| The evolutionarily conserved serine/threonine protein kinase mammalian target of rapamycin (mTOR) belongs to the phosphoinositide-3-kinase-related kinase family and is a master regulator of cell growth, proliferation and differentiation. It senses nutrient availability, growth factor and energy levels through PI3K/Akt/mTOR, amino, nutrients and environmental stress pathway. mTOR forms two structurally and functionally distinct complexs:mTOR complex1(mTORC1) and mTOR complex2(mTORC2). Much is known about the functions of mTORCl, consisting of the conserved components mTOR, Raptor, Deptor, PRAS40and mLST8. It responds to the presence of nutrients and energy sources to control protein synthesis and is related to cell growth, autophagy, apoptosis. However, studies that define mTORC2in cellular functions and signaling have lagged behind. mTORC2is a multiprotein complex composed of subunits including mTOR, PRR5, mSinl, mLST8and Rictor. Rictor is an essential component of mTORC2, which is required for its full function. Furthermore, mTORC2has been implicated in cytoskeletal organization and cells deficient in the mTORC2-specific protein rictor display altered morphology with impaired migration. Meanwhile, Rictor/mTORC2also plays a critical role in the formation of cardiovascular, cardioprotection induced by ischaemic, myocardialatrophy and is indispensible in embryonic development and embryoid body formation. However, Rictor/mTORC2involving in cardiomyocyte differentiation from mouse Embryonic Stem (ES) cells has not been reported. ES cells can be cultured and maintained in undifferentiated state in vitro. Accumulated evidences suggest that cardiomyocyte differentiation of ES cells in vitro faithfully recapitulates cardiomyocyte differentiation of ES cells in vivo. Thus cardiomyocyte differentiation of ES cells is considered as an efficiency model in vitro for cardiac developmental biology, cardiac pathology and new drug discovery. The roles of Rictor/mTORC2are still not understood in cardiomyocyte differentiation of ES cells.Cardiac cell-cell junctions play a key role in consistent cellular activity, information transmission, differentiation and development of cells. Connexin43(Cx43), N-cadherin and Desmoplakin are the main cell-cell junction in cardiomyocyte. Usually, the expression changing, structural confusion and different distributions of Cx43, N-cadherin and Desmoplakin can induce heart disease, as arrhythmias and cardiac remodeling. On the other hand, the action potential of cardiomyocyte is spreaded through Gap junction. Cx43couples neighboring cardiomyocytes and involves in synchronize heart contraction. The expression and distribution of Cx43are committed to synchronous electrical activity contraction. Rictor/mTORC2regulates blood-testis barrier dynamics via its effects on the expression and distributions of Cx43and mediates the expression of VE-cadherin in vascular endothelial cells to promote the embryonic angiogenesis formation. However, it is never reported that the connection between Rictor/mTORC2and Cx43in embryo cardiac development.Cardiomyocytes with electrophysiological properties characteristic of pacemaker-ventricular-, atrial-, purkinje-like cells could be differentiated from mouse and human ES cells. The direct differentiation of mouse ES cells into specific cardiac subtypes was mediated by some drugs or small molecules. For example, suramin and IWR-1enhanced cardiac specialization into sinus node-like and ventricular-like cells respectively.But whether Rictor/mTORC2involved in the differentiation into the specific cardiac subtypes has not been uncovered.In our study, the expression and regulation of Rictor/mTORC2was investigated in cardiomyocyte differentiation from mouse ES cells. We explored whether Rictor/mTORC2involved in the differentiation into the specific cardiac subtypes, and clarified whether Rictor/mTORC2mediated the cell-cell junction proteins expressions to affect the electrical activity of the cardiomycyte function. It can provide the promising new target of myocardial differentiation inducer and therapeutic potential to heart disease. Aim:To investigate the expression and regulation of Rictor/mTORC2in cardiomyocyte differentiation from mouse ES cells. To illuminate whether Rictor/mTORC2involved in the differentiation into the specific cardiac subtypes, and clarified whether Rictor/mTORC2mediated the cell-cell junction proteins expressions to affect the electrical activity of the cardiomycyte function.. Methods:At first, using the hanging drop culture method, mouse ES cells were differentiated into cardiomyocytes. PP242, an ATP-competive dual mTORC1/mTORC2inhibitor, was added into each sample from the day0, or the shRNA-Rictor was transfected in the ES cells. The percentage of beating embryoid bodies (EBs) after shRNA-Rictor was detected. The function of Rictor/mTORC2in cardiomyocyte differentiation was assessed by western blot and immunofluorescent analysis. As a further confirmation, mouse ES cells-derived cardiomyocytes (ES-CMs) were purified. The relationship between Rictor/mTORC2and cell-cell junctions were discoved by western blot and immunofluorescent analysis. Finally, the action potentials (APs) were recorded from spontaneously beating ES-CMs using the whole-cell patch-clamp. According to the action potentials parameters, the role of shRNA-Rictor in cardiac electrical activity was confirmed.Results:1. The cardiomyocyte differentiation from mouse ES cells was inhibited by100nmol/L PP242. The phosphorylation of p-p70S6K (Thr389) and p-Akt (Ser473), as the substrate of mTORC1and mTORC2respectively, were decreased. PP242could remarkably inhibit the spontaneous cardiomyocyte differentiation, leading to decrease the expression of Brachyury (mesodermal marker), Nkx2.5(cardiac progenitor cells marker) and a-Actinin in cardiomyocyte differentiation from day3to day5+3. The19±3.0%of embryoid bodies (EBs) using PP242were beating, while35±4%of EBs in the contorl at day5+3(P<0.01). Flow cytometry analysis showed5.2±1.6%of cells stained for a-Actinin in the control at day5+3, while15.2±2.6%of cells treated with PP242. Immunocytoehemistry analysis showed disorderly sarcomeric structure for a-Actinin in mouse ES cells treated with PP242.Rictor/mTORC2played a critical role in controlling the cardiomyocyte differentiation from mouse ES cells. The absence of the mTORC2binding partner Rictor could selectively blocked the cardiogenesis. Meanwhile, knockdown of Rictor inhibit the spontaneous cardiomyocyte differentiation, leading to decrease the expression of Brachyury, Nkx2.5and a-Actinin in cardiomyocyte differentiation from day3to day5+3. On differentiating day5+3, the percentage of beating EBs with shRNA-Rictor was reduced by42±6%compared with shRNA-Con (70±6%). Flow cytometry analysis demonstrated only6±2%of cells stained for the cardiac specific protein a-Actinin in shRNA-Rictor group, while12±2%in the control were a-Actinin-positive cardiomyocytes. shRNA-Rictor mouse ES cells showed a Rictor down-expression accompanied by disorderly sarcomeric structure for a-Actinin.2. shRNA-Rictor significantly decreased the proteins expressions of Rictor, p-Akt (Ser473), but could not effect Raptor/mTORCl and p-p70S6K (Thr389) protein expression. Interestingly, shRNA-Raptor could significantly decreased the proteins expressions of Raptor/mTORC1, p-p70S6K (Thr389), p-Rictor (Thr1135), and Rictor, p-Akt (Ser473) protein expression were increased, leading to promote the differentiation from mouse ES cells into cardiomyocyte. Above all, Rictor/mTORC2mediated the direct differentiation from mouse ES cells into cardiomyocyte of shRNA-Raptor.3. The expression of Cx43, N-cadherin, Desmoplakin were significantly decreased in mouse ES Cell-derived cardiomyocytes (ES-CMs) transferred by shRNA-Rictor as assessed by western bolt and immunocytoehemistry. The co-expression of N-cadherin, Desmoplakin with sarcomeric a-Actinin were decreased by shRNA-Rictor, and the co-expression coefficient were0.63±0.03,0.74±0.03respectively, while0.25±0.01,0.34±0.09in shRNA-Con. Cx43was located in cell to cell in mouse ES-CMs transferred by shRNA-Con, contrarily shRNA-Rictor disordered the cell to cell location of Cx43.4. The patch-clamp analysis demonstrated that shRNA-Rictor increased the proportion of ventricular-like myocytes differentiation from mouse ES cells, but yielded a nonsignificant effect on nodal-like, atrial-like myocytes. However, the APA, Vmax were decreased and the APD50, APD90were prolonged by shRNA-Rictor in ventricular-like myocytes, leading to DAD. shRNA-Rictor also decreased the APA, but increased the decay slope of fourth phase repolarisation, which enhanced antomaticity, and induced the happening of EAD, DAD in nodal-like myocytes. Meaningwhile, the APD50, APD90were also prolonged by shRNA-Rictor in atrial-like myocytes.Conclusion:1. Knockdown of Rictor inhibited the spontaneous cardiomyocyte differentiation. The EBs showed a markedly decreased. Knockdown of Rictor inhibited the Brachyury, Nkx2.5and a-Actinin protein expressions in cardiomyocyte differentiation. Meaningwhile shRNA-Rictor decreased the quantity of myocytes accompanied by disorderly sarcomeric structure for a-Actinin.2. Rictor/mTORC2mediated the effect of Raptor/mTORC1in cardiomyocyte differentiation from mouse ES cells through changing the expression of Rictor.3. Knockdown Rictor/mTORC2decreased the expression of N-cadherin, Desmoplakin, Cx43and disordered the location in cell to cell.4. shRNA-Rictor could increase the direct differentiation of ventricular-like myocytes from mouse ES cells and the quantity of myocytes with symptom of arrhythmias. The APD50, APD90of ventricular-like and atrial-like myocytes were prolonged. The APA of ventricular-like and nodal-like myocytes were decreased irregularly, which induced rhythmic disorders and the happening of DAD. The decay slope of fourth phase repolarisation was increased, which enhanced antomaticity and induced the happening of EAD, DAD in nodal-like myocytes. |
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