Integrin Response To Altered Actin-Myosin Mechanochemistry In Cardiac Myocytes

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Altered contractility in cardiac myocytes associated with motor protein mutations has been implicated in several pathologies such as familial hypertrophic (FHC) and dilated (DCM) cardiomyopathies. The mechanism(s) by which these altered contractile forces result in cell phenotype changes are poorly understood. However, they are generally believed to involve mechanotransduction of force by cell adhesion molecules such as the integrin class of proteins. Here we investigate the use of blebbistatin and other small molecule effectors of actin-myosin mechanochemistry as a chemical model for the reduced contractile force associated with the pathogenesis of DCM. In order to establish intercalated-disk cell associations in vitro that are representative of in vivo conditions, we employ micropatterned cardiac myocyte cell culture, where the extracellular matrix proteins collagen and laminin are printed onto the culture vessel surface in 10μm wide lines. Fluorescent polymer microspheres (0.25μm dia.) are embedded in the collagen layer. As the beads move from myocyte contraction, the movement amplitude is measured by nanometer-resolution position analysis, both in the presence of blebbistatin and in the rescue state where blebbistatin is removed. Relative force generation is calculated from the position analysis data and matrix modulus. Cells are then fixed and prepared with fluorescent antibodies for observation of connexin-43 and β1 integrin expression and localization by confocal microscopy. By employing micropatterned cardiac myocyte cell culture, optical contractile force measurement, and subsequent observation of the expression and localization of connexin-43 and β1 integrin, we assess the changes in integrin activity due to blebbistatin-induced reduction in contractile force.