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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.
Neonatal brachial plexus injury (NBPI), a leading cause of pediatric upper limb paralysis, results in disabling and incurable muscle contractures that are driven by impaired longitudinal growth of denervated muscles. A rare form of NBPI, which maintains both afferent and sympathetic muscle innervation despite motor denervation, protects against contractures. We have previously ruled out a role for NRG/ErbB signaling, the predominant pathway governing antegrade afferent neuromuscular transmission, in modulating the formation of contractures. Our current study therefore investigated the contributions of sympathetic innervation of skeletal muscle in modulating NBPI‐induced contractures. Through chemical sympathectomy and pharmacologic modification with a β2‐adrenergic agonist, we discovered that sympathetic innervation alone is neither required nor sufficient to modulate contracture formation in neonatal mice. Despite this, sympathetic innervation plays an intriguing sex‐specific role in mediating neonatal muscle growth, as the cross‐sectional area (CSA) and volume of normally innervated male muscles were diminished by ablation of sympathetic neurons and increased by β‐adrenergic stimulation. Intriguingly, the robust alterations in CSA occurred with minimal changes to normal longitudinal muscle growth as determined by sarcomere length. Instead, β‐adrenergic stimulation exacerbated sarcomere overstretch in denervated male muscles, indicating potentially discrete regulation of muscle width and length. Future investigations into the mechanistic underpinnings of these distinct aspects of muscle growth are thus essential for improving clinical outcomes in patients affected by muscle disorders in which both length and width are affected.
Purpose: Neonatal brachial plexus injury (NBPI) causes disabling and incurable muscle contractures, arising from impaired longitudinal growth of denervated muscles. This growth impairment is caused by dysregulation of muscle proteostasis characterized by increased proteasome-mediated protein degradation. Myostatin (MSTN), a secreted growth differentiation factor and prominent muscle-specific negative regulator of proteostasis, drives muscle protein degradation. MSTN signaling is thus a possible pathway through which denervation impairs muscle growth and a potential muscle-specific molecular target for preventing contractures. This study uses pharmacologic inhibition of MSTN signaling in a mouse model of NBPI to test the hypothesis that NBPI induces contractures through MSTN-dependent impairment of longitudinal muscle growth. Methods: Unilateral NBPI was generated in postnatal day (P)5 mice. Mice were treated weekly with Dulbecco’s phosphate-buffered saline (DPBS) or ACVR2B-Fc, a soluble inhibitory decoy receptor for MSTN, starting at P4. Mice were sacrificed at P33, whereupon elbow and shoulder range of motion were measured to assess contractures. Brachialis muscles were prepared for microCT assessment of cross-sectional area (CSA) and volume, then processed for sarcomere length measurement as a readout of longitudinal muscle growth. Total protein content in biceps was assessed via Bradford assay. Protein degradation in triceps was quantified via 20S proteasome β1 subunit activity assay. Results: In female mice, myostatin inhibition with ACVR2B-Fc enhanced muscle weight and total protein content of denervated biceps (Figure 1), increased cross-sectional and volumetric growth of denervated brachialis (Figure 2), preserved longitudinal growth in denervated brachialis (Figure 3), and reduced elbow and shoulder contracture severity in the denervated limbs (Figure 4). Analysis of protein dynamics demonstrates the improvements in female denervated muscles were associated with a reduction in proteasome β1 subunit activity (Figure 5). However, no such improvements in denervated muscles occurred in male mice with ACVR2B-Fc. Conversely, myostatin inhibition increased muscle weight, CSA, volume, and total protein content of the normally innervated, contralateral control muscles in both male and female mice (Figures 1 and 2). Conclusion: Our findings reveal MSTN as a driver of impaired muscle growth and contracture formation in female but not male mice. This sex dimorphism lies in the pathophysiology of denervation-induced contractures rather than drug pharmacodynamics, given the sex-independent effects of MSTN inhibition on normally innervated muscle. Future studies must rigorously interrogate the contributions of sex in contracture pathophysiology. Significance: Our discovery identifies MSTN as a signaling target for modulating contractures in females and highlights the overlooked role of sex in the pathophysiology of acquired neuromuscular disorders. Level of Evidence: Not Applicable FDA: The FDA has not cleared the following pharmaceuticals and/or medical devices for the use described in this presentation.
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