Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models

Abstract

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in dystrophin that compromise sarcolemma integrity. Currently, there is no treatment for DMD. Mutations in transient receptor potential mucolipin 1 (ML1), a lysosomal Ca²⁺ channel required for lysosomal exocytosis, produce a DMD-like phenotype. Here, we show that transgenic overexpression or pharmacological activation of ML1 in vivo facilitates sarcolemma repair and alleviates the dystrophic phenotypes in both skeletal and cardiac muscles of mdx mice (a mouse model of DMD). Hallmark dystrophic features of DMD, including myofiber necrosis, central nucleation, fibrosis, elevated serum creatine kinase levels, reduced muscle force, impaired motor ability, and dilated cardiomyopathies, were all ameliorated by increasing ML1 activity. ML1-dependent activation of transcription factor EB (TFEB) corrects lysosomal insufficiency to diminish muscle damage. Hence, targeting lysosomal Ca²⁺ channels may represent a promising approach to treat DMD and related muscle diseases.

Fig. 1. Muscle-specific transgenic overexpression of ML1.

(A) PCR genotyping of the mdx mutation, GCaMP3-ML1 transgene, and MCK-Cre. (B) Western blotting with anti-ML1 antibody in brain and various skeletal muscle tissues, including GAS, TA, and DIA from WT, ML1 ROSA-lSl;MCK Cre (ML1^MCK), mdx, and mdx;ML1^MCK mice (see fig. S1B for the source files). GAPDH served as the loading control. Note that MCK-Cre is selectively expressed in differentiated myotubes (16), and its expression in the muscle tissue might be affected by the degree of dystrophies. (C) Immunofluorescence analysis of TA, GAS, and DIA cryosections from various transgenic mice. Scale bar, 10 μm. (D) Immunofluorescence analysis of primary myotubes isolated from ML1^MCK mice. Scale bar, 10 μm. (E) Whole-endolysosome ML1 currents (I_ML1) were activated by ML-SA1 (20 μM), a synthetic agonist of ML1, in primary myotubes harvested from WT, ML1^MCK, mdx, and mdx;ML1^MCK mice. Currents were stimulated with a ramp voltage protocol from −120 to +60 mV. Holding potential = 0 mV. (F) I_ML1 current densities of myotubes from (E). Each open circle represents one cell/patch. (G) Structure of ML-SA5. (H and I) ML-SA5–induced lysosomal Ca²⁺ release, measured with GCaMP3 imaging, in primary myotubes isolated from ML1^MCK mice. GPN (glycyl-l-phenylalanine 2-naphthylamide), a dipeptide causing osmotic lysis of lysosomes, was used as a negative (depleting lysosomal Ca²⁺ stores) control. (J) Representative images showing H&E staining of GAS isolated from 2-month-old ML1^−/− and ML1^−/−;ML1^MCK mice. Arrows label necrotic areas, and asterisks show centrally nucleated myofibers. Scale bar, 100 μm. (K and L) Quantification of necrosis (K) and central nucleation (L) in GAS sections from 2-month-old ML1^−/− and ML1^−/−;ML1^MCK mice. All data are means ± SEM; *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. Transgenic overexpression of ML1 reduces muscle pathologies in young mdx mice.

(A) H&E staining of TA sections from WT, ML1^MCK, mdx, and mdx;ML1^MCK mice at the age of 1 month, before (rest) and after treadmill exercise. Both male and female mice were randomly assigned into different groups. Arrows label necrotic areas and asterisks show centrally nucleated myofibers. Scale bar, 500 μm or 50 μm (zoom-in images). (B) Percentage of necrotic area in TA muscles from various transgenic mice. Each datum (n indicates the number of the muscle) represents the averaged result from at least five representative images randomly selected from at least three sections. Statistical analyses were performed by experimenters who were blind to animal genotypes. (C) Percentage of centrally nucleated fibers in TA muscles from different transgenic mice. (D) Serum CK levels in 1-month-old WT, ML1^MCK, mdx, and mdx;ML1^MCK mice before and after treadmill exercise. (E) Specific force test of GAS from multiple 1-month-old mice. (F) Body weight measurements of WT, utrophin^−/−;mdx (utrn^−/−;mdx), and utrn^−/−;mdx;ML1^MCK male mice at the age of 1 month. (G) Effect of ML1 overexpression on histology of TA muscles isolated from 2-month-old utrn^−/−;mdx mice. Scale bar, 50 μm. (H) Quantification on central nucleation of muscle histology from (G). All data are means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 3. Transgenic ML1 overexpression ameliorates myopathies in aged mdx mice.

(A) H&E staining of DIA isolated from mdx and mdx;ML1^MCK mice at the age of 1, 4, and 10 months. Both male and female mice were used in this experiment. Scale bar, 50 μm. (B) Age-dependent progressive fibrosis in DIA muscles isolated from mdx and mdx;ML1^MCK mice. (C) Trichrome collagen staining of DIA from 4-month-old mice. Scale bar, 100 μm. (D) Quantification of results (n indicates the number of the animal) averaged from multiple randomly selected images as shown in (C). (E and F) Thickness of IVS was measured by echocardiography (see fig. S2H) at the end diastole (E) and end systole (F) from 13- to 15-month-old WT, mdx, and mdx;ML1^MCK male mice. The echocardiographer was blind to the mouse genotype. (G) Calculated left ventricle mass in 13- to 15-month-old WT, mdx, and mdx;ML1^MCK male mice. (H) Peak velocity of E wave measured by LV pulse-wave Doppler (see fig. S2I) in 13- to 15-month-old WT, mdx, and mdx;ML1^MCK male mice. (I) Ratios between peak velocity of E and A waves (see fig. S2I) in WT, mdx, and mdx;ML1^MCK male mice. (J) Quantification for the percentage of fibrotic area in WT, mdx, and mdx;ML1^MCK mice at the age of 13 to 15 months (see fig. S2J). All data are means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4. Small-molecule ML1 agonists ameliorate muscular dystrophies in mdx mice.

(A) ML-SA1 and ML-SA5 dose-dependently activated whole-endolysosomal ML1 currents in DMD myoblasts. (B) H&E staining of TA from 1-month-old mdx mice that received daily intraperitoneal injection of ML-SA5 (2 mg/kg) for 14 days starting at P14. Arrows and asterisks indicate necrotic areas and centrally nucleated fibers, respectively. Scale bar, 500 or 50 μm (zoom in). (C) Percentages of necrotic area in ML-SA5–injected mice. Each datum (n indicates the number of the muscle; n ≥ 4) represents the averaged result from at least three representative images randomly selected from at least three sections. Statistical analyses were performed by experimenters who were blind to treatment conditions. (D) Percentage of centrally nucleated fibers in ML-SA5–injected mice. (E) Serum CK levels in ML-SA5–treated mdx mice at the age of 1 month before and after treadmill exercise. (F and G) Effect of ML-SA5 intraperitoneal injection on TA from 2-month-old ML1 KO mice. Injection starts from P14. N.S., not significant. (H) Treadmill exhaustion time of mdx mice treated with ML-SA5 versus vehicle control. N, number of tested animals. The experimenter was blind to the treatment conditions. In the experiments shown in this figure, both male and female mice were randomly assigned into different treatment groups. All data are means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 5. ML1 activation facilitates sarcolemma repair to reduce muscle damage in mdx mice.

(A) Representative images of EB dye uptake in GAS isolated from WT, mdx, and mdx;ML1^MCK mice at the age of 1 month, before (rest) and after treadmill exercise. Scale bar, 10 μm. GCaMP3-ML1 expression was detected using an anti-GFP antibody. (B) Quantification of EB-positive fibers from (A). Each datum (n, number of the muscle) represents the averaged result from at least five representative images selected from at least three sections. (C) EB dye uptake in GAS isolated from ML-SA5–treated mdx mice. Scale bar, 10 μm. (D) Quantification of EB dye uptake in GAS from ML-SA5–treated mdx mice. (E and F) EB dye uptake in cardiac muscles isolated from 2-month-old ML-SA5 (2 mg/kg)–injected mdx mice that were stimulated with β-isoproterenol to cause cardiac damage. Injection of ML-SA5 started from P14. Scale bar, 500 μm. (G) Muscle force deficit of mechanically stretched GAS from 1-month-old mice. (H) Representative images of laser damage–induced FM dye accumulation in isolated FDB fibers. Arrows highlight damage sites. Scale bar, 20 μm. (I) Time-dependent laser damage–induced FM dye accumulation in FDB fibers isolated from WT, mdx, and mdx;ML1^MCK mice at the age of 1 month. n indicates the number of the FDB fibers for each genotype. In all the experiments shown in this figure, both male and female mice were randomly assigned into different experimental groups. All data are means ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6. ML1 agonist activates TFEB to correct lysosomal insufficiency in mdx muscles.

(A) Lamp1 immunofluorescence staining of GAS from WT, ML1^MCK, mdx, and mdx;ML1^MCK mice at the age of 1 month. GCaMP3-ML1 expression was detected using an anti-GFP antibody. Scale bar, 100 μm. Both male and female mice were used in the experiments shown in this figure. (B) Quantitative analyses of images in (A). For each datum representing each muscle, at least five representative images from at least three sections were analyzed. (C) Western blotting analysis of Lamp1 protein expression in GAS and DIA from 1-month-old mice. (D) Quantitation of Western blotting results in (C). (E) TFEB immunolabeling in GAS from ML-SA5–treated mice at the age of 1 month. (F) Quantitative analysis of nuclear versus total TFEB ratio from (G). n indicates muscle fibers in randomly selected images from at least four muscles in each group. (G) Lamp1 immunostaining in GAS from ML-SA5 (2 mg/kg)–treated mdx mice at the age of 1 month. Scale bar, 100 μm. (H) Quantification of Lamp1 immunolabeling in GAS from ML-SA5–treated mdx mice. For each datum representing each muscle, at least five representative images from at least three sections were analyzed. (I) Genetic or pharmacological up-regulation of ML1 ameliorates myopathies in vivo through TFEB-dependent lysosome biogenesis, Ca²⁺-dependent lysosomal exocytosis, and sarcolemma repair. Muscle damage may generate a yet-to-be-defined signal (e.g., reactive oxygen species) (18) to activate ML1 channels on lysosome membranes. Subsequent lysosomal Ca²⁺ release triggers Ca²⁺-dependent lysosomal exocytosis and nuclear translocation of TFEB, which then activates transcription of a unique set of genes related to lysosome biogenesis. Subsequently, lysosome function is boosted and sarcolemma repair may be facilitated to reduce muscle damage in DMD cells in vitro and in vivo. All data are means ± SEM; *P < 0.05 and **P < 0.01.