The role of cAMP/PKA pathway in the differentiation of embryonic stem cells into cardiac myocytes

The role of cAMP/PKA pathway in the differentiation of embryonic stem cells into cardiac myocytes

Tomohide Takaya1, Teruhisa Kawamura1, Koh Ono1, Kyoko Hidaka2, Takayuki Morisaki2, Toshio Heike3, Tatsutoshi Nakahata3, Toru Kita4, Koji Hasegawa1.

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

第70回日本循環器学会学術集会 (名古屋), 2006/03/24 (ポスター).

Abstract

Differentiation of embryonic stem (ES) cells into cardiac myocytes involves multiple signaling steps. In order to identify novel pathways required for myocardial cell differentiation, we used retrovirus insertion-mediated random mutagenesis to generate mouse ES cell lines that lose their ability to differentiate into cardiac myocytes. The disrupted genes were identified by 3'-rapid amplification of cDNA end. For high throughput screening, we have established a concise ES cell differentiation system in 96-well plates. Extent of myocardial differentiation was quantified by flow cytometry in an ES cell line, in which GFP is expressed under the control of the cardiac-specific Nkx-2.5 promoter. We have obtained several clones in which myocardial differentiation was apparently reduced. In one of these clones, PRKAR1&beta:, regulatory subunit type 1β of cAMP-dependent protein kinase (PKA), was disrupted. Since PRKAR1β represses catalytic unit of PKA at a basal state 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-methylxanthine for 24-48 hours at early stages of the differentiation. The administration repressed the rate of myocardial cell differentiation. These findings suggest that PRKAR1β is required for myocardial cell differentiation and that activation of cAMP/PKA pathway at early stages inhibits the differentiation.