PT356 VCP746, a novel A1/A2 adenosine receptor agonist, reduces hypertrophy in cardiac myocytes and collagen synthesis in cardiac fibroblasts and renal mesangial cells

Abstract

Introduction: The establishment of induced pluripotent stem cells (iPSCs) potentially provides a unique resource for generating patient specific cardiomyocytes to for cardiac repair. However, the poor efficiency and yield of cardiomyocytes currently limits the application of this technology. Insulin-like growth factor binding protein 4 (IGFBP4) has been reported to play critical role in cardiomyocytes differentiation of embryonic stem cells (ESCs). But whether it promotes iPSCs into cardiomyocytes is unclear. Objectives: In the present study, we aim to explore the role of IGFBP4 in the cardiogenesis of mouse iPSCs. Methods: For differentiation, iPSCs were trypsinized and EBs were generated by the threedimensional hanging-drop method. EBs were grown in hanging drops for three days (day 0 to day 3), each drop initially consisted of 1000 cells in 30 mL of differentiation medium (growth medium without LIF). IGFBP4 (Sino Biological Inc.) was added at day 3-8,day 3-6 and day 6-8 period under 1 mg/ mL to determine the effective time-window. Results: We observed that IGFBP4 treatment at late-stage during differentiation process of mouse iPSCs greatly enhanced the beating frequency of embryoid bodies (EBs). The expressions of Nkx2.5 (cardiac specific transcription factor), a-MHC, a-actinin and Troponin I (cardiac specific protein) were significantly enhanced by IGFBP4 treatment. Immunostaining analysis showed that a-MHC, TNNT2 and connex43, typical cardiac markers, were obviously expressed in isolated cardiomyocytes from iPSCs with or without IGFBP4 treatment. Further study revealed that IGFBP4 had little effect on the apoptosis of EBs. But it significantly promoted the proliferation of cardiomycytes from iPSCs chracterized by higher ratio BrdU positive cells in differentiated cardiomyocytes. We next observed that IGFBP4 inhibted b-catenin expression in cytosol of EBs at late-stage during differentiation of iPSCs. Knockdown of b-catenin using a siRNA technique promoted the proliferation of differentiated cardiomyocytes and enhancing cardiomyocytes induction of iPSCs, suggesting that the effect of IGFBP4 on cardiomyocyte differentiation of iPSCs has relationship with b-catenin signaling pathway. Conclusion: In conclusion, IGFBP4 promotes cardiogenesis of iPSCs by enhancing the proliferation of differentiated cardiomyocytes through b-catenin signaling. Disclosure of Interest: None Declared