Cardiac Stromal Cell Reprogramming to Functional Cardiovascular Precursors via Epigenetic Intervention

INTRODUCTION:Cardiac mesenchymal stromal cells (CStC) are more efficient than the bone marrow counterpart differentiating into adult cardiomyocytes. These cells, however, do not express cardiac stem cell markers and the molecular basis of their regeneration capacity are currently unknown. To molecularly characterize CStC differentiation a series of biological and biochemical investigations were performed after treatment with epigenetically active molecules able of cellular reprogramming.


METHODS AND RESULTS: A series of experiments were performed in which CStC were cultured in IMDM added of 20% fetal bovine serum (FBS) or exposed to low serum (5% FBS) in the presence or absence of 5 mM all-trans retinoic acid (ATRA), 5 mM phenylbutyrate (PB) and 200 mM diethylenetriamine/nitric oxide (DETA/NO), realizing an unprecedented epigenetic reprogramming coktail (Epic). While significantly inhibiting cell proliferation and preserving vitality, Epic, strongly up-regulated the expression of cardiac-resident stem cell markers including c-Kit, MDR-1, Notch, Jagged-1 and Numb. The expression of the pluripotency factors Oct4 and Nanog was negative and Nucleostemin down-regulated. In reprogrammed cells, the MDR-1 transporter was functionally active as indicated by the rhodamine extrusion assay. Further analyses revealed that Epic increased a large number of cardiovascular progenitors and precursors markers including Nkx2.5, GATA4, Flk-1, GATA6 and a-sarcomeric actin along with the expression of miR-1 and miR-499. A fraction of epic-treated cells also exhibited a small sodium current completely blocked by 10µM TTX. Remarkably, Epic treatment determined global histone modifications, characterized by an increased H3K4me3 and H4K16Ac density typically associated to a transcriptionally competent chromatin conformation often observed in cells with a high developmental potential.
CONCLUSIONS: Altogether these data indicate that CStC may be epigenetically reprogrammed to acquire a reversible cardiovascular niche-like phenotype