Abstract 20687: The Histone Methyltransferase Smyd3 Promotes the Myocyte Commitment of Human Cardiac Stem Cells
The phenotypic properties of embryonic and adult stem cells are largely mediated by epigenetic modifications which control stemness, multipotentiality and fate specification. Our objective was to identify whether similar molecular mechanisms regulate the primitive state of human cardiac stem cells (hCSCs) and their commitment to cardiac lineages. Undifferentiated hCSCs showed a bivalent chromatin configuration similar to that found in embryonic stem cells. Genome-wide expression of H3K27me3, H3K4me2 and H3K9me2 was detected by Western blotting and immunolabeling in c-kit-positive hCSCs. This chromatin conformation may represent a condition in which, following the removal of the repressive function of H3K27me3, myocyte lineage-related genes can be transcriptionally activated by H3K4me2/3. To test this possibility, we assessed the function of the histone methyltransferase Smyd3 in hCSCs. This enzyme was chosen because of its selective di- and tri-methylation of lysine 4 at histone H3. Also, non-functional mutations of the c-kit receptor in CSCs resulted in inadequate myocyte generation and Smyd3 downregulation. To determine the role of Smyd3 in hCSCs, a loss and gain of function approach was implemented. By siRNA strategy, the transcript and protein levels of Smyd3 were ∼3.5-fold lower than in control cells. Inhibition of Smyd3 decreased by 60% the fraction of cycling hCSCs and increased 2.5-fold apoptosis. Attenuation of hCSC growth was coupled with a decline in the expression of telomerase, TERT, and the myocyte specific transcription factor Nkx2.5. By immunolabeling, siRNA for Smyd3 resulted in a 60% decrease in the fraction of hCSCs expressing Smyd3 in their nuclei. Parallel changes were seen for TERT and Nkx2.5. Importantly, these three proteins showed a consistent nuclear co-localization in control and Smyd3-inhibited hCSCs. Smyd3 downregulation led to a marked decrease in di- and tri-methylated lysine 4 at H3 suggesting that reduced methylation of the promoter regions of TERT and Nkx2.5 was involved in the changes in their. Conversely, transfection of hCSCs with a plasmid carrying Smyd3 was coupled with upregulation of TERT and Nkx2.5. Thus, Smyd3 represents a crucial epigenetic modulator of hCSC commitment to the myocyte lineage.
- © 2010 by American Heart Association, Inc.