cAMP/PKA signaling pathway affects differentiation of mouse embryonic stem cells into cardiac myocytes

cAMP/PKA signaling pathway affects differentiation of mouse embryonic stem cells into cardiac myocytes

Tomohide Takaya1, Teruhisa Kawamura1, Koh Ono2, Kyoko Hidaka3, Takayuki Morisaki3, Toshio Heike4, Tatsutoshi Nakahata4, Toru Kita2, Koji Hasegawa1.

  1. Division of Translational Research, Kyoto Medical Center, National Hospital Organization, Kyoto, Japan.
  2. Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  3. Department of Bioscience, National Cardiovascular Center Research Institute, Osaka, Japan.
  4. Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan.

The 1st International Congress of Cardiomyopathies and Heart Failure (Kyoto, Japan), 2007/03/12 (Poster).

Abstract

Background: Differentiation of embryonic stem (ES) cells into cardiac myocytes involves multiple signaling steps. We performed comprehensive mutagenesis assay to identify novel pathways required for myocardial cell differentiation. In this study, we found that cAMP/PKA signaling affects differentiation of mouse ES cells into cardiac myocytes.

Methods and Results: We performed retrovirus insertion-mediated random mutagenesis to generated mouse ES cell lines that lose their ability to differentiate into cardiac myocytes. Extents of myocardial differentiation of mutant ES cell lines were quantified by flow cytometry as a positive-rate of GFP expressed by Nkx-2.5 promoter. Myocardial cell differentiation was induced by trichostatin A (TSA), a specific histone deacetylases inhibitor. We have obtained several clones in which myocardial cell differentiation was apparently reduced. The disrupted genes were identified by 3'-RACE. In one of these clones, PRKARIb, regulatory subunit type Ib of cAMP-dependent protein kinase (PKA), was disrupted. Since PRKARIb represses catalytic unit of PKA at a basal stae and is involved in the inhibition of cAMP/PKA signaling, we examined whether activation of cAMP/PKA will repress the differentiation of ES cells into cardiac myocytes. In an ES cell model of developing embryoid bodies, we administrated a cAMP activator, 3-isobutyl-1-metylxanthine (IBMX), at early stages of the differentiation. The administration of IBMX repressed TSA-induced expression of Nkx-2.5 and myocardial differentiation in wild-type ES cells.

Conclusion: These findings suggest that PRKARIb is required for myocardial cell differentiation, and that activation of cAMP/PKA pathway at early stages inhibits differentiation of mouse ES cells.