Mouse embryonic stem cell-derived cardiomyocytes express functional adrenoceptors

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Abstract

The cardiogenic capacity of embryonic stem (ES) cells has been well-investigated. However, little is known about the development of adrenoceptor (AR) systems during the process of ES cell differentiation, which are critically important in cardiac physiology and pharmacology. In this present study, we investigated the expression profile of adrenoceptor subtypes, β-adrenergic modulation of muscarinic receptors and adrenoceptor-related signaling in cardiomyocytes derived from ES cells (ESCMs).

Reverse transcription-polymerase chain reaction revealed that undifferentiated mouse ES cells expressed α1A-, α1B-, α1D- and β2-AR mRNA. However, β1-AR was only expressed after vitamin C induction. The expressions of α1A-, α1D- and β1-ARs increased significantly while α1B- and β2-ARs showed no significant change during the differentiation process. Furthermore, we detected the expression of tyrosine hydroxylase. Both α1-AR and β-AR could activate extracellular responsive kinase in ESCMs. Isoprenaline could inhibit the expression of M2 muscarinic receptor protein. CGP20712A, a β1-AR antagonist, up-regulated the expression of M2 muscarinic receptor while ICI118551, a β2-AR antagonist, showed no effect.

These results indicated that functional adrenoceptors and tyrosine hydroxylase, a critical enzyme in catecholamine biosynthesis, were differentially expressed in ESCMs. Adrenoceptor-related signaling pathways and β-adrenergic modulation of muscarinic receptors were established during differentiation.

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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.

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