Abstract 19792: Fibronectin-Induced Mechanotransduction Mediated by alpha (5) beta (1) Integrin Requires Calcium and Vinculin in Mouse Cardiomyocytes
Introduction: Transmembrane integrins that connect the extracellular matrix (ECM) and intracellular cytoskeleton are important for mechanotransduction in cardiomyocytes. This signaling pathway is altered in cardiomyopathy, however, the mechanism linking mechanotransduction with cardiac function is not understood. Designing treatments to target this pathway will require identification of the important cytoskeletal components and cell signals.
Hypothesis: Since vinculin is a major costameric protein associated with α5β1 integrin and Ca2+ is an important contractile signal, we hypothesized that fibronectin (FN) binding to this integrin would increase myocardial mechanical stiffness through vinculin and modulation of intracellular Ca2+ ([Ca2+]i).
Methods: To test this hypothesis, we used atomic force microscopy (AFM) in contact mode and force-volume imaging combined with fluorescence microscopy for measuring cell stiffness and adhesion force between integrin and FN and [Ca2+]i in adult mouse cardiomyocytes. Myocytes from normal (vin +/+) and heterozygous vinculin knockout mice (vin +/−) were used.
Results: Elasticity mapping of the myocyte surface showed that stiffness of the costameric region was greater than non-costameric regions. Vinculin expression at costameres and the intercalated disc was lower in the vin +/− myocytes. Also, adhesion to FN and stiffness in cardiomyocytes from vin +/− mice was decreased by −32%, and −45%, respectively, compared to vin +/+ myocytes (n=10). Rescue of vinculin to the vin +/− cardiomyocytes by transfecting with a vinculin expression plasmid restored FN adhesion and stiffness to control levels (n=10). Application of a FN coated AFM probe to the myocyte surface caused a 26% increase in cell stiffness with a corresponding 17% increase in [Ca2+]i (n=6) which could be prevented by pre-treatment of the cardiomyocytes with a function blocking α5β1 integrin antibody, but not by pretreatment with a function blocking α3β1 integrin antibody.
Conclusions: Thus, our data demonstrate that vinculin is important for myocyte stiffness and FN adhesion, and that FN acting via α5β1 integrin increases cardiac myocyte stiffness and [Ca2+]i suggesting potential importance of this pathway for therapeutic development.
- © 2010 by American Heart Association, Inc.