Coordination of Myofibroblast Contraction in Vitro

The aim of this thesis is to elucidate the common mechanisms that regulate myofibroblast contraction and intercellular communication, with a particular focus on the role of Ca2+ signalling. Synthesis and remodelling of collagenous matrix by myofibroblasts are key elements in the healing process of injured organs. Deregulation of myofibroblast activity and excessive remodelling result in hypertrophic scarring and organ fibrosis. Myofibroblasts are specialised fibroblasts with particularly contractile stress fibres and features that resemble smooth muscle cells. Although myofibroblast contraction is acknowledged being responsible for wound closure and tissue contracture, the underlying regulation mechanisms are unclear. In particular, it remains elusive to what extent changes in the cytosolic Ca2+, a central regulator of smooth muscle contraction, regulate myofibroblast activities. Previous studies have revealed that cultured fibroblasts and myofibroblasts exhibit spontaneous cytosolic Ca2+ oscillations but a link with cell contraction has not been established. In the first part of this thesis, we are testing the hypothesis that cytosolic Ca2+ oscillations regulate myofibroblast contraction. For this, we use analysis of Ca2+ dynamics with fluorescent indicators, tracking of stress-fibre-linked microbeads on the myofibroblast surface and quantification of subcellular pulling events with atomic force microscopy. This combined approach demonstrates that myofibroblasts exhibit periodic (∼100 seconds) Ca2+ oscillations that control small (∼400 nm) and weak (∼100 pN) contractions. Using deformable culture substrate to assess cell isometric tension, we further demonstrate that myofibroblast contraction is regulated at two levels: variations of intracellular Ca2+ mediate contractions of dorsal stress fibres whereas the Ca2+-independent Rho kinase pathway is responsible for maintaining overall isometric cell tension. In the second part of this thesis, we investigate whether the observed subcellular contractions and Ca2+ oscillations may play a role in coordinating the activities between directly contacting myofibroblasts. Rational for this hypothesis is that myofibroblasts in densely populated wound granulation tissue form two types of intercellular junctions: adherens junctions that mechanically couple stress fibres between cells, and gap junctions that provide electrochemical coupling. Both junctions have been shown to play a role in modulating the contraction of wound tissue and of myofibroblast collagen gel cultures but their precise roles are still elusive. To fill this gap in knowledge, we analyse the coordination of Ca2+ oscillations between adjacent fibroblasts and myofibroblasts, respectively. Intercellular communication is modulated with pharmacological treatments acting on either mechanical or electrochemical coupling, as well as on the cell's contractile apparatus and mechanosensitive membrane channels. Our results demonstrate that stress-fibre associated adherens junctions mechanically coordinate myofibroblast activities within a cell population. This mechanism implies that the transmission of contractile events from one cell induces a Ca2+ influx through mechanosensitive ion channels in its neighbour and there triggers a new contraction. Based on these results we suggest that, at the tissue level, myofibroblasts remodel collagen by Ca2+-dependent micro-contractile events that add up to macroscopic tissue contracture, whereas Rho kinase maintains cell isometric tension. Mechanical coupling via adherens junctions may simultaneously improve the remodelling of cell-dense tissue by coordinating the activity of myofibroblasts.


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