Regulation of PTEN/Akt Pathway Enhances Cardiomyogenesis and Attenuates Adverse Left Ventricular Remodeling following Thymosin beta 4 Overexpressing Embryonic Stem Cell Transplantation in the Infarcted Heart

Abstract

Thymosin beta 4 (T beta 4), a small G-actin sequestering peptide, mediates cell proliferation, migration, and angiogenesis. Whether embryonic stem (ES) cells, overexpressing T beta 4, readily differentiate into cardiac myocytes in vitro and in vivo and enhance cardioprotection following transplantation post myocardial infarction (MI) remains unknown. Accordingly, we established stable mouse ES cell lines, RFP-ESCs and T beta 4-ESCs, expressing RFP and an RFP-T beta 4 fusion protein, respectively. In vitro, the number of spontaneously beating embryoid bodies (EBs) was significantly increased in T beta 4-ESCs at day 9, 12 and 15, compared with RFP-ESCs. Enhanced expression of cardiac transcriptional factors GATA-4, Mef2c and Txb6 in T beta 4-EBs, as confirmed with real time-PCR analysis, was accompanied by the increased number of EB areas stained positive for sarcomeric alpha-actin in T beta 4-EBs, compared with the RFP control, suggesting a significant increase in functional cardiac myocytes. Furthermore, we transplanted T beta 4-ESCs into the infarcted mouse heart and performed morphological and functional analysis 2 weeks after MI. There was a significant increase in newly formed cardiac myocytes associated with the Notch pathway, a decrease in apoptotic nuclei mediated by an increase in Akt and a decrease in levels of PTEN. Cardiac fibrosis was significantly reduced, and left ventricular function was significantly augmented in the T beta 4-ESC transplanted group, compared with controls. It is concluded that genetically modified T beta 4-ESCs, potentiates their ability to turn into cardiac myocytes in vitro as well as in vivo. Moreover, we also demonstrate that there was a significant decrease in both cardiac apoptosis and fibrosis, thus improving cardiac function in the infarcted heart