Overexpression of Cardiac-Specific Kinase TNNI3K Promotes Mouse Embryonic Stem Cells Differentiation into Cardiomyocytes

Abstract

Backgroud/Aims: The biological function of cardiac troponin I-interacting kinase (TNNI3K), a cardiac-specific functional kinase, is largely unknown. We investigated the effect of human TNNI3K (hTNNI3K) on the differentiation of mouse embryonic stem cells (mESCs) into cardiomyocytes. Methods: First, the time-space expression of endogenous Tnni3k was detected by real-time polymerase chain reaction (PCR) and western blotting at 16 different time-points over a period of 28 days. Further, action potentials and calcium current with/without 5 µM nifedipine were measured by patch clamp for mESC-derived cardiomyocytes. HTNNI3K and mouse-derived siRNA were transfected into mESC using lentivirus vector to induce hTNNI3K overexpression and knock-down, respectively. Results: The number of troponin-T (cTnT) positive cells was greater in the group with TNNI3K overexpression as compared to that in control group, while less such cells were detected in the mTnni3k knock-down group as evaluated on flow cytometry (FCM) and ImageXpress Micro system. After upregulation of connexin43, cardiac troponin-I (Ctni), Ctni, Gata4 were detected in mESCs with TNNI3K overexpression; however, overexpression of α-Actinin and Mlc2v was not detected. Interestingly, Ctnt, connexin40 and connexin45, the markers of ventricular, atrial, and pacemaker cells, respectively, were detected in by real-time PCR in TNNI3K overexpression group. Conclusion: our study indicated that TNNI3K overexpression promoted mESC differentiating into beating cardiomyocytes and induced up-regulating expression of cTnT by PKCε signal pathway, which suggested a modulation of TNNI3K activity as a potential therapeutic approach for ischemic cardiac disease.

Sequencing of Supernumerary Chromosomes of Red Fox and Raccoon Dog Confirms a Non-Random Gene Acquisition by B Chromosomes

“…Still, genes involved in cell differentiation and proliferation did not form a significant cluster, although genes involved in these processes were found on Bs in all species studied. Although not automatically classified, TNNI3K , the first gene identified in Siberian roe deer Bs and the only confirmed example of transcribed B chromosomal gene in mammals [ 37 ], was included in this list based on the report of its role in cardiomyocyte differentiation from embryonic stem cells [ 38 ].…”

“…Similar tendencies were observed in mammals, both in terms of gene function enrichment and reuses (genes encoding cell-cycle related signaling protein kinases— KIT , RET and VRK1 , as well as splicing regulatory KHDRBS3 ). The only species with Bs seemingly lacking genes related to cell cycle or early development is the Siberian roe deer, in which the first gene described and shown to be transcribed, TNNI3K , [ 37 ] has been recently associated with cardiovascular cell differentiation [ 38 ].…”

Preconditioned and Genetically Modified Stem Cells for Myocardial Infarction Treatment

“…Overexpression of the cardiac troponin I-interacting kinase (TNNI3K) gene enhanced the differentiation of ESCs toward cardiomyocytes as demonstrated by the increased levels of cardiac lineage markers (i.e., α-actinin, MLC2v, MHC6, cx43, cTnl, and cTnT) and transcription factors (i.e., GATA4 and Nkx2.5). The underlying mechanism includes suppression of the ERK pathway for p38 as well as c-Jun-NH(2)-terminal kinase (JNK)-modulated apoptosis and regulation of PKCε/p-PKCε to overproduce cTnT-positive cells [ 120 ]. Moreover, the treatment of mouse BMSCs with miR1-2 increased the differentiation of BMSCs into cardiomyocytes through the activation of the Wnt/β-catenin signaling pathway.…”

Recent Progress in Stem Cell Modification for Cardiac Regeneration

“…Other promising targets, which also have demonstrated a positive effect on SC viability, are the apoptosis-regulating protein BCL-2 and the channel-forming connexin43 [ 95 , 145 ]. Moreover, overexpression of several proteins, including NKX2.5, TNNIK, hypoxia-inducible factor-1, CXCR4, and AKT1, has been proven to enhance homing, survival, and differentiation of SC [ 95 , 146 , 147 ].…”