5-HT2 receptors enable cardiac differentiation of mouse embryonic stem cells

Identification of factors able to promote cardiac differentiation of embryonic stem cells (ES) is essential to envisage cardiac ES cell therapy. During heart development , serotonin (5HT) acts as morphogen via type 2 receptors (5HT2). The role of 5HT2 during cardiac differentiation of ES is unknown. Mouse ES (mES) differentiated into cardiomyocytes via embryoid bodies (EBs) formation within medium containing 5 microM 5-HT, as assessed by HPLC. PCR analysis detected 5HT2A, 2B and 2C receptors in cardiac differentiated EBs only , receptors being absent in undifferentiated mES. Functional coupling of 5HT2 was tested by microelectrode technique on beating EBs. Application of alphaMe-5HT (5HT2 selective agonist) decreased frequency of spontaneous firing; this effect was accompanied by a decreased frequency of intracellular calcium oscillation, determined by fluorescence levels, and by a reduction of ICa caused by exposure to 5HT of patch–clamped cells. Contribution of 5HT2 to cardiac differentiation was assessed by using 5HT2 antagonists: mianserin (non selective), SB215505 (2B selective) and ketanserine (2A selective). At 7 days of differentiation antagonists significantly reduced pulsating activity of EBs with respect to control. Accordingly, antagonists impaired mRNA expression of cardiac transcription factors NK2.5, GATA4 and MEF2C and of ventricular myosin light chain (mlc-2v). In conclusion 5HT2 receptors are selectively expressed in cardiomyocytes differentiated from mES, functionally coupled to intracellular calcium handling and essential to enable cardiac specification of the undifferentiated cells.