Mechanical Tension Decreases Fibrillin-1 Calcium Binding And Stiffness

Introduction: Aneurysm pathogenesis involves a complex interplay between vascular tissue remolding and hemodynamics. In Marfan syndrome, vascular tissues fail to remodel appropriately, causing aneurysms. The root cause for this is mutations in the extracellular matrix protein fibrillin-1. However, the precise molecular mechanisms through which fibrillin-1 contributes to aneurysm formation remain unclear. Hypothesis: We hypothesize that fibrillin-1 calcium binding, which is known to regulate extracellular proteolysis and growth factor sequestration, decreases under mechanical tension. Methods: Recombinant fragments of human fibrillin-1 containing the hypothesized force-sensitive calcium binding domains were genetically engineered with unique N- and C-terminal peptide tags (Fig. 1A). The C-terminal tag was used to irreversibly attach the protein to an atomically flat surface (Fig. 1B) The N-terminal tag was then used to reversibly bind a functionalized atomic force microscopy (AFM) probe (Fig. 1C), thereby facilitating force spectroscopy experiments to measure the unfolding dynamics of fibrillin-1 calcium binding under tension (Fig. 1D). Results: Force spectroscopy experiments measured a 27% decrease in peak force required to unfold fibrillin-1 calcium binding domains following calcium removal (112 ± 25 pN vs 93 ± 16 pN; p < 0.031; n = 12). This correlates extremely well with the 25% decrease in peak force predicted from our previously published molecular dynamics simulations. Moreover, characteristic force profiles were observed which support force induced calcium unbinding. Conclusions: Force-sensitive calcium binding represents a novel mechanism to explore fibrillin-1 as an extracellular tension sensor in Marfan syndrome related aneurysm pathogenesis. Targeting extracellular mechanotransduction could thus provide a new avenue for regenerative medicine research in vascular disease.