Myofilament lengths determine the extent of myofilament overlap and, hence, sarcomeric force generation at a specified sarcomere length. While thick filament lengths are constant (~1.65 m) in all muscle types and species examined, thin filament lengths vary widely (from 0.95-1.40 m) across muscle types and species. I previously demonstrated that, in skeletal muscle, thin filament lengths are determined via the thin filament's bipartite architecture, which consists of two structurally distinct segments: (i) a nebulin-coated proximal segment whose barbed end is capped by CapZ at the Z- line and whose length is constant in all mammalian muscles; (ii) a nebulin-free distal segment whose pointed end is capped by two sarcomeric tropomodulin isoforms (Tmod1 and Tmod4) and whose length varies both across muscles and across muscles of a single species. Thus, variability in overall thin filament lengths is specifically dictated by variability in distal segment lengths and not proximal segment lengths. Nevertheless, little information is available regarding how distal segment lengths are specified during postnatal muscle development or remodeled after muscle injury, and whether the ultrastructure of the thin filament pointed end can provide a molecular mechanism for actin subunit exchange at the pointed end.
The first aim of this proposed project is to investigate the biological roles of sarcomeric Tmods in distal segment remodeling during skeletal muscle maturation, injury, and pathology. Here, I will compare wild-type (WT), Tmod1-/-, and Tmod4-/- mice under a variety of experimental conditions. First, confocal fluorescence microscopy and Distributed Deconvolution (DDecon) analysis will be used to measure distal segment lengths at different postnatal ages, after acute mechanical injury (eccentric exercise), and after chemical injury (cardiotoxin injection). Next, I will acquire sarcomere length-tension curves and X-ray diffraction patterns of skinned fibers from WT, Tmod1-/-, and Tmod4-/- mice (with J. Ochala). Finally, to study distal segment remodeling in dystrophic muscle with elevated calpain- mediated proteolysis, I will measure Tmod levels and distal segment lengths in the mdx and mdx/mTR mouse models of mild and severe Duchenne muscular dystrophy, respectively (with A. Sacco).
The second aim of this proposed project is to characterize the length variability and molecular architecture of the distal segment at nanometer-scale resolution. To understand the mechanisms of distal segment length variability and remodeling, I will perform nanometer-resolution imaging and computational techniques to examine how the molecular architecture of the distal segment provides a structural basis for distal segment length variability and remodeling. I will use stochastic optical reconstruction microscopy (STORM) and electron tomography to quantify distal segment length variability in WT, Tmod1-/-, and Tmod4-/- mice; these analyses will be followed by single-particle cryoelectron microscopy of isolated thin filaments to solve the 3D structure of the Tmod/tropomyosin/actin complex at the pointed end and ascertain a structural mechanism for actin subunit dynamics at the pointed end (with B. Carragher and C.S. Potter).
Muscle force production and joint movement depend on the precise specification of the lengths of the distal segments of the thin filaments, which vary in a muscle-specific manner and can become misspecified in myopathic muscles. This proposed project seeks to elucidate how sarcomeric tropomodulin (Tmod) proteins control distal segment lengths, remodeling, and molecular organization in health, adaptation, and disease. These studies will provide new insights into myofibrillar adaptation and skeletal muscle plasticity, with implications for surgical reattachment of muscles in orthopaedic reconstructive procedures as well as understanding the pathogenesis of hereditary myopathies.
