Abstract 18631: The Histone Methyltransferase Smyd3 Modulates Growth and Differentiation of Human Cardiac Stem Cells

Abstract

Stemness and pluripotency of embryonic stem cells (ESCs) are largely mediated by epigenetic modifications. The objective of this study was to determine whether growth and commitment of human cardiac stem cells (hCSCs) are similarly regulated. Discarded surgical specimens were enzymatically digested and c-kit-positive hCSCs were sorted by FACS. Genome-wide analysis of histone modifications documented that adult hCSCs are characterized by a bivalent chromatin configuration similar to that of ESCs with repressive and activating marks at lysine residues of histones H3 and H4. The high levels of di- (H3K4me2) and tri-methylation (H3K4me3) at lysine 4 of histone H3 prompted us to test the possibility that the histone methyltransferase Smyd3 is involved in the epigenetic control of hCSC function. Smyd3 downregulation by siRNA decreased the levels of H3K4me2 and H3K4me3 and resulted in a marked attenuation in hCSC replication and differentiation into myocytes and vascular cells. In contrast, Smyd3 overexpression led to a 1.6-fold increase in dividing hCSCs and a 2-fold increase in hCSCs committed to the myocyte lineage. Thus, we studied whether Smyd3 regulates a group of genes controlling the growth of hCSCs and their specification to the myocyte and vascular fate: telomerase (TERT), Nkx2.5, and GATA6. Downregulation of Smyd3 decreased the activity of telomerase, and the expression of Nkx2.5 and GATA6. By immunolabeling, Smyd3 co-localized with TERT, Nkx2.5 and GATA6 in hCSC nuclei. To document the role of Smyd3 in the transcription of TERT, Nkx2.5, and GATA6, ChIP assays with antibodies against Smyd3 and H3K4me3 were performed in hCSCs. Amplification and sequencing of the precipitated DNA fragments demonstrated that Smyd3 occupied the TERT, Nkx2.5, and GATA6 promoter regions in which its consensus sites are located. Gene reporter assays with plasmids carrying the 5’ flanking regions of TERT or Nkx2.5 containing mutated Smyd3 binding sites led to a marked decrease in promoter activity of the two genes. Importantly, high levels of expression of Smyd3 were found in the regenerated myocytes derived from hCSCs injected in immunosuppressed infarcted rats. Thus, Smyd3 plays a central role in the modulation of growth and differentiation of hCSCs in vitro and in vivo.