Epigenetic control of cardiac differentiation in mouse embryonic stem cells: Role of thyroid hormone in pacemaker cell commitment.

Heart transplantation is the usual solution for severe heart failure although it is limited by the number of donors and by restrictive inclusion criteria. These limits prompted research into stem cell-based alternatives, restricted however by a scarce source of adult stem cells and their relatively inefficient contribution to heart regeneration. Embryonic stem cells (ES) retain great promise as unlimited source of pluripotent progenitors for myocardial regeneration, but their therapeutics use is still impaired by ethical concerns and incomplete understanding of factors governing cardiomyocytes differentiation. We aimed at creating ES-derived cardiomyocytes for experimental cell transplantation therapies in mice. Mouse ES cells (mES) were treated with Triiodothyronine (T3) and/or anacardic acid (AA), a natural epigenetic drug inhibiting histone acetylases, to investigate whether and how efficiently cardiac cell differentiation occurred. For easier identification of differentiated cells, engineered mES expressing red fluorescent protein (RFP) under the NCX1 gene promoter, an early cardiac differentiation marker, were used. The hanging-drop embryoid body (EB) technique was adopted to reproduce in vitro an embryo-like architecture. mES-derived RFP-positive cardiomyocytes were collected and analysed by RT-PCR, WB, electrophysiology and FACS. Single (AA or T3) or combined treatments (AA+T3) promoted cardiac differentiation, anticipating EBs beating and increasing beating areas. Mechanistically, T3 did not affect acetylation whereas AA decreased lysine acetylation of histonic and non histonic proteins. In these conditions, RT-PCR showed decreased stemness genes expression in EBs. Specifically, AA increased expression of Nkx2.5, a cardiac differentiation key gene while T3 treatment repressed miR133a expression, a specific miR known to regulate potassium voltage-gate channels (KCNA/D family), a feature of cardiac pacemaker phenotype. This finding was confirmed by electrophysiology showing increased spontaneous firing, in support of T3-inducing pacemaker-cell-like differentiation. Our data indicate T3 and AA as important controllers of multiple epigenetic signals and provide evidence for the production of specialized functional cardiac cells suitable for therapeutic intervention.