Third party funded individual grant
Start date : 01.01.2019
End date : 31.12.2020
The extracellular matrix (ECM) provides an intercellular connecting scaffold that provides not only stability to a multicellular unit within an organ, but also crucially provides the structural correlate for an optimum cellular function. In skeletal muscle, in particular, the highly linear and hierarchical arrangement of parallel myofibers alongside with their capability to shorten or to elongate by synchronized degrees upon activation or passive stretch, respectively, must be ensured by appropriate inter-element anchorage. For myofibrils, the intermediate filament network (e.g. filamin C, plectin) links individual fibrils together at the level of the z-discs, while for myofibers, intercellular linkage is realized through the extracellular matrix (collagen, merosin, etc.), linking to the intracellular cytoskeleton via focal adhesion complexes (FACs) or the dystrophin-associated glycoprotein complex (DAG). One major mechanical stabilizer molecule underneath the sarcolemma providing ‘shock absorber’ stability to the muscle cell is dystrophin which is completely absent in Duchenne muscular dystrophy (DMD) or the murine mdx model. Increased collagen cross-linking has been shown to be a signature in dystrophic muscle (Smith et al. 2016, Muscle & Nerve). However, the questions of (i) how the resulting three-dimensional arrangement of collagen fibrils within the fibrotic tissue around the muscle fibres impact on the distribution of traction forces along the axial and normal fiber direction, (ii) how FACs remodel through the presence of increased fibrosis and (iii) how the fibrotic collagen network correlates to altered biomechanical properties of single fibers in resting and eccentrically exercised muscle, are still unanswered. A profound insight into the interplay between acto-passive biomechanics of muscle fibers and their surrounding embedding ECM would be of fundamental importance to understand structure-related muscle weakness through connective tissue, not only applicable to the setting of DMD but also extending to chronic inflammatory myopathies associated with augmented collagen matrix.
The specific scientific goals during this collaboration initiative are:
1) To assess the 3D cytoarchitecture of the myofibrillar lattice and the ECM collagen-I distribution in optically cleared EDL and diaphragm muscle from adult (8-12 mo) and old (2 yrs) dystrophin-deficient mdx mice using Second Harmonic Generation (SHG) microscopy. The ultrastructural parameters cosine angle sum (CAS) and Vernier Density (VD) will be assessed within extended 3D volumes (up to 1 mm³) to represent the myofibrillar cytoarchitecture from forward scattered SHG, and the backward scattered SHG signal will serve to analyse the 3D arrangement of interconnecting collagen-I network (for which angular isotropy will be assessed). Age-matched wt animals will serve as controls.
2) To assess 3D cytoarchitecture and intercellular collagen fibril distribution in mechanically dissected small fiber bundles (containing interlinking ECM) and single fibers (no ECM) at given sarcomere lengths using a novel combined SHG-biomechatronics device (MechaMorph), developed by the German team.
3) To assess resting length-tension curves and strain-stress relationships in small fiber bundles (EDL, diaphragm) and single fibers of mdx and wt mice to correlate structural cytoarchitecture data with direct passive biomechanics parameters of steady-state compliance and stiffness. The biomechanics parameters will be assessed prior to and following eccentric contractions induced by Ca2+ saturating maximum isometric contractions with suddenly superposed 20% stretches.
4) To assess FAC density and localization patterns through integrin and vinculin immunofluorescence microscopy of single fibers within small fiber bundles.