Embryonic stem cells (ESCs) can differentiate into functional cardiomyocytes and 
thus represent a promising cell source for cardiac regenerative therapy. 
Nevertheless, the therapeutic application of ESC-derived cardiomyocytes is 
limited by the low efficacy of the current protocol for cardiac differentiation 
and their immature phenotypes. Although thyroid hormone is essential for normal 
cardiac development and function, its role in the cardiac differentiation of 
ESCs, as well as the maturation of ESC-derived cardiomyocytes, remains unclear. 
In this study, we examined the cardiac differentiation of murine ESCs in the 
presence of T(3) for 7 d using flow cytometry, RT-PCR, cellular 
electrophysiology study, and confocal calcium imaging. Compared with control 
conditions, T(3) supplementation increased the number of ESC-derived 
cardiomyocytes and was accompanied by up-regulation of a panel of cardiac 
markers, including Nkx2.5, myosin light chain-2V, as well as alpha- and 
beta-myosin heavy chain. More importantly, electrophysiological study revealed 
that ESC-derived cardiomyocytes exhibited more adult-like phenotypes after T(3) 
supplementation based on action potential characteristics. They also exhibited 
more adult-like calcium homeostasis properties. These phenotypic changes were 
associated with up-regulation of sarco(endo)plasmic reticulum calcium ATPase-2a 
and ryanodine receptor-2 expression. In addition, the classical (genomic) 
pathway was shown to be involved in T(3)-induced cardiac differentiation of 
ESCs. Our results show that T(3) supplementation promotes cardiac 
differentiation of ESCs and enhances maturation of electrophysiological, as well 
as calcium homeostasis, properties of ESC-derived cardiomyocytes.