNA-Rictor cells and 18.26±1.06%in shRNA-Rictor+mitoCx43 cells).These results indicated that overexpression of mitochondrial Cx43 could reverse shRNA-Rictor induced mitochondrial function damage and cardiomyocyte differentiation prevention.Furthermore,the activities of mitochondrial respiratory chain complex?,? and ? were markedly reduced after knockdown of Rictor and the complex?and ? activities could be reversed by overexpression of mitochondrial Cx43.The opening of mPTP was significantly increased in shRNA-Rictor treated cells and overexpression of mitochondrial Cx43 could prevent it.Knockdown of Rictor down-regulated the protein levels of p-AktSer473,p-GSK3?Ser9 and inhibited the binding of p-GSK3?Ser9,Cx43,p-Cx43Ser368,ANT and CypD.These results indicated that shRNA-Rictor inhibited the activities of respiratory chain complex I and IV in ESC-CMs via reducing the expression of mitochondrial Cx43.On the other hand,loss of Rictor inhibited Akt-GSK3? signaling pathway,inhibiting the association of Cx43 with ANT and CypD,resulting in the opening of mPTP,then damaging mitochondrial respiratory chain and disturbing mitochondrial function.Conclusions:1.Rictor/mTORC2 was up-regulated during the differentiation of mouse ES cells into cardiomyocytes and mailnly localized at ER and MAM in ESC-CMs.Knockdown of Rictor prevented cardiomyocyte differentiation from mouse ES cells.Rictor knockdown inhibited the activity of mTORC2 complex and destroyed its integrity.Loss of Rictor lead to the reduction of mitochondrial membrane potential and swollen mitochondria in ESC-CMs.Meanwhile,knockdown of Rictor destroyed the integrity of MAM structure,leading to the decrease of IP3R-mediated mitochondrial Ca2+ transient,which finally resulted in the decrease of ATP production and disturbed mitochondrial function,while the cytochrome c was not change and did not induce mitochondria-dependent apoptosis.2.One of the possible mechanisms of shRNA-Rictor induced damaged mitochondrial function is that the decrease of mitochondrial Cx43 translocation lead to inhibited activities of mitochondrial respiratory chain complex ? and ? in ESC-CMs.On the other hand,knockdown of Rictor inhibited the Akt-GSK3? signaling pathway,preventing the association of Cx43 binding to ANT and CypD,resulting the opening of mPTP.