Embryonic stem cells and Cardioprotection

Discovery of human ES cells and their potential to differentiate into cardiac myocytes has provided a foundation for future explorations to identify various growth factors and signaling molecules that enhance cardiac myocyte differentiation. Transplanted ES cells in the infarcted mouse and rat hearts can differentiate into cardiac myocytes, however, data obtained from these studies raise major concerns regarding the limited capacity of cardiac myocyte differentiation following ES cell transplantation. In this regard, identification of appropriate factors, which will enhance cardiac myocyte differentiation from ES cells, has gained recent significant attention. In fact, various factors such as TGFβ2, BMP4, vitamin C, etc. have been reported to promote cardiac myocyte differentiation from ES cells. Thymosin β4 (Tβ4), a highly conserved, 43-amino acid peptide, is the most abundant member of the β–thymosin family in a majority of mammalian tissues. Tβ4 has been shown to play an important role in the regulation of cell proliferation, migration, and angiogenesis. Recently, Tβ4 has been found to protect against myocardial injury via inhibiting apoptosis and reducing infarct size with improved myocardial function. Moreover, knockdown of Tβ4 by shRNA deprives embryonic endothelial progenitor cells of their capacity to preserve myocardial function in the ischemic heart. In contrast, Tβ4 overexpression in endothelial cells inhibits apoptosis after hypoxia-reoxygenation and also decreases the adhesion of inflammatory cells to an endothelial cell layer in vitro. Furthermore, Tβ4 stimulates the migration of hair follicle stem cells, resulting in hair growth, and promotes differentiation of epicardial progenitor cells to vascular cells [9].

It has been suggested that Tβ4 is a novel signaling molecule, which activates various cell type differentiation. It was observed a beating EBs derived from stably transfected RFP-ES cells at D9 and this was further enhanced at D12 and D15. Tβ4-ES cells had a significant increase in the number of beating EBs, compared with RFP-ES cells. We further confirmed the presence of cardiac myocytes in the beating EBs using immunofluorescent staining as well as western blot data for S-actin. Similarly, it has been reported that Tβ4 plays a significant role in the heart development at embryonic stage (E10) and at E14.5 because expression of Tβ4 was widely observed in the heart. Cell culture data demonstrating increased beating cardiac myocytes derived from Tβ4-EBs at D9 is in the agreement of the role played by Tβ4 in the developing heart. Furthermore, our real time PCR data from Tβ4 derived EBs revealed significant up regulation of mRNA levels of cardiac transcription factors, GATA-4, Mef2c and Tbx6, suggesting a possible role of up regulating these early transcriptional factors in cardiac myocyte differentiation. Tβ4-ESCs would promote cardiac myocyte differentiation and provide indigenous cardioprotection, thus protecting left ventricular function in the post-MI heart. Previous data suggests that Tβ4-ESCs generate significant numbers of new cardiac myocytes in the injured heart compared to all other experimental and control groups. Pretreatment of mice with Tβ4 peptide promoted cardiac repair, which was attributed to the differentiation of epicardial cells into cardiac myocytes. However, the administration of Tβ4 after MI did not reprogram epicardial cells into cardiac myocytes, and moreover, the native Tβ4 peptide has been the subject of a clinical trial for the treatment of MI.

The Notch pathway plays a pivotal role in a myriad of cardiac developmental and pathological scenarios, including regulation of cardiac progenitor cell fate and the proliferation of cardiac myocytes. It was also clear that transplanted Tβ4-ESCs differentiate into cardiac myocytes and these newly formed cells have increased expression of Notch-1 in the post-MI heart, suggesting Notch-1 is also regulated in the derivation of cardiac myocytes from the transplanted cells. It has been reported that Tβ4 reduces cardiac myocyte death in the cell culture system after hypoxia and also in the adult mouse heart after MI, regardless of intra-myocardial or intraperitoneal systemic application. It was found that transplantation of unmodified ES cell reduces to some extent cardiac myocyte apoptosis in the MI model. In particular, transplanted Tβ4-ES cells provided significantly greater protection against cardiac myocyte apoptosis in the infarcted heart than RFP-ES cells.

The Akt signaling pathway plays a pivotal role in a host of cellular processes including growth, proliferation, and survival. Phosphatase and tensin homolog (PTEN) inhibits PI3K phosphorylation and subsequent phosphorylation and activation of Akt. Recent data suggests a significant increase in PTEN activity following MI, which is corroborated in other myocardial injury studies. Following transplantation of either Tβ4-ES cells or RFP-ES cells, we report a significant decrease in PTEN expression, compared with the MI group. Downregulation of PTEN would lead to increased Akt activity, which would account for the diminished cardiac myocyte apoptosis and abrogated adverse cardiac remodeling. The later data suggests that Tβ4-ES cells enhance cardioprotection against MI at least in part through inhibition of apoptosis and activation of the Akt pathway. Moreover, cardiac myocyte apoptosis and fibrosis formation contribute significantly to the pathological cardiac remodeling following MI. A dramatic loss of cardiac myocytes in the infarcted heart results in subsequent replacement and proliferation of non-myocytes, such as cardiac fibroblasts, forming a fibrotic network to rescue the architecture of injured myocardium. This process involves metalloproteinase (MMP) activation, extracellular matrix (ECM) degradation and collagen deposition in the infarcted heart, leading to interstitial and vascular fibrosis, scar formation, and eventual cardiac dysfunction. In agreement to previous studies transplanted ES cells (Tβ4-ES or RFP-ES cells) inhibit the formation of fibrosis in the MI model. Furthermore, there is a significant decrease in fibrosis in the infarcted heart transplanted with Tβ4-ES cells, compared with the RFP-ES cell group.

Transplanted ES cells overexpressing Tβ4 significantly improve cardiac function 2 weeks after MI, as demonstrated by increased fractional shortening and ejection fraction. On this basis it was suggested that enhanced left ventricular output following transplantation of Tβ4-ESCs is indicative of enhanced cardiac myocyte differentiation and augmented cardioprotection provided by these cells in the injured myocardium [9].