The Role And Mechanism Of Cardiac-specific Kinase TNNI3K Overexpression In Differentiation Ofmouse Embryonic Stem Cells Into Cardiomyocytes

Backgroud and objectiveThe cardiac troponin I-interacting kinase (TNNI3K), named for its interaction with the cardiac troponin-I, is a cardiac specific kinase, and belongs to the MAPK family. Over the past decades, studies have shown that, on the one hand, TNNI3K might be involved in some pathological process, such as promoting a concentric hypertrophy, prolonging the PR interval and accelerating the progression of dilated cardiomyopathy, et al. On the other hand, TNNI3K played a protective role in attenuating ischemia-induced ventricular remodeling, improving cardiac performance, inducing cardiomyocyte differentiation, and promoting cardiac myogenesis, et al. According to the positioning of TNNI3K gene on chromosome, it was found that it closed nearly to the region of susceptibility genes of heart ventricular septal defect. Therefor, we speculated that TNNI3K participated in the normal heart development. This study aims to explore the role and mechanism of TNNI3K gene in mESC differentiation into myocardial cell in vitro.MethodsTo prove our conjecture, first of all, we started from appraisal mESC totipotency through morphology and totipottential surface markers, SSEA-1 and Oct-4, alkaline phosphatase positive test and HE staining. And then, we adopted traditional hanging drop method to form EB, removed LIF to make it beat spontaneously, and further detected the relative expressions of genes related with cardiomyocytes by real-time PCR, the relative expressions of cardiac-specific protein markers by western blotting, the position of cardiac-specific protein expression by immunofluorescence and cardiomyocyte ultrastructure specific marker, muscle fibers by TEM, which proved the viable method that can differentiate mESC into cardiomyocyte. Furthermore, we permanently transfected hTNNI3K gene and siRNA into mESC through lentivirus and differentiated into cardiomyocytes, through real-time PCR to detect relative expression of cardiac-specific genes, constituting western blotting to detect relative expression of cardiac-specific protein markers, and immunofluorescence co-positioning to detect relative expression of time and space of cardiac-specific protein, as well as the differentiatial rate of cTnT+ cells by FCM to prove the effect of hTNNI3K on myogenesis. Furthermore, we detected the relative expressions of Connexin45, Connexin40, and Ctnt, which is the marker gene of sinoatrial node cells, atrial cells and ventricular cells, respectively by real-time PCR. Finally, we detected the relative expressions of MAPK signaling pathways related proteins by western blotting in hTNNI3K group, shRNA group and respective control group.ResultsThus, it can be seen that surface protein molecules of mESC, SSEA-1 and Oct-4, were expressed positively (green fluorescence), and mESC presented blue-violet by ALP test, as well as nuclear/cytoplasmic ratios >> 1 by HE staining. The cardiac-related genes and proteins expression of normal self-differentiatial cells presented characteristics of time and space. Cardiac-specific proteins, MLC2, cTnI and cTnT and a-actinin, expressed in cytoplasm (green fluorescence) and sarcomeres were clearly visible by immunofluorescence, as well as muscle fibers by TEM on day 16. Cardiac-related gene expressions were remarkable in hTNNI3K goup (p<0.05). Western blotting showed that in hTNNI3K group, cardiac-specific proteins, MLC2, cTnT, cTnI and a-actinin, were significantly higher than that in control group (p<0.05), and co-immunofluorescence showed MHC6 protein expression in advance. FCM showed in hTNNI3K group, the rate of cTnT+positive cells was significantly higher than that in control group (p<0.05). In shRNA group, not only was the rate of cTnT+positive cells significantly lower than that in control group (p<0.05), but also cardiac-specific proteins were significantly lower than that in control group (p<0.05), as well as MHC6 protein expression was delayed. What’s more, the overexpression of TNNI3K gene induced the differentiation of sinoatrial node cells, atrial cells and ventricular cells through detecting the relative expressions of Connexin45, Connexin40 and Ctnt by real-time PCR (P>0.05). Interestingly, we found that P-p38, P-ERK and P-JNK were suppressed, as well as P-p38 and P-JNK more remarkably decreased in hTNNI3K group, while Flag-only group, shRNA group and non-sense RNA signaling pathways protein expressions were normal through testing the MAPK signaling pathways related proteins.ConclusionsTo sum up, TNNI3K gene induced myogenesis, and promoted mESC differentiation into cardiomyocytes. TNNI3K gene induced mESC differentiation into atrial cells and ventricular cells and sinoatrial node cells via MAPK signaling pathway.