Serotonin 5-HT2 receptors are essential for in-vitro cardiac differentiation of mouse embryonic stem cells.

Background: Identification of factors regulating cardiac differentiation of embryonic stem cells (ES) is important to obtain a cell source suitable for cardiac cell transplantation therapy. In the developing heart, serotonin (5-HT) plays a crucial role during embryogenesis via type 2 receptor subtypes (5-HT2). The role of 5-HT2 during cardiac differentiation of ES cells is unknown. Methods and results: Mouse ES (mES) spontaneously differentiate toward a cardiac phenotype in-vitro via formation of embryoid bodies (EBs); 5-HT content in the differentiation medium assayed by HPLC was about 5 µM. RT-PCR analysis of 5-HT2 revealed that 2A, 2B and 2C isoforms are selectively expressed during cardiac differentiation, while absent in undifferentiated mES. Functional coupling of 5-HT2 was tested by fluorescence detection of intracellular calcium levels at the confocal microscope. In myocytes isolated from EBs, exposure to either 5-HT (1 µM) or alphaMe-5-HT (100 nM, a 5-HT2 selective agonist) decreased intracellular calcium. This effect was accompanied by a reduction of T-type Ca-current caused by acute exposure to 5-HT (1 µM) of patch–clamped cells. To establish the contribution of 5-HT2 to cardiac differentiation, mES were grown in the presence of mianserin (1-10 µM, a 5-HT2 antagonist) or SB215505 (1 µM, a selective 5-HT2B antagonist). Clusters of pulsating cardiomyocytes were observed in >20% of EBs at day 7 of differentiation. In contrast, only 6% of EBs grown in the presence of 1 µM mianserin showed beating foci. No pulsating activity was detected if mianserin was raised to 10 µM. SB215505 (1 µM) reduced the appearance of beating foci to 16%. Accordingly, 5-HT2 blockade impaired expression of cardiac transcription factor MEF2C, which was present in control conditions (mRNA relative expression/GADPH 0.18%±0.033) and undetectable in the mianserin-treated EBs. Finally, expression of structural cardiac genes was markedly affected, being ventricular myosin light chain present in control condition (mRNA relative expression/GADPH 1.2%±0.23), and undetectable in mianserin-treated EBs. Conclusions: these data show for the first time that 5-HT2 isoforms are selectively expressed in cardiomyocytes differentiated from mES and have a functional role in modulating intracellular calcium handling. Their pharmacological blockade during differentiation suppresses/reduces the occurrence of spontaneous contractile activity, the expression of cardiac specific transcription factors and sarcomeric proteins. Thus, 5-HT via 5-HT2 stimulation plays a key role in cardiomyocyte development.