Journal: Nature medicine
Article Title: An Intracellular Ca2+ Channel is Required For Sarcolemma Repair to Prevent Muscular Dystrophy
Figure Lengend Snippet: Defective membrane repair capacity in ML1 KO muscle (a) Immunofluorescence of dystrophin, β-dystroglycan (β-DG), integrin β1, laminin, caveolin-3 (Cav-3), and dysferlin in ML1-null Gastroc muscle. Scale bar = 10 µm. (b) Western blotting analysis of the DGC components, Cav-3, dysferlin, and MG53 in ML1 KO mice. Myosin served as a loading control. (c) A membrane repair assay performed on single FDB muscle fibers isolated from WT and ML1 KO mice. Membrane damage was induced with a two-photon laser at t = 0 s. Scale bar = 10 µm. The right panel shows the time-dependent changes (ΔF) in FM1–43 fluorescence intensity normalized to the basal fluorescence (F 0 ) for WT (blue) and ML1 KO (red) fibers. (d) Representative images of in vitro differentiated myotubes in response to mechanical damage elicited by a microelectrode (arrows). The lower panel shows the percentage of “surviving” myotubes in response to microelectrode penetration. The experiments were performed in the presence of extracellular Ca 2+ , and defective membrane resealing led to excessive Ca 2+ influx that triggered prolonged muscle contraction. The “surviving” cells are those without prolonged contraction. Scale bar = 50 µm. (e) Responses of C2C12-derived myotubes to microelectrode penetration in Tyrode's solution (2 mM Ca 2+ ) in the presence of DMSO (0.1%; 1 st and 2 nd penetration), ML-SI3 (20 µM), BAPTA-AM (20 µM), and GPN (200 µM). The right panels show the time course of FM4–64 accumulation (ΔF/F 0 ) at injury sites following microelectrode penetration. Scale bar = 50 µm. Note that the same set of DMSO control data was compared with all groups of drug treatment. (f) The effect of ML-SI3, a TRPML-specific synthetic inhibitor, on EB dye uptake in WT Gastroc muscles injected with the cardiotoxin VII4 (CTX), a cytolytic toxin that disrupts cell membrane in living animals 24 . The lower panel shows that intramuscular coinjection of ML-SI3 with CTX on EB dye uptake in WT and ML1 KO muscle. Scale bar = 10 µm. Data are presented as the mean ± s.e.m, n = 3 animals for each condition.
Article Snippet: Myofiber damage assay After mice were sacrificed by cervical dislocation, FDB muscles were surgically removed to be digested in a Tyrode's solution containing type I collagenase (2 mg/ml; Sigma) at 37 °C for 60 min. After re-suspension in the Tyrode's solution, single FDB fibers were mounted on a glass bottom chamber in Tyrode's or zero Ca2+ solution in the presence of 2.5 µM green-colored FM1–43 dye (Molecular Probes).
Techniques: Immunofluorescence, Western Blot, Mouse Assay, Isolation, Fluorescence, In Vitro, Derivative Assay, Injection