Biochemical and Biophysical Research Communications
Mouse embryonic stem cell-derived cardiomyocytes express functional adrenoceptors
Section snippets
Materials and methods
Materials. All agonists and antagonists, mitomycin C, β-mercaptoethanol, vitamin C and anti-mouse α-actinin monoclonal antibody were purchased from Sigma Chemical (St. Louis, MO, USA). Phospho-p44/p42 MAP Kinase (Thr202/Tyr204) antibody and p44/p42 MAP kinase (ERK1/2) antibody were purchased from Cell Signaling Technology (Danvers, MA, USA). Leukemia inhibitory factor (LIF), anti-mouse tyrosine hydroxylase monoclonal antibody and rabbit anti-mouse M2 muscarinic acetylcholine receptor affinity
ES cells differentiated into cardiomyocytes after induction
ES cells were propagated on feeder cells for two days. Cardiomyocyte differentiation was initiated by inducing EB formation from undifferentiated ES cells. After five days suspension in culture, EBs were allowed to differentiate further in adherent cultures. Spontaneously contracting EBs appeared at differentiation day 10. The number of beating EBs in 24-well-plates was recorded at different time points. Spontaneously contracting cells appeared as clusters and were identified in approximately
Discussion
Previous studies have investigated the function of adrenoceptor signaling pathways in adult and neonatal cardiomyocytes. However, the changes in adrenoceptor expression in the early development of cardiomyocytes were unclear. Our present study has detected the changes in expression of five main adrenoceptor subtypes (α1A, α1B, α1D, β1 and β2) during the differentiation of ESCMs. Our results demonstrate that during the development of the β-AR system, β2-AR emerges earlier than β1-AR but its
Acknowledgments
We are grateful to Dr. Jason WONG, University of Cambridge, UK, for his kind help in the preparation of this manuscription. We thank Prof. Youyi Zhang and Dr. Ming Xu, Institute of Vascular Medicine, Peking University Third Hospital, for their helpful suggestions during the work. This work was supported by the National Natural Sciences Foundation of China (30570711).
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These authors contributed equally to this work.