Sex-specific role of myostatin signaling in neonatal muscle growth, denervation atrophy, and neuromuscular contractures
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Curated by eLife
Evaluation Summary:
This paper will be of interest to scientists within the field of neuromuscular disorders and has potential clinical relevance. It reveals a novel targeted strategy to improve the pathophysiology of children with neonatal brachial plexus injury. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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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.
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Author Response
Reviewer #1 (Public Review):
“This manuscript reports the results of studies on the effects of an ActRIIB-Fc ligand trap inhibitor of myostatin on muscle contractures that develop when brachial plexus nerve roots are severed at 6 after birth. One component of this pathological response seems to be a failure to add sarcomeres as the skeleton grows resulting in short muscles. The authors use a carefully performed set of animal studies to test the effects of the ligand trap on denervation-induced limitations in range of motion in young mice. They also investigate several biochemical mechanisms that might contribute to contractures and be modified by the ligand trap. Finally, the test for gender discordance in the protective effect of a proteasome inhibitor against contractures. The major finding of these studies is that …
Author Response
Reviewer #1 (Public Review):
“This manuscript reports the results of studies on the effects of an ActRIIB-Fc ligand trap inhibitor of myostatin on muscle contractures that develop when brachial plexus nerve roots are severed at 6 after birth. One component of this pathological response seems to be a failure to add sarcomeres as the skeleton grows resulting in short muscles. The authors use a carefully performed set of animal studies to test the effects of the ligand trap on denervation-induced limitations in range of motion in young mice. They also investigate several biochemical mechanisms that might contribute to contractures and be modified by the ligand trap. Finally, the test for gender discordance in the protective effect of a proteasome inhibitor against contractures. The major finding of these studies is that the ligand trap improved the range of motion at the elbow and shoulder in female mice but not in males. The major caveat to interpreting the data is that group sizes are relatively small such that the study may have been underpowered to detect smaller effects on a range of motion and biochemical endpoints.”
Thank you very much for your thoughtful review of our manuscript. We have taken your feedback regarding the interpretation of our data into consideration, and revised our manuscript accordingly.
We appreciate the reviewer’s careful scrutiny of our group sizes. As mentioned in the Statistical analysis section of our Materials and Methods, we included at least 6 mice per group for all range of motion and physiological endpoints. Based on an a priori power analysis, this is the number of mice per group necessary to detect a 10° difference in contractures and a 0.2 µm difference in sarcomere lengths at 80% power between experimental conditions. However, the small size of the forelimb muscles, especially following denervation, precluded the investigation of all biochemical parameters in each muscle. Therefore, we used we used smaller subgroup sizes for certain biochemical endpoints (Akt, Smad2/3, and Atrogin-1). In our revised Discussion, we acknowledge our study may be underpowered to detect smaller effects in these parameters of protein dynamics.
Discussion (lines #461-468): First, the small size of our denervated muscles precluded the use of the same muscles for all analyses, instead requiring smaller subgroup sizes as well as different muscles for certain biochemical endpoints (Akt, Smad2/3, MuRF1, and Atrogin-1). We therefore acknowledge that our study may be underpowered to detect smaller effects in certain parameters of protein dynamics, specifically signaling proteins and ubiquitin ligases. We also acknowledge that the precision of our findings would be further enhanced with the use of the same muscle type across all of our morphological, physiological, and biochemical analyses.
Reviewer #2 (Public Review):
“The manuscript by Emmert et al. describes an original and straightforward study demonstrating the utility of targeted therapy in a neonatal brachial plexus injury (NBPI) mouse model. The authors sought to investigate whether pharmacologic inhibition of MSTN signaling using a soluble decoy receptor (ACVR2B-Fc) could preserve longitudinal muscle growth and prevent contractures after NBPI. More specifically, through in vivo experiments using wild-type female and male mice, the authors assessed the impact of inhibiting the MSTN signaling in basal and pathophysiological conditions, on developmental, morphological, and biomechanical parameters, and on several biochemical markers of protein synthesis, protein degradation, and their associated signaling pathways, in forelimb skeletal muscle.
The authors provide multiple lines of compelling evidence that ACVR2B-Fc improves skeletal muscle biology and function in NBPI mice, provokes hypertrophy, rescues longitudinal growth, and impedes neuromuscular contractures in denervated muscles. Rather than improving the condition independently of the sex, it appears selective to the muscles of female mice showing thus a sex-specific improvement, and therefore the discovery of a sex dimorphism. The experiments also try to provide a mechanistic explanation, though it is incompletely clear why and how it is happening at the end.
Overall, the study details a promising intervention in NBPI mice and begins to highlight a pathway that can be exploited for this goal. While the reviewer did enjoy the manuscript, and the conclusions of this paper are mostly well supported by data, there are certain deficiencies that cannot be overlooked.
Strengths:
A) This study includes a clear-cut demonstration leading to a coherent narrative of a potential intervention for children affected by NBPI, which is well supported by prior literature mentioning the effects of palliative mechanical solutions and investigating the effects of pharmacologic strategies for the prevention of muscle contractures.
B) This study uses a pharmacologic chronic treatment, in vivo, on female and male neonatal mice to investigate the effects and relevant mechanisms of the MSTN signaling inhibition, using a soluble decoy receptor (ACVR2B-Fc), from the whole organism into the skeletal muscle and further into cellular signaling pathways.
C) This study provides promising data about the effects of the MSTN signaling inhibition on developmental, morphological, and biomechanical parameters, as well as biochemical markers in the NBPI mice.
D) This study underlines the importance of using female and male mice during experimental procedures, clearly showing that sex dimorphism can produce very different results.
E) The manuscript is well written, well organized, and cogent.
Weaknesses and Limitations:
A) This study attempts to provide mechanistic information to support and explain the results observed. However, the analysis remains superficial and should go further into detail especially in investigating completely the different molecular pathways considered, and the non-canonical alternatives.
B) The use of different muscles for biochemical analyses compared to the muscles used for developmental, morphological, and biomechanical parameters limits the interpretation of data, which could be due to muscle differences instead for example.
C) The interpretation of the findings should be done carefully, knowing that it is an MSTN/Activin A signaling blockade and not an MSTN inhibition alone.
D) The conclusion would be reinforced with data obtained at later time points (8 and/or 12 weeks).”
Thank you very much for your comprehensive and insightful review. Your detailed comments and suggestions have not only allowed us to improve our current manuscript with greater clarity and additional data, but they also reinforce our plan to elucidate biochemical mechanisms more completely in future studies. In this revision, we have provided additional experiments to strengthen our analysis of known pathways downstream of MSTN signaling, addressed the use of different muscles as well as the four-week time point, and discussed the potential implications stemming from the broad specificity of the ligand trap. We certainly share your enthusiasm about dissecting the different molecular pathways and non-canonical alternatives. Indeed, we intend to interrogate these mechanistic underpinnings with the same rigor with which we obtained our physiological and translational findings, which cannot be completed within the scope of the current study. Follow-up studies will focus on exploring non-canonical alternatives and investigating long-term effects at skeletal maturity and beyond.
Reviewer #3 (Public Review):
“This timely manuscript describes the sex dimorphisms in neonatal development as it applies to muscle injury and denervation. More and more studies are identifying sex differences in skeletal muscle function and dysfunction. This is one more study to point out differences. A missing piece to the field and this study are the mechanistic links between skeletal muscle function/dysfunction and sex differences. This paper starts to point to a mechanism highlighting the non-canonical AKT pathway. This is a very wellwritten manuscript with a clear experimental plan and workflow. I have no major concerns.
My biggest question is the molecular mechanism linking sex differences and skeletal muscle function and dysfunction. However, this is perhaps a follow-up study to the already complete study the authors present.”
Thank you very much for your kind words and enthusiasm! We likewise find it important to improve our understanding of sex differences in muscle function/dysfunction, and are committed to unraveling the molecular mechanism(s) that link them in future studies.
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Evaluation Summary:
This paper will be of interest to scientists within the field of neuromuscular disorders and has potential clinical relevance. It reveals a novel targeted strategy to improve the pathophysiology of children with neonatal brachial plexus injury. The key claims of the manuscript are well supported by the data, and the approaches used are thoughtful and rigorous.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
This manuscript reports the results of studies on the effects of an ActRIIB-Fc ligand trap inhibitor of myostatin on muscle contractures that develop when brachial plexus nerve roots are severed at 6 after birth. One component of this pathological response seems to be a failure to add sarcomeres as the skeleton grows resulting in short muscles. The authors use a carefully performed set of animal studies to test the effects of the ligand trap on denervation-induced limitations in range of motion in young mice. They also investigate several biochemical mechanisms that might contribute to contractures and be modified by the ligand trap. Finally, the test for gender discordance in the protective effect of a proteasome inhibitor against contractures. The major finding of these studies is that the ligand trap …
Reviewer #1 (Public Review):
This manuscript reports the results of studies on the effects of an ActRIIB-Fc ligand trap inhibitor of myostatin on muscle contractures that develop when brachial plexus nerve roots are severed at 6 after birth. One component of this pathological response seems to be a failure to add sarcomeres as the skeleton grows resulting in short muscles. The authors use a carefully performed set of animal studies to test the effects of the ligand trap on denervation-induced limitations in range of motion in young mice. They also investigate several biochemical mechanisms that might contribute to contractures and be modified by the ligand trap. Finally, the test for gender discordance in the protective effect of a proteasome inhibitor against contractures. The major finding of these studies is that the ligand trap improved the range of motion at the elbow and shoulder in female mice but not in males. The major caveat to interpreting the data is that group sizes are relatively small such that the study may have been underpowered to detect smaller effects on a range of motion and biochemical endpoints.
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Reviewer #2 (Public Review):
The manuscript by Emmert et al. describes an original and straightforward study demonstrating the utility of targeted therapy in a neonatal brachial plexus injury (NBPI) mouse model. The authors sought to investigate whether pharmacologic inhibition of MSTN signaling using a soluble decoy receptor (ACVR2B-Fc) could preserve longitudinal muscle growth and prevent contractures after NBPI. More specifically, through in vivo experiments using wild-type female and male mice, the authors assessed the impact of inhibiting the MSTN signaling in basal and pathophysiological conditions, on developmental, morphological, and biomechanical parameters, and on several biochemical markers of protein synthesis, protein degradation, and their associated signaling pathways, in forelimb skeletal muscle.
The authors provide …
Reviewer #2 (Public Review):
The manuscript by Emmert et al. describes an original and straightforward study demonstrating the utility of targeted therapy in a neonatal brachial plexus injury (NBPI) mouse model. The authors sought to investigate whether pharmacologic inhibition of MSTN signaling using a soluble decoy receptor (ACVR2B-Fc) could preserve longitudinal muscle growth and prevent contractures after NBPI. More specifically, through in vivo experiments using wild-type female and male mice, the authors assessed the impact of inhibiting the MSTN signaling in basal and pathophysiological conditions, on developmental, morphological, and biomechanical parameters, and on several biochemical markers of protein synthesis, protein degradation, and their associated signaling pathways, in forelimb skeletal muscle.
The authors provide multiple lines of compelling evidence that ACVR2B-Fc improves skeletal muscle biology and function in NBPI mice, provokes hypertrophy, rescues longitudinal growth, and impedes neuromuscular contractures in denervated muscles. Rather than improving the condition independently of the sex, it appears selective to the muscles of female mice showing thus a sex-specific improvement, and therefore the discovery of a sex dimorphism. The experiments also try to provide a mechanistic explanation, though it is incompletely clear why and how it is happening at the end.
Overall, the study details a promising intervention in NBPI mice and begins to highlight a pathway that can be exploited for this goal. While the reviewer did enjoy the manuscript, and the conclusions of this paper are mostly well supported by data, there are certain deficiencies that cannot be overlooked.
Strengths:
A. This study includes a clear-cut demonstration leading to a coherent narrative of a potential intervention for children affected by NBPI, which is well supported by prior literature mentioning the effects of palliative mechanical solutions and investigating the effects of pharmacologic strategies for the prevention of muscle contractures.
B. This study uses a pharmacologic chronic treatment, in vivo, on female and male neonatal mice to investigate the effects and relevant mechanisms of the MSTN signaling inhibition, using a soluble decoy receptor (ACVR2B-Fc), from the whole organism into the skeletal muscle and further into cellular signaling pathways.
C. This study provides promising data about the effects of the MSTN signaling inhibition on developmental, morphological, and biomechanical parameters, as well as biochemical markers in the NBPI mice.
D. This study underlines the importance of using female and male mice during experimental procedures, clearly showing that sex dimorphism can produce very different results.
E. The manuscript is well written, well organized, and cogent.Weaknesses and Limitations:
A. This study attempts to provide mechanistic information to support and explain the results observed. However, the analysis remains superficial and should go further into detail especially in investigating completely the different molecular pathways considered, and the non-canonical alternatives.
B. The use of different muscles for biochemical analyses compared to the muscles used for developmental, morphological, and biomechanical parameters limits the interpretation of data, which could be due to muscle differences instead for example.
C. The interpretation of the findings should be done carefully, knowing that it is an MSTN/Activin A signaling blockade and not an MSTN inhibition alone.
D. The conclusion would be reinforced with data obtained at later time points (8 and/or 12 weeks). -
Reviewer #3 (Public Review):
This timely manuscript describes the sex dimorphisms in neonatal development as it applies to muscle injury and denervation. More and more studies are identifying sex differences in skeletal muscle function and dysfunction. This is one more study to point out differences. A missing piece to the field and this study are the mechanistic links between skeletal muscle function/dysfunction and sex differences. This paper starts to point to a mechanism highlighting the non-canonical AKT pathway. This is a very well-written manuscript with a clear experimental plan and workflow. I have no major concerns.
My biggest question is the molecular mechanism linking sex differences and skeletal muscle function and dysfunction. However, this is perhaps a follow-up study to the already complete study the authors present.
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