Effect Of Autophagy On Cardiomyocytes Differentiation Of Human Induced Pluripotent Stem Cells

Background: Cardiovascular diseases(CVD) become the main cause of mobidity and mortality because of the changes of eating habit and lifestyle. Among them, coronary artery heart disease(CHD) poses the most threat to human life. Current treatment, including medicine treatment, percutaneous coronary intervention(PCI) and coronary artery bypass grafting(CABG) significantly improve the outcome and quality of life of CHD patients by improving myocardial blood supply, saving dying cardiomyocytes, delaying the occurrence of left ventricular remodeling. However, aforementioned methods have no effect on rescuing cardiomyocytes which are already dead because of ischemia for a long time. For patients who suffered massive myocardial infarction, a great amount of dead cardiomycytes were replaced by fibrous tissues, which show no contractibility. The fibrous tissues form scars in the heart and eventually, lead to heart failure. The rise of stem cell therapy provides favorable cell source for cardiac regenerative therapy. So far, several types of stem cells have been applied to animal and pre-clinical trials of for autologous/ allogeneic transplantation to treat acute myocardial infarction, some of them have shown prominent effect.Terminally differentiated somatic cells can be reprogrammed into a pluripotent state by ectopic expression of several stem cell transcriptional factors. Such induced pluripotent stem cells, termed as i PSCs, shares the similar properties with embryonic stem cells(ESCs) such as self-renewal and multipotent differentiation, which could theoretically differentiate into all three germ layer cell type including cardiomyocytes. In addition, it also overcomes the immune and ethical problem caused by the ESC research. Therefore, i PSC has become the hot spot in the area of stem cell research. In recent years, i PSCs have obtain great development in the field of cardiac regenerative therapy, building individualized patient-specific disease model, drug cardiac toxicity screening.Autophagy is a reparative, life-sustaining process by which cytoplasmic long live protein and other components are sequestered in double-membrane vesicles and degraded on fusion with lysosomal compartments. As a defense mechanism against adverse microenvironment, autophagy is important for maintaining cellular homeostasis. Recent studies indicate autophagy was involved not only in the embryonic cardiogenesis, but in cardiac differentiation of stem cell as well. However, the specific effect of autophagy on cardiomyocytes differentiation and the corresponding mechanism was still unknown.Aims: 1. To observe the regulatory effect of autophagy on cardiomyocytes differentiation of human induced pluripotent stem cells.2. To xplore the mechanisms of autophagy-mediated cardiomyocytes differentiation of hi PSCs.Methods: 1. In vitro cultivation of i PSCs and verify its pluripotency using immunofluorescent(IF) staining for stem cell marker, Oct-4.2. To optimize the temporal and concentrate condition for regulating autophagy in human embryoid bodies(h EBs), rapamycin(Rapa) and ammonium chloride(NH4Cl) were used as autophagy activator and inhibitor, respectively. Human EB were induced using suspension method, experimental groups were divided into(1) control group;(2) rapamycin treated group(Rapa group);(3) rapamycin combined with NH4 Cl treated group(NH4Cl). Autophagic level was evaluated by detecting the expression of autophagic-related protein LC3 and autophagic substrate P62, observing autophagosome using transmission electron microscopy(TEM) and immunofuolrescence staining for LC3.3. Observe the specific effect of autophagy on cardiac differentiation of hi PSCs. Cells were divided into 3 groups:(1) control group;(2) Rapa group;(3)R+N group. Each group was treated with corresponded agent at optimal time window during the differentiation process. Expression of cardiac specific genes(GATA-4, NKX2-5, TBX5, c TNT) and proteins(Mef-2c, c Tn I), number of beating EBs and percentage of cardiac troponin I positive cells were examined. The percentage of myocardial protein MEF-2c and c Tn I were observed by IF staining and flow cytomytry analysis(FCM).4. For mechanism study, the activation level of wnt/β-catenin signaling pathway was assessed in each group by detecting the expression of well-characterized canonical wnt/β-catenin target genes CYCLIN-D1, AXIN2, C-MYC. Gene and protein expression of dishevelled(Dvl), a key molecule of wnt/β-catenin pathway, were examined using Westernblot analysis. GSK-3β inhibitor CHIR-99021 was used to activate canonical wnt/β-catenin signaling pathway and the number of beating EBs in each group were counted to assess the myocardial differentiation efficiency.Results: 1. Human i PSC clones were in compactly suborbicular shape with uniform cell composition, and have distinct boundaries between i PS clones and feeder cells. IF staining showed that hi PSCs were stained positive for pluripotency marker Oct-4.2. The optimal treatment condition for cardiomyocyte differentiation of hi PSCs was established with ideal concentration(1μmol/ml) and duration(from day 4 to day 6 after the differentiation was initiated) of rapamycin administration.3. Our results demonstrated that rapamycin significantly increased the number of beating EBs as well as enhanced expression of cardiac specific genes((GATA-4, NKX2-5, TBX5, c TNT) and myocardial proteins. In addition, elevated amount of cardiac troponin I(c Tn I) positive cells were detected in Rapa group using IF staining and FCM analysis. Such effects were completely blocked with the addition of NH4 Cl.4. Increased level of autophagy significantly suppress the expression of Wnt/β-catenin target genes(CYCLIN-D1, AXIN2, C-MYC), while NH4 Cl could augment the expression of these genes, furthermore, our results found that the protein expression level of disheveled 2(dvl2), a key molecule of canonical wnt/β-catenin pathway, was significantly decrease in Rapa group, indicating autophagy may inhibit wnt/β-catenin pathway by eliminating cytoplasmic dvl2, such effect was partly blocked by the addition of NH4 Cl. Finally, our results showed that the addition of CHIR99021 to activate Wnt/β-catenin pathway could partly block the autophagy-mediated cardiac differentiation of hi PSCs and resulted in a decreased number of beating EBs.Conclusions: Our results suggest that autophagy could enhanced the cardiomyocytes differentiation of human induced pluripotent stem cells by negative regulation of canonical wnt/β-catenin signally pathway. These results may provide us a better understanding of the role that autophagy played during the cardiomyocyte differentiation of i PSCs.