Allosteric Modulation of GSK-3β as a New Therapeutic Approach in Limb Girdle Muscular Dystrophy R1 Calpain 3-Related

Abstract

Limb-girdle muscular dystrophy R1 calpain 3-related (LGMDR1) is an autosomal recessive muscular dystrophy produced by mutations in the CAPN3 gene. It is a rare disease and there is no cure or treatment for the disease while the pathophysiological mechanism by which the absence of calpain 3 provokes the dystrophy in muscles is not clear. However, key proteins implicated in Wnt and mTOR signaling pathways, which regulate muscle homeostasis, showed a considerable reduction in their expression and in their phosphorylation in LGMDR1 patients' muscles. Finally, the administration of tideglusib and VP0.7, ATP non-competitive inhibitors of glycogen synthase kinase 3β (GSK-3β), restore the expression and phosphorylation of these proteins in LGMDR1 cells, opening the possibility of their use as therapeutic options.

Keywords: CAPN3; GSK-3β; LGMDR1; VP0.7; Wnt; limb-girdle muscular dystrophy; mTOR; tideglusib.

Figure 1

Western blot (A) and densitometry analysis (B) of Wnt signaling pathway proteins in samples from muscles of LGMDR1 and controls: total GSK-3β, P-GSK-3β (Ser9), P-GSK-3β/GSK-3β, β-catenin and Active β-catenin. Control samples (n = 4), symptomatic patients (n = 3) and pseudo-asymptomatic patients (n = 2) (05 and 07-109). All error bars represent standard error of the mean (SEM). Each circle represents a sample. Loading control: GAPDH.

Figure 2

Western blot (A) and densitometry analysis (B) mTOR signaling pathway proteins in samples from muscles of LGMR1 and controls: mTOR, P-mTOR (Ser2448), mTOR/P-mTOR (Ser2448), p70S6K, P-p70S6K (Thr389), P-p70S6K (Thr421/Ser424), p70S6K/P-p70S6K (Thr421/Ser424), P-RPS6 (Ser235/Ser236) and AMPK. P-RPS6 is statistically significant in symptomatic patients (p = 0.0159). 05, 09 and 07-109: pseudo-asymptomatic patients. Control samples (n = 4) for mTOR, P-mTOR, p70S6K and AMPK and (n = 5) for P-p70S6K -Thr389 and Thr421/Ser424- and P-RPS6. Symptomatic patients (n = 3) for mTOR, P-mTOR, p70S6K, P-p70S6K (Thr389), P-RPS6 and AMPK, (n = 2) for P-p70S6K (Thr421/Ser424) and (n = 1) for p70S6K/P-p70S6K (Thr421/Ser424). Pseudo-asymptomatic patients (n = 3) for AMPK, (n = 2) for mTOR, P-mTOR, p70S6K and P-p70S6K (Thr421/Ser424) and (n = 1) for P-p70S6K (Thr389), P-RPS6. All error bars represent standard error of the mean (SEM). *: Statistically significant. Each circle represents a sample. Loading control: GAPDH.

Figure 3

Schematic representation of the altered protein expression or phosphorylations in Wnt and mTOR signaling pathways in LGMDR1 muscle. Green: Upregulated expression or phosphorylation in LGMDR1. Red: Downregulated expression in LGMDR1.

Figure 4

Activation of the Wnt pathway in human myotubes on day 8 of differentiation and after 48 h of treatment with Tideglusib and VP0.7 at three concentrations (1.2, 3.0 and 15.0 µM) and LiCl in control (n = 1) and patient LGMDR1 (n = 1). (A) Wnt signaling pathway activation: western blot of total GSK-3β, P-GSK-3β (Ser9), P-GSK-3β/GSK-3β, total β-catenin and active β-catenin. (B) Expression of CAPN3, FOS, ANOS1 and ITGB1BP2 genes in LGMDR1 myotubes (n = 1 with technical triplicates). (C) Western blot of structural proteins: ITGβ1D and melusin. In red, lowest concentration used in the LGMDR1 myotubes (1.2 µM) for densitometry analysis.

Figure 5

Activation of the Akt/mTOR pathway in human myotubes on day 8 of differentiation and after 48 h of treatment with tideglusib and VP0.7 at three concentrations (1.2, 3.0 and 15.0 µM) in control (n = 1) and patient LGMDR1 (n = 1): (A) western blot of AKT, P-AKT (Ser473), mTOR, P-mTOR (Ser2448 and Ser2481), p70S6K, P-p70S6K (Thr421/Ser424), P-p70S6K (Thr389), P-RPS6 (Ser235/Ser236) and P-AMPK (Thr172). (B) Effect of FRZB silencing on the mTOR pathway in controls’ (n = 2) and LGMDR1 patients’ (n = 3) myotubes. The red squares in the WB show the lowest concentration used in patients (1.2 µM) for densitometry analysis. For the quantification of total AMPKα in LGMDR1 patient’s myotubes, only the lowest dose was analyzed.

Figure 6

Analysis of fibroblasts of controls (n = 3) and LGMDR1 patients (n = 3) treated with LiCl.

Figure 7

Analysis of fibroblasts of controls (n = 3) and LGMDR1 patients (n = 3) treated with the GSK-3 inhibitors, tideglusib and VP0.7.

Figure 8

Analysis of CD56- cells treated with (A) LiCl (controls, n = 2 and LGMDR1 patients, n = 3) and (B) Tideglusib and VP0.7 (controls, n = 3 and LGMDR1 patients, n = 3).