Sex-specific role of myostatin signaling in neonatal muscle growth, denervation atrophy, and neuromuscular contractures

Abstract

Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures that result from impaired longitudinal growth of denervated muscles. This deficit in muscle growth is driven by increased proteasome-mediated protein degradation, suggesting a dysregulation of muscle proteostasis. The myostatin (MSTN) pathway, a prominent muscle-specific regulator of proteostasis, is a putative signaling mechanism by which neonatal denervation could impair longitudinal muscle growth, and thus a potential target to prevent NBPI-induced contractures. Through a mouse model of NBPI, our present study revealed that pharmacologic inhibition of MSTN signaling induces hypertrophy, restores longitudinal growth, and prevents contractures in denervated muscles of female but not male mice, despite inducing hypertrophy of normally innervated muscles in both sexes. Additionally, the MSTN-dependent impairment of longitudinal muscle growth after NBPI in female mice is associated with perturbation of 20S proteasome activity, but not through alterations in canonical MSTN signaling pathways. These findings reveal a sex dimorphism in the regulation of neonatal longitudinal muscle growth and contractures, thereby providing insights into contracture pathophysiology, identifying a potential muscle-specific therapeutic target for contracture prevention, and underscoring the importance of sex as a biological variable in the pathophysiology of neuromuscular disorders.

Keywords: developmental biology; mouse; muscle atrophy; muscle length; myostatin; neonatal denervation; neuromuscular contractures; sex dimorphisms.

Figure 1.. Pharmacologic inhibition of myostatin (MSTN) enhances neonatal skeletal muscle growth.

(A) Schematic depiction of ACVR2B-Fc treatment to inhibit MSTN in neonatal mice prior to and after unilateral brachial plexus injury (NBPI) at P5. Representative MicroCT images in (B) transverse and three-dimensional views revealed increased growth of non-denervated brachialis muscles in both female and male mice following 4 weeks of pharmacologic MSTN inhibition with ACVR2B-Fc. Quantitative analyses of (C) cross-sectional area and (D) whole-muscle volume in control brachialis muscles confirmed that neonatal MSTN inhibition enhances skeletal muscle growth. Neonatal MSTN inhibition further enhances (F) muscle weight and (G) total protein content of non-denervated biceps muscles in both sexes. Despite this, the increases in (E) muscle volume, and (H) muscle weight and protein levels were larger in females than males when compared to their respective DPBS controls. Data are presented as mean ± standard deviation (SD), n = 7–10 independent mice. Statistical analyses: (C, D, F, G) two-way analysis of variance (ANOVA) for sex and treatment with a Bonferroni correction for multiple comparisons, (E, H) unpaired two-tailed Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 1000 µm.

Figure 1—figure supplement 1.. Off-target effects of pharmacologic myostatin (MSTN) inhibition.

(A) MSTN inhibition does not alter protein levels in normally innervated biceps when normalized to muscle weight. Treatment with ACVR2B-Fc (B) does not promote additional weight gains in male mice, but instead, attenuates skeletal growth of non-denervated forelimbs and (D) reduces heart size. In contrast, ACVR2B-Fc treatment (E) reduces spleen size only in female mice. Data are presented as mean ± standard deviation (SD), n = 7–10 independent mice. Statistical analyses: (A–E) two-way analysis of variance (ANOVA) for sex and treatment with a Bonferroni correction for multiple comparisons. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 2.. Pharmacologic myostatin (MSTN) inhibition reduces denervation-induced muscle atrophy in neonatal female mice.

Representative MicroCT images in (A) transverse and three-dimensional views revealed larger denervated brachialis muscles in female mice following MSTN inhibition. Quantitative analyses of (B) brachialis cross-sectional area and (C) whole-muscle volume confirmed MSTN inhibition reduces denervation-induced atrophy only in female mice. This partial rescue in growth of the denervated brachialis muscles is accompanied by increased (D) muscle weight and (E) total protein content of denervated biceps muscles in female mice only. Data are presented as mean ± standard deviation (SD), n = 7–10 independent mice. Statistical analyses: (B–E) two-way analysis of variance (ANOVA) for sex and treatment with a Bonferroni correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar: 1000 µm.

Figure 2—figure supplement 1.. Effect of pharmacologic myostatin (MSTN) inhibition on normalized protein levels and skeletal growth in denervated forelimb.

(A) MSTN inhibition does not alter protein levels in denervated biceps when normalized to muscle weight. (B) Treatment with ACVR2B-Fc attenuates skeletal growth of denervated forelimbs only in male mice. Data are presented as mean ± standard deviation (SD), n = 7–10 independent mice. Statistical analyses: (A, B) two-way analysis of variance (ANOVA) for sex and treatment with a Bonferroni correction for multiple comparisons. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 3.. Pharmacologic myostatin (MSTN) inhibition reduces neonatal denervation-induced contractures in neonatal female mice.

(A) Representative images of denervated (neonatal brachial plexus injury, NBPI) and contralateral forelimbs, and quantitation of both (B) elbow joint extension range of motion and (C) contracture severity revealed that MSTN inhibition reduces the formation of elbow flexion contractures after 4 weeks of neonatal denervation in female, but not male mice. Similarly, (D) representative images of bilateral forelimbs, and quantitation of both (E) shoulder joint external rotation range of motion and (F) contracture severity demonstrated improvements in shoulder rotation only in female mice. In (C, F), elbow and shoulder contracture severity is calculated as the difference in passive elbow extension and shoulder rotation, respectively, between the NBPI side and the contralateral side. Data are presented as mean ± standard deviation (SD), n = 7–10 independent mice. Statistical analyses: (B, E) three-way analysis of variance (ANOVA) for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons, (C, F) two-way ANOVA for sex and treatment with a Bonferroni correction for multiple comparisons. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 3—figure supplement 1.. Nonparametric analysis in female mice.

(A) Quantitation of elbow joint extension range of motion in female mice only. Data are presented as mean ± standard deviation (SD), n = 7–10 independent mice. Statistical analyses: unpaired, Mann–Whitney U-tests between groups and paired, Wilcoxon signed rank tests between limbs of mice in each group. *p < 0.05, **p < 0.01, ****p < 0.0001.

Figure 4.. Pharmacologic myostatin (MSTN) inhibition preserves longitudinal muscle growth of denervated muscles in neonatal female mice.

(A) Representative differential interference contrast (DIC) images of sarcomeres, and (B) quantitation of sarcomere length showed that MSTN inhibition preserves sarcomere length of denervated muscles after neonatal denervation in female mice, whereas the sarcomeres of denervated muscles in male mice remain overstretched. (C) Sarcomere length on the neonatal brachial plexus injury (NBPI) side is normalized to the contralateral side to generate a sarcomere length ratio. This normalized sarcomere length between control and NBPI limbs further confirmed that MSTN inhibition improves functional length of denervated muscles only in female mice. Data are presented as mean ± standard deviation (SD), n = 7–10 independent mice. Statistical analyses: (B) three-way analysis of variance (ANOVA) for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons, (C) two-way ANOVA for sex and treatment with a Bonferroni correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 10 µm.

Figure 5.. Sex-specific differences in myostatin (MSTN)-dependent contracture formation is not mediated through protein synthesis.

(A) Representative western blots of puromycin incorporation and (B) quantitative analysis of optical densities revealed MSTN inhibition does not further alter the denervation-induced increases in whole-muscle protein synthesis in biceps muscles of both female and male mice. n = 7–10 independent mice. (C) Representative western blots and quantitative analyses of (D) pAkt (Ser473) and (E) total Akt similarly showed that ACVR2B-Fc treatment does not lead to additional increases in activity and translation of Akt after denervation in both sexes. (F) Quantification of the western signal for pAkt normalized to total protein levels further indicated that MSTN inhibition does not alter Akt/mTOR signaling in neonatally denervated muscles. n = 4–6 independent mice. Statistical analyses: (B, D–F) three-way analysis of variance (ANOVA) for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons. *p < 0.05, **p < 0.01. 7d MOV = adult mouse plantaris muscle that had been subjected to 7 days of mechanical overload.

Figure 6.. Sex-specific differences in myostatin (MSTN)-dependent contracture formation is mediated through proteasome activity.

(A) Representative western blots of K48-linked polyubiquitin and (B) quantitative analysis of optical densities showed similar levels of ubiquitination in control and denervated biceps muscles of neonatal female and male mice. Despite this, (C) in-depth analyses of optical densities discovered that denervation increases ubiquitination levels of higher molecular weight proteins >40 kDa, (D) but not lower molecular proteins <40 kDa in both sexes. Importantly, MSTN inhibition does not alter levels of K48 polyubiquitination across the different molecular weights after denervation. n = 4–7 independent mice. (E, F) Assessment of proteasome activity in triceps muscles revealed that MSTN inhibition blunts the denervation-induced increase in β5 but not β1 constitutive proteasome activity solely in female mice. n = 7 independent mice. Data are presented as mean ± standard deviation (SD). Statistical analyses: (B–F) three-way analysis of variance (ANOVA) for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. 7d MOV = adult mouse plantaris muscle that had been subjected to 7 days of mechanical overload.

Figure 6—figure supplement 1.. Assessment of muscle-specific ubiquitin ligases.

(A) Representative western blots and quantitative analyses of (B) MuRF1, and (C) Atrogin-1 showed myostatin (MSTN) inhibition does not alter protein levels in denervated (neonatal brachial plexus injury, NBPI) or contralateral triceps muscles. Data are presented as mean ± standard deviation (SD), n = 3 independent mice. (B, C) Three-way analysis of variance (ANOVA) for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons. *p < 0.05.

Figure 7.. Sex-specific differences in myostatin (MSTN)-mediated proteostasis dysregulation occurs independent of Smad2/3 signaling.

(A) Representative western blots and quantitative analyses of (B) pSmad2, (C) total Smad2, (E) pSmad3, and (F) total Smad3 revealed that ACVR2B-Fc treatment does not blunt the denervation-induced increase in activity and translation of Smad2 and Smad3 in triceps muscles of both sexes. Quantification of the western signal for (D) pSmad2 and (G) pSmad3 normalized to total protein levels further indicated that MSTN inhibition does not alter Smad2/3 signaling in neonatally denervated muscles. n = 3–6 independent mice. Data are presented as mean ± standard deviation (SD). Statistical analyses: (B–G) three-way analysis of variance (ANOVA) for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons. *p < 0.05, **p < 0.01.

Figure 8.. Proteasome inhibition with bortezomib prevents contractures and restores proteostasis preferentially in denervated muscles of neonatal male mice.

(A) Schematic depiction of bortezomib treatment and assessment of outcomes at 4, 8, or 12 weeks post-neonatal brachial plexus injury (NBPI). (B, C) Continuous bortezomib treatment preferentially prevents elbow and shoulder contractures in male mice throughout postnatal development and into skeletal maturity (n = 3–10 independent mice). (D) Despite this, long-term bortezomib does not confer sex-specific improvements in longitudinal muscle growth, as sarcomere length was preserved in denervated brachialis muscles of both female and male mice (n = 3–10 independent mice). (E–H) As opposed to myostatin (MSTN) inhibition, the rescue in long-term contractures with continuous bortezomib treatment in male mice at 8 and 12 weeks post-NBPI is associated with a decrease in β1 proteasome subunit activity, rather than β5 activity of denervated biceps muscles (n = 3 independent mice). In (B, C), elbow and shoulder contracture severity is calculated as the difference in passive elbow extension and shoulder rotation, respectively, between the denervated (NBPI) side and the contralateral control side. In (D), sarcomere length on the NBPI side is normalized to the contralateral side to generate a sarcomere length ratio. Data are presented as mean ± standard deviation (SD). Statistical analyses: (B–D) two-way analysis of variance (ANOVA) for sex and treatment with a Bonferroni correction for multiple comparisons at each time point, (E–H) three-way ANOVA for sex, treatment, and denervation (repeated measures between forelimbs) with a Bonferroni correction for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. This figure is generated from primary data reported in Goh et al., 2021.