Pharmacological Inhibition of Myostatin in a Mouse Model of Typical Nemaline Myopathy Increases Muscle Size and Force

Abstract

Nemaline myopathy is one of the most common non-dystrophic congenital myopathies. Individuals affected by this condition experience muscle weakness and muscle smallness, often requiring supportive measures like wheelchairs or respiratory support. A significant proportion of patients, approximately one-third, exhibit compound heterozygous nebulin mutations, which usually give rise to the typical form of the disease. Currently, there are no approved treatments available for nemaline myopathy. Our research explored the modulation of myostatin, a negative regulator of muscle mass, in combating the muscle smallness associated with the disease. To investigate the effect of myostatin inhibition, we employed a mouse model with compound heterozygous nebulin mutations that mimic the typical form of the disease. The mice were treated with mRK35, a myostatin antibody, through weekly intraperitoneal injections of 10 mg/kg mRK35, commencing at two weeks of age and continuing until the mice reached four months of age. The treatment resulted in an increase in body weight and an approximate 20% muscle weight gain across most skeletal muscles, without affecting the heart. The minimum Feret diameter of type IIA and IIB fibers exhibited an increase in compound heterozygous mice, while only type IIB fibers demonstrated an increase in wild-type mice. In vitro mechanical experiments conducted on intact extensor digitorum longus muscle revealed that mRK35 augmented the physiological cross-sectional area of muscle fibers and enhanced absolute tetanic force in both wild-type and compound heterozygous mice. Furthermore, mRK35 administration improved grip strength in treated mice. Collectively, these findings indicate that inhibiting myostatin can mitigate the muscle deficits in nebulin-based typical nemaline myopathy, potentially serving as a much-needed therapeutic option.

Keywords: myostatin; nebulin; nemaline myopathy.

Figure 1

mRK35 treatment increases body weights in both wild-type (A) and Compound-Het (B) mice. (C) Growth curves for all groups overlayed. mRK35 increased the body weights of the Compound-Het mice to that of untreated wild-type mice. A Gompertz’s growth curve was used to fit body weights. **** p < 0.0001 indicates significantly different curves vs. vehicle-treated mice of the same genotype. #### p < 0.0001 indicates a significantly different curve vs. vehicle-treated wild-type mice. N-values: WT Vehicle: 14; WT mRK35: 10; Compound-Het Vehicle: 11; Compound-Het mRK: 8.

Figure 2

mRK35 treatment resulted in hypertrophy of multiple skeletal muscles in both wild-type (A) and Compound-Het (B) mice. Graphs show the relative change in muscle weights normalized to tibia lengths. The dashed line in B indicates vehicle-treated wild-type mice. Two-way ANOVA with Tukey’s post hoc test was used for statistical testing. *** p < 0.001 and **** p < 0.0001 vs. vehicle-treated mice of the same genotype. ### p < 0.001 and #### p < 0.0001 vs. vehicle-treated wild-type.

Figure 3

No effect of mRK35 treatment on cardiac muscle tissue weights in wild-type (A) and Compound-Het (B) mice. Graphs show the relative change in muscle weights normalized to tibia lengths. The dashed line in B indicates vehicle-treated wild-type mice. Two-way ANOVA with Tukey’s post hoc test was used for statistical testing.

Figure 4

mR35 treatment increases primarily the minFeret diameter of type Iib fibers. (A) Immunofluorescence images of the different fiber types found in EDL muscles. Top row; red: laminin; blue: type I; green: type IIx exclusion (non-stained fibers are type IIx). Bottom row; red: laminin; green: type IIa; blue: type IIb. (B) MinFeret diameter in wild-types. (C) MinFeret diameter in Compound-Hets. Two-way ANOVA with Šídák’s post hoc test was used for statistical testing. * p < 0.05, *** p < 0.001 and **** p < 0.0001 vs. vehicle-treated animals of same genotype. Scale bar: 50 µm.

Figure 5

mRK35 treatment enlarges the physiological cross-sectional area of EDL muscles and increases force production. (A) Optimal length for force production. p < 0.01 for treatment effect in a two-way ANOVA. (B) Physiological cross-sectional area (PCSA) at optimal length. (C) Absolute force. (D) Specific force (absolute force normalized to cross-sectional area). Two-way ANOVA with Tukey’s post hoc test was used for statistical testing. ** p < 0.01, *** p < 0.001 and **** p < 0.0001.

Figure 6

Increased grip strength in mRK35-treated mice. (A) In wild-type mice, mRK35 only increased grip strength at 4 months of life. (B) From two months of life and onwards Compound-Het mice treated with mRK35 displayed greater grip strength. (C) Grip strength for all groups at all time points. Two-way ANOVA with Šídák’s post hoc test was used for statistical testing. In C, grip strength at the plateau of the fitted curves is compared (see methods for details). * p < 0.05, ** p < 0.01, and **** p < 0.0001 vs. vehicle-treated mice of the same genotype. ## p < 0.01, ### p < 0.001, and #### p < 0.0001 for vehicle-treated Compound-Het vs. vehicle-treated wild-type.