title: The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system.
abstract: Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low-resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell-to-cell communication pathways, resulting in an inability to co-ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 μm (P < 0.05), while not affecting cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in-cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non-cytotoxic concentrations (≥2000 μm), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 μm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.
authors: National Center for Computational Toxicology (B205-01), US Environmental Protection Agency, Research Triangle Park, NC, USA.
authors: Bhavesh K
authors: BK
authors: Ahir
authors: School of Life Sciences, Queen's Medical Centre, University of Nottingham, Nottingham, UK.
authors: Margaret K
authors: MK
authors: Pratten
mesh_terms: D000818:Animals; D002110:Caffeine / Q000008:administration & dosage / Q000633:toxicity*; D002478:Cells, Cultured; D002642:Chick Embryo; D018031:Connexin 43 / Q000235:genetics / Q000378:metabolism*; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D023381:Endpoint Determination; D017629:Gap Junctions / Q000187:drug effects / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D010766:Phosphorylation; D000097003:Gap Junction alpha-5 Protein
chemical_list: D002110:Caffeine; D018031:Connexin 43; D017630:Connexins; D000097003:Gap Junction alpha-5 Protein
keywords: Caffeine; Congenital heart defects; Embryonic chick cardiomyocytes micromass (MM) culture; Embryotoxicity/Teratogenicity; Gap junction proteins (Cx40, Cx43, Cx45)