Abstract 15396: HASF, a Novel Paracrine Stem Cell Factor Protein Stimulates Neonatal Cardiomyocyte Cell Cycle Progression Through PI3K-Akt-CDK7 Pathway
Introduction The regenerative capacity of the mouse heart is lost during the first few days after birth coinciding with withdrawal of cardiomyocytes from cell cycle. Consequently, the adult mammalian heart has limited capacity to regenerate after injury. Identification of factors that induces cardiomyocyte proliferation is of high interest and the focus of extensive investigation. While, it is established that stem cells can enhance cardiac repair by affecting cell survival/ angiogenesis via paracrine mechanisms, few studies indicate their role in cardiomyocyte proliferation. We have recently identified C3orf58 as a novel mesenchymal stem cell paracrine factor which we named Hypoxia regulated Akt mediated Stem cell Factor (HASF). Here, we tested the hypothesis that HASF can induce cardiomyocyte division and proliferation and determined the signaling pathway mediating its paracrine effect.
Methods Neonatal ventricular cardiomyocytes from 2-3 day old rats were stimulated in culture for 7 days with 10-100 nM HASF. BrdU incorporation was analyzed by Elisa and FACS assays. Immunofluoresence (IF) staining was used for BrdU+/cTnT+, Ki67+/cTnT+ cells. Expression of cell cycle and signaling marker proteins was assessed by western blot.
Results HASF-treated neonatal cardiomyocytes exhibited 60% (P=0.01) increase in BrdU incorporation as compared to control untreated cells. These results were confirmed by IF staining showing 50-100% increase in the BrdU+ or Ki67+ cells coexpressing cTnT. Moreover, the cell cycle regulators CDK7 and cyclin H increased in HASF treated cells, highlighting the proliferative effects of the HASF protein. HASF upregulated the P-Akt levels. Moreover, the presence of specific Akt pathway inhibitors; LY294002 and Rapamycin decreased both the BrdU uptake and the levels of CDK7 and cyclin H. Importantly, in vivo cardiac overexpression of HASF using a transgenic mouse model led to enhanced neonatal cardiomyocyte proliferation.
Conclusion Our studies support the hypothesis that HASF, a novel stem cell paracrine factor stimulates DNA synthesis and induces proliferation in mammalian neonatal cardiomyocyte, in vitro and in vivo. The implications of this finding in cardiac regeneration biology and therapeutics are significant.
- © 2011 by American Heart Association, Inc.