Fig 2: Lack of BIN1 muscle-specific isoforms correlates with normal histology and triad structure. (A) Histological features in Bin1ex11−/− mice at 12 weeks and 12 months of age with H&E or SDH. (B) Localization of BIN1 without the PI domain and triad markers in isolated fibers stained with BIN1 (pan-isoform antibody), RYR1 (sarcoplasmic reticula), or DHPR (T-tubules) antibodies. (C) Electron micrograph of 12-week-old wild-type (WT) and Bin1ex11−/− muscles labeled with potassium ferrocyanide for T-tubules. Scale bars: 200 µm (A); 10 µm (B); 0.5 µm (C).

Fig 3: Muscle regeneration is altered in Bin1ex11−/− mice. Right TA muscles of 12 weeks mice were injected with notexin and left TA muscles were used as control. (A) Regenerative capacity, estimated through normalization of the muscle mass of injected leg to uninjected contralateral leg at different time points; represented as the percentage of recovery. (B,C) TA muscle fiber cross-sectional area at 14 days (B) and 28 days (C) after notexin injection. Only regenerating fibers (with centralized nuclei) were taken into account and fiber cross-sectional area is grouped into 200 µm² intervals, and represented as the percentage of total fibers (n=5-7 mice). (D) Specific maximal force of TA muscles expressed as the ratio between regenerating and control legs. Unlike wild-type (WT), Bin1ex11−/− muscle force does not increase with time. (E) Bin1 iso8 (muscle-specific) mRNA expression in wild-type mice in non-injured muscle and during regeneration. (F) Fusion index 14 days after notexin injection in wild-type and Bin1ex11−/− TA muscles. Number of nuclei per fiber was counted on transversal sections. n=3 mice per group. Data are mean±s.e.m. Unpaired Student's t-test. *P<0.05,**P<0.01,***P<0.001.

Fig 4: BIN1 expression and localization. (A) In situ Bin1 labeling in wild-type mouse at E14.5 and E18.5. (B) Immunogold labeling using anti-PI domain-specific antibody on a TA muscle from a wild-type adult mouse showing specific triad localization. Scale bars: 500 nm (left panel); 100 nm (right panel). (C) BIN1 functional domains encompass the N-BAR (N-terminal amphipathic helix with Bin-Amph-Rvs sequences), the PI (phosphoinositides binding), and the SH3 (src homology) domains. Exon 11 encodes the muscle-specific PI domain, whereas exon 20 encodes the second half of the ubiquitous SH3 domain. Missense mutations in the N-BAR (vertical bars) or truncation of the SH3 domains (cross) lead to recessive CNM, whereas skipping of the PI domain is linked to highly progressive CNM and DM. Bottom: Bin1ex20−/− mice express a strongly reduced and truncated BIN1, whereas Bin1ex11−/− mice express the ubiquitous isoforms in muscle. B, brain; D, diaphragm; E, eye; H, heart; Li, liver; Lu, lung, M, mitochondria; SR, sarcoplasmic reticulum; T, T-tubule.

Fig 5: Defects in T-tubule and triad formation. T-tubule and BIN1 localization finish to mature around birth, and BIN1 is still partially longitudinal in newborn mice. (A) BIN1 detected with a pan-isoform antibody localizes on intracellular longitudinal tubules in newborn isolated myofibers, a pattern lost in Bin1ex20−/− mice. Signal is shown in black over white. (B) DHPR aggregation in Bin1ex20−/− isolated myofibers from newborn mice. (C) MTM1 localization was disrupted in Bin1ex20−/− isolated myofibers. (D) Primary muscle cells differentiated into myotubes in culture and with FM4-64 staining of sarcolemma-connected membrane tubules; Bin1ex20−/− myotubes lack the dense tubules network. Scale bars: 5 µm.