Abstract 17819: The Histone Methyltransferase Smyd3 Regulates Cardiac Differentiation of Human Cardiac Progenitor Cells by Directly Regulating the Expression of Nkx2.5

The balance between self-renewal and differentiation of adult stem cells is largely controlled by epigenetic modifications that enhance or repress gene expression. The objective of this study was to identify epigenetic regulators of human cardiac differentiation. To this effect, we established an in vitro system using cells derived from c-kit positive human cardiac progenitor cells (hCPCs) in the process of lineage specification. These early committed cells (ECCs) express proteins specific to cardiac cell classes, including Nkx2.5, α-sarcomeric actin, von Willebrand factor and α-smooth muscle actin. We focused on one of the most well described activating modifications, the di-methylation and tri-methylation of histone H3 in the residue of lysine K4, i.e., H3k4me2 and H3K4me3. These modifications are established by the histone methyltransferase Smyd, which has multiple isoforms; the prevalent Smyd isoform expressed in ECCs is Smyd3. Silencing of Smyd3 leads to a reduction in the levels of H3K4me2 and H3K4me3, indicating its role in maintaining these activating epigenetic marks in ECCs. The loss of Smyd3 also results in a loss of proliferative potential, reflected in a reduction of the number of cells positive for Ki67. In development, cardiomyocytes express the protein telomerase (Tert) which is implicated in the preservation of telomere length. Smyd3 was found to enhance the expression of Tert by directly binding the promoter region of the human Tert gene. More importantly, the transcription factor Nkx2.5, activated during the commitment of hCSCs to the myocyte lineage, is directly regulated by Smyd3. Smyd3 binds the promoter of Nkx2.5 increasing Nkx2.5 expression. By silencing Smyd3, we observed a drastic reduction in the number of cells expressing Nkx2.5 and, thereby, in the number of forming myocytes. To confirm the role of Smyd3 in the generation of human cardiomyocytes, we performed immunohistochemistry on sections of tissue obtained from the left ventricle of patients with ischemic cardiomyopathy. Expression of Smyd3, together with Nkx2.5, was found in ECCs throughout the non-infarcted myocardium. In conclusion, Smyd3 appears to be essential for the commitment of hCPCs to the cardiomyocyte lineage in vitro and in vivo.