Abstract 536: Mechanical Properties of the Interaction Between Fibronectin and α5β1-Integrin on Cardiomyocytes Studied by Atomic Force Microscopy
The molecular mechanisms that lead to hypertrophic cardiomyopathy are poorly understood. Integrins that link the extracellular matrix (ECM) and intracellular cytoskeleton function as mechanotransducers to translate the mechanical force to intracellular signals. It has been suggested that integrin-mediated signaling events are involved in the hypertrophic remodeling processes. In this study, we characterized the unbinding force and adhesion events of the ECM protein, fibronectin (FN)-α5β1 integrin interactions on the membrane of isolated mouse cardiomyocytes using atomic force microscopy (AFM), which determines mechanical changes at nano-molecular resolution. It is postulated that these initial binding events are important to the subsequent myofilament activation processes in the cardiomyocytes. AFM probes were coated with FN (2.7 μM) and used to measure unbinding force between FN and integrins by quantifying the unbinding force required to break single or multiple FN-integrin bonds. FN-cardiomyocyte (integrin) adhesion events were selectively blocked by 59% or 50% in the cells pretreated with anti-α5β1 integrin (100 nM) or RGD peptide (100 μM) (P<0.05), respectively. FN-integrin adhesion events were not inhibited by pretreated with anti-α3β1 integrin (100 nM) or RAD (100 μM, control peptide for RGD). Binding probability was calculated as number of force curves with adhesion / number of total force curves sampled. FN binding probability was significantly reduced by 35% or 39% in the presence of anti-α5β1 or RGD (P<0.05), respectively. However, the FN-α5β1 first peak unbinding force (represented as detachment of FN from α5β1, 36.5±0.3 (SEM) pN) was not significantly changed by any pre-treatments. When AFM probes were coated with anti-rat MHC mAb (MHC, 1.7 μM), the MHC-cardiomyocyte adhesion events were 61% less than and first unbinding force was 20% smaller than FN probes (P<0.05). These results indicate that FN-adhesive activities are functional specific and primarily bound to α5β1 integrin in cardiomyocytes. Thus, the use of AFM and its specific single-molecular force measurements makes it as a powerful tool for investigating the mechanical properties of integrin-ECM interactions and their regulation in the hypertrophic remodeling.