Lithium chloride corrects weakness and myopathology in a preclinical model of LGMD1D

Figures

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  Figure 1. DNAJB6 knockout results in accumulation of sarcomeric proteins and altered myofibrillogenesis (A) Western blot demonstrating the absence of both DNAJB6 isoforms in Crispr/Cas9-generated DNAJB6 KO C2C12 myoblasts and during differentiation into myotubes. (B) Bright-field image demonstrating normal morphology of DNAJB6 KO myoblasts. (C) Flowchart of forward and reverse SILAC labeling combined with LC-MS/MS for comparative analysis of protein expression in WT and DNAJB6 KO myoblasts. (D) Quantitation overlap of the detected proteins in the 2 forward and 2 reverse SILAC labeling experiments and those that differentially accumulated in WT vs DNAJB6 KO C2C12 cells. (E) Ontological analysis of proteins increased in DNAJB6 KO myoblasts. (F) Ontological analysis of proteins decreased in DNAJB6 KO myoblasts. (G) Western blot of WT and KO myoblasts confirming increased levels of several proteins identified in SILAC analysis. (H) Differentiation of KO myoblasts into myotubes reveals altered myofibrillar organization on myosin staining and electron microscopy. White arrows demonstrate Z discs in WT myotubes with well-organized sarcomeres. Dark arrows demonstrate Z discs in KO myotubes with…

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  Figure 2. Absence of DNAJB6 impairs GSK3β dephosphorylation and enhances myotube formation (A) C2C12 WT and DNAJB6 KO myoblasts differentiated into myotubes for 6 days and stained for myosin. DNAJB6 KO myoblasts fused to form large myotubes with enhanced fusion index (B). Error bars represent the standard error of 3 independent experiments. (C) Western blot confirmed increased inactive GSK3β-P(ser-9) in KO myoblasts. GSK3β-P(ser-9)/GSK3β-total ratio is given below western blot. (D) Increased β-catenin transcriptional activity in DNAJB6 KO myoblasts compared with WT myoblasts measured by TOPFLASH luciferase assay. (E) Increased NFATc3 transcriptional activity in DNAJB6 KO myoblasts compared with WT myoblasts. Error bars represent the standard error of at least 2 independent experiments. GSK3β = glycogen synthase kinase-β; KO = knockout; NFATc3 = nuclear factor of activated T cells cytoplasmic 3; WT = wild type.

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  Figure 3. DNAJB6 disease mutation impact on GSK3β dephosphorylation and downstream signaling pathways (A) Western blot of skeletal muscle lysates from 3-month old LGMD1D mutant mice (F93Lb, n = 2) vs control (WTb, n = 2) demonstrating significantly reduced inactive GSK3β-P(ser-9) in mutant mice. Error bars represent the standard error. (B) Quantitation of western blot. Dual-luciferase assay demonstrating reduced β-catenin (C) and NFATc3 (D) transcriptional activity in HeLa cells transfected with mutant DNAJB6b constructs compared with unstimulated controls. β-catenin transcriptional activity was stimulated via treatment with 20 mM LiCl for 12 hours. NFATc3 transcriptional activity was stimulated by overexpression of NFATc3 via transient transection. Error bars in C and D represent the standard error from 3 separate experiments. (E) Proposed model for DNAJB6's spectrum of impact on GSK3β activation and myogenesis. GSK3β = glycogen synthase kinase-β; LGMD1D = limb-girdle muscular dystrophy 1D; NFATc3 = nuclear factor of activated T cells cytoplasmic 3; WT = wild type.

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  Figure 4. GSK3β inhibition improves muscle strength in LGMD1D mice (A) Diagram demonstrating DNAJB6's role in chaperoning the dephosphorylation of GSK3β-P(ser-9) and treatment strategy with GSK3β inhibitor, LiCl. (B) Functional testing (wire hang and grip strength) of 3-month-old DNAJ6b-WT and DNAJB6b-F93L mice treated daily with LiCl vs no treatment (NT). Two independent experiments were performed for a total of 8 mice per condition. Strength of DNAJB6b-F93L mice treated with LiCl significantly improves over the course of the month and reaches levels similar to DNAJB6b-WT mice. Error bars represent the standard error. *p < 0.05, **p< 0.01, ***p < 0.005. GSK3β = glycogen synthase kinase-β; WT = wild type.

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  Figure 5. GSK3β inhibition improves muscle size in LGMD1D mice (A) Mass of selected muscles after 1 month of treatment. Note visible difference in the size of F93L muscles treated with LiCl. (B) H&E staining of TA from LiCl treated or untreated F93L mice illustrating increased muscle fiber size and reduced fiber size variability. Two representative examples are provided from the tibialis anterior. (C) Quantitation of fiber CSA profile demonstrates an overall increase in the muscle fiber size after LiCl treatment in F93L mice and no change in LiCl-treated control mice. (D) Average muscle fiber cross-sectional area of LiCl-treated F93L mice significantly improves but does not reach control levels. LiCl does not alter the muscle fiber size of control mice. Error bars from A and D represent the standard error. (E) Western blot of skeletal muscle lysates in LiCl-treated and untreated mice illustrates no improvement in accumulated sarcomeric proteins or RNA-binding proteins in F93L mice. WT = wild type.