Treatment with galectin-1 improves myogenic potential and membrane repair in dysferlin-deficient models

Abstract

Limb-girdle muscular dystrophy type 2B (LGMD2B) is caused by mutations in the dysferlin gene, resulting in non-functional dysferlin, a key protein found in muscle membrane. Treatment options available for patients are chiefly palliative in nature and focus on maintaining ambulation. Our hypothesis is that galectin-1 (Gal-1), a soluble carbohydrate binding protein, increases membrane repair capacity and myogenic potential of dysferlin-deficient muscle cells and muscle fibers. To test this hypothesis, we used recombinant human galectin-1 (rHsGal-1) to treat dysferlin-deficient models. We show that rHsGal-1 treatments of 48 h-72 h promotes myogenic maturation as indicated through improvements in size, myotube alignment, myoblast migration, and membrane repair capacity in dysferlin-deficient myotubes and myofibers. Furthermore, increased membrane repair capacity of dysferlin-deficient myotubes, independent of increased myogenic maturation is apparent and co-localizes on the membrane of myotubes after a brief 10min treatment with labeled rHsGal-1. We show the carbohydrate recognition domain of Gal-1 is necessary for observed membrane repair. Improvements in membrane repair after only a 10 min rHsGal-1treatment suggest mechanical stabilization of the membrane due to interaction with glycosylated membrane bound, ECM or yet to be identified ligands through the CDR domain of Gal-1. rHsGal-1 shows calcium-independent membrane repair in dysferlin-deficient and wild-type myotubes and myofibers. Together our novel results reveal Gal-1 mediates disease pathologies through both changes in integral myogenic protein expression and mechanical membrane stabilization.

Fig 1. rHsGal-1 increases myogenic regulatory factors in A/J^-/- myotubes.

A. Quantification of myogenin after 72h treatment with varying concentrations of rHsGal-1. B. Western blot images of myogenin at different rHsGal-1 treatments. C-F. Quantification of myogenic markers MHC(C), Pax7(D), MyoD(E), and Myf5(F) in A/J^-/- myotubes after 72h treatment with 0.11μM rHsGal-1. G-H. Quantification of Gal-1(G) and His.H8(H)in A/J^-/- myotubes after 72h treatment with 0.11μM rHsGal-1. I. Western blot images of myogenic markers (Pax7, Myf5, MyoD, and MHC) and of mouse Gal-1 and His Tagged rHsGal-1. J. RT-qPCR quantification of LGALS1 transcript between A/J WT, A/J ^-/- NT, and A/J ^-/- 0.11μM rHsGal-1 treated myotubes. p values are measured by Tukey’s multiple comparison test and indicated by *p< 0.05, **p< 0.01, ****p< 0.0001 (n = 3 for each group). Error bars represent SEM.

Fig 2. rHsGal-1 treatment increases levels of fusion index and myotube maturity.

A. Representative images of A/J cells cultured and immunostained with Phalloidin (red) and DAPI (blue). B. Representative images of A/J cells cultured and immunostained with MHC (red) and DAPI. C. Representative images of A/J cells cultured and immunostained with Myf5 (green), Phalloidin, and DAPI. D. Average number of nuclei per myotube compared between WT (n = 1608 nuclei, 187 myotubes, 10 fields), NT (n = 1587 nuclei, 215 myotubes, 9 fields), and 0.11 μM rHsGal-1 treated (n = 2476 nuclei, 166 myotubes, 13 fields) groups E. Fusion index between WT, NT, and 0.11μM rHsGal-1 treated myotube groups. F. Myotube alignment along the major axis compared between WT (n = 50 myotubes, 10 fields), NT (n = 49 myotubes, 9 fields) and 0.11 μM rHsGal-1 treated (n = 75 myotubes, 13 fields) myotubes. G. Minimum Feret’s diameter measurements between WT (n = 30 myotubes, 10 fields), NT (n = 34 myotubes, 9 fields), and 0.11μM rHsGal-1 treated (n = 36 myotubes, 13 fields) myotubes. H. Rate of migration between WT, NT, and 0.11μM rHsGal-1 treated myoblast groups. p values are measured by Tukey’s multiple comparison test and indicated by **p<0.01 and ****p< 0.0001. Error bars represent SEM. Scale bar = 100 μm.

Fig 3. rHsGal-1 treatment increases membrane repair capacity in A/J^-/- and A/J^+/+ myotubes dependent on CRD activity.

A. Representative images of FM 1–43 dye accumulation in NT and 48h 0.11 μM rHsGal-1 treated A/J^-/- myotubes after injury with UV laser. White arrows indicate site of injury. B. Quantification of the change in fluorescent intensity inside A/J^-/- myotubes following laser injury when treated with 0.11μM rHsGal-1 for 10min and 48h compared to WT A/J^+/+ and NT A/J^-/- myotubes. C. Change in the fluorescent intensity in 0.11μM rHsGal-1 treated A/J^-/- myotubes supplemented with lactose and sucrose compared to NT A/J^-/- myotubes. D. Change in the fluoresce intensity in 0.11μM rHsGal-1 treated A/J^+/+ myotubes supplemented with or without EGTA and rHsGal-1 compared to WT A/J^+/+ and NT A/J^-/- myotubes. Values were measured by Tukey’s multiple comparison test and indicated by in B. ****p< 0.0001 A/J^-/- NT vs A/J WT, ####p <0.0001 between A/J^-/- NT vs A/J^-/- 10min rHsGal-1, $ $ $ $p<0.0001 between A/J^-/- NT vs A/J^-/- 48hr rHsGal-1, %p<0.05 between A/J^-/- 48hr rHsGal-1 and A/J WT, and & p<0.05 between 10min and 48h. C. *p<0.05, **p<0.01, ***p<0.001. and ****p< 0.0001 between A/J^-/- 0.11μM rHsGal-1 20mM sucrose vs. A/J^-/- 0.11μM rHsGal-1 20mM lactose and A/J^-/- NT. D. at least significance of **p<0.01 between A/J WT, AJ WT + EGTA + 0.11μM rHsGal-1, and A/J^-/- 0.11μM rHsGal-1 + EGTA vs. A/J^-/- + EGTA and A/J WT + EGTA Scale bars = 50 μm. Error bars represent SEM. n ≥ 11 from 2 independent experiments for each group.

Fig 4. rHsGal-1 treatment positively effects sarcolemma and membrane repair in a Ca²⁺ depleted environment.

A. Representative images at time points 0s, 30s, 60s, 90s of FM1-43 dye accumulation in Bla/J mouse fibers upon laser injury with a 405nm laser. White arrows indicate site of injury. B. Quantification of the total change of fluorescence in Bla/J myofibers. C. Quantification of the total change of fluorescence in Dysf^-/- myofibers post injury. D. Quantification of the change in fluorescence in C57BL/6-WT myofibers after treatment with or without rHsGal-1. E. Quantification of the change in fluorescence in C57BL/6-WT myofibers after treatment with or without EGTA and rHsGal-1. p values were measured by Tukey’s multiple comparison test and indicated by *p< 0.05, **p< 0.01, and ***p< 0.001. Scale bars = 50 μm. Error bars represent SEM. n≥ 15 myofibers per condition.

Fig 5. rHsGal-1 localizes at the site of injury and is found in intra and extracellular spaces.

A–B. Representative images of laser ablation assay with labeled 647rHsGal-1 (green) and FM1-43 Dye (red) with treatment time of 10min(A) and 48h(B). White arrows indicate site of injury. C. Quantification of the average ratio of corrected total cell 647rHsGal-1 fluorescence between inside and outside of the cell. D-I. Confocal images of A/J ^-/- myoblasts/myotubes treated and differentiated with 0.11 μM rHsGal-1 at varying time points: 10min (D), 4 h (E), 8 h (F), 24 h (G), 48 h (H), with labeled rHsGal-1(647rHsGal-1) (green) showing nucleus (blue) and membrane (red) identifying critical structures pertaining to rHsGal-1 location. 1 and 2 represents vesicle localization (I). Zoom: rHsGal-1 encapsulated in vesicles.

Fig 6. Shotgun proteomic analyses (MS/MS) of AJ WT, AJ^-/- NT and 0.11 μM rHsGal-1 treated myotubes.

A. Heatmap of top 20 and bottom 20 expressed proteins for WT myotubes based on Log₂ fold change (FC) from the mean value all conditions. Box denotes cluster of proteins that had similar FC values between WT and rHsGal-1 treatment. B. GO-Term Slim analysis of rescued proteins. C. Relative amount of proteins found in the selected GO-Terms(B). D. Log₂FC of Annexin family proteins (calculated based on WT and Gal-1 FC from NT). E. Quantification of ANXA6 after 48h differentiation in WT, NT, and 0.11 μM rHsGal-1 treated myotubes. F. Quantification of ANXA1 after 48h in WT, NT, and 0.11 μM rHsGal-1 treated myotubes. G. Western blot images of ANXA1 and ANXA6. p values were measured by 2-way ANOVA multiple comparison test and indicated by *p< 0.05, **p< 0.01, and ***p< 0.001. Error bars represent SEM. n = 3. All proteins were preselected based on protein ID significance values of ≥20% (p≤ 0.01, FDR≤0.05).