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.

Effects of the Herbal Product Curcumin on Cardiomyocytes in Micromass Culture and the Potential Role It May Play in Pregnancy and Development

“…The cell density was always adjusted to 3 x 106 cells/mL. Cell density of 3x106 cells/ml was used, as this was identified as optimal in previous studies [ 10 ]. Cells were seeded as 20μl drops into a 24-well plate (Nunclon, UK).…”

“…Previous studies in this laboratory have demonstrated the presence of cardiac muscle cells as opposed to skeletal muscle cells in micromass culture [ 4 ]. In addition, more detailed immunocytochemical studies in similar cultures performed by Ahir et al demonstrated the presence of predominantly cardiomyocytes and the role of connexins in the mediation of the effects of caffeine on chick cardiomyocytes in micromass culture [ 10 ]. Immunohistochemistry was used to show that cells within the chick MM culture employed in this study primarily yielded cardiomyocytes, showing positive staining for cardiac-specific α- and β-myosin heavy chains and cardiac troponin T antibodies.…”

Elementary calcium signaling in arterial smooth muscle

“…RyRs may modulate gap junction formation during stress and aging. The pan RyR activator caffeine decreased the expression of gap junction forming proteins, the connexins Cx40, Cx43, and Cx45, and reduced the contractility of cardiomyocytes [74]. These effects have been attributed to long-lasting activated ryanodine receptors by caffeine.…”