Injury to the brachial plexus at birth (neonatal brachial plexus injury – NBPI) is the most common cause of upper limb paralysis in children (Foad et al., 2008), occurring in approximately 1.5 of every 1000 live births, in both females and males at an equal rate (McLaren et al., 2019). This initial nerve injury leads to permanent neurologic deficits in 20–40% of affected children (Govindan and Burrows, 2019; Pondaag et al., 2004), and results in the secondary formation of disabling and incurable muscle contractures, or joint stiffness. Contractures severely impede range of motion and functional use of the involved limbs, ultimately resulting in skeletal deformity that further worsens limb dysfunction (Hale et al., 2010). However, current strategies are insufficient in restoring muscle function and joint range of motion once contractures have developed, and may even worsen function by further weakening abnormal muscles (Newman et al., 2006; Pedowitz et al., 2007; van der Sluijs et al., 2004). To develop effective strategies for treating contractures, it is therefore important to establish greater insights into the pathophysiology of contracture formation.

Using a mouse model of NBPI, we previously discovered that contractures result from impaired longitudinal muscle growth, as characterized by the overstretch/elongation of sarcomeres in denervated muscles (Nikolaou et al., 2011; Weekley et al., 2012; Nikolaou et al., 2014; Nikolaou et al., 2015). Such deficits in muscle length are independent of satellite cell-mediated myonuclear accretion, but are instead caused by aberrant levels of muscle protein degradation associated with increased catalytic activity of the 20S proteasome (Nikolaou et al., 2019). These results posit a critical mechanistic role for proteasome-mediated dysregulation of muscle proteostasis in driving the impairment of longitudinal muscle growth that ultimately causes contractures. As proof of concept, we showed that inhibition of the proteasome with an FDA-approved proteasome inhibitor, bortezomib, restores sarcomere length and reduces contracture formation (Nikolaou et al., 2019). Our collective findings therefore establish that the biomechanical contracture phenotype can be attributed to a biological defect, and that contractures can be corrected by targeting their causative mechanism. However, despite the effectiveness of proteasome inhibition for preventing contractures, this pharmacologic strategy cannot be easily translated to children. We recently reported that bortezomib treatment is required throughout postnatal development to prevent contractures at skeletal maturity, and that its efficacy is diminished beyond the neonatal period (Goh et al., 2021). This need for chronic bortezomib treatment is problematic. Prolonged administration of proteasome inhibitors results in potential cumulative toxicity as these drugs nonspecifically block degradation and cause tissue damage to many organs, such as the brain (Nikolaou et al., 2019), and even impede musculoskeletal development (Goh et al., 2021). Due to these concerns, our laboratory seeks to identify safer strategies to prevent denervation-induced contractures by targeting skeletal muscle-specific regulators of proteostasis to reduce the elevated protein degradation responsible for contractures.

One such signaling mechanism specific to skeletal muscle is the myostatin (MSTN) pathway. MSTN, also known as growth differentiation factor-8 (GDF8), is a myokine and a member of the transforming growth factor-β (TGF-β) superfamily (Lee, 2004). In normally innervated skeletal muscles, ligand binding of MSTN to its Type 2 receptors, ACVR2 (ActRIIA) and ACVR2B (ActRIIB), activates the downstream Smad proteins 2 and 3 (Sartori et al., 2009; Trendelenburg et al., 2009). These signaling events regulate muscle homeostasis by increasing degradation and decreasing Akt/mTOR-mediated synthesis, ultimately attenuating muscle size and limiting aberrant growth (Lee, 2004; Lee, 2012; Lee, 2021). MSTN thereby functions as a negative regulator of muscle mass and protein balance. Given its prominent role in muscle proteostasis, it is possible that MSTN is a signaling pathway by which denervation impairs muscle growth. As mentioned, we previously discovered that neonatal denervation increases protein degradation in denervated muscles (Nikolaou et al., 2019), which leads to impaired longitudinal muscle growth that ultimately causes contractures (Nikolaou et al., 2011; Weekley et al., 2012; Nikolaou et al., 2014; Nikolaou et al., 2015). However, the mechanism by which neonatal denervation increases protein degradation is unknown. Given that MSTN is a potent inhibitor of muscle growth through its mediation of muscle proteostasis, we speculate that MSTN signaling may be a mechanistic pathway linking denervation with protein degradation, leading to impaired muscle growth and ultimately contractures. If contractures are indeed mediated through MSTN signaling, it could lead to a potential breakthrough in identifying a muscle-specific target for contracture prevention.

Hence, in the current study, we seek to elucidate the role of MSTN in the formation of denervation-induced muscle contractures. Using a soluble decoy receptor (ACVR2B-Fc) to inhibit ligand binding of MSTN to ACVR2B (Lee et al., 2005; Lee et al., 2012; Goh et al., 2019; Lee et al., 2020), we specifically investigated whether pharmacologic inhibition of MSTN signaling preserves longitudinal muscle growth and prevents contractures after NBPI. Our collective results establish several novel insights in skeletal muscle biology and contracture pathophysiology. First, we show that pharmacologic MSTN inhibition is efficacious in augmenting neonatal growth of normally innervated skeletal muscles in both female and male mice. Second, MSTN inhibition effectively restores sarcomere length and reduces contracture severity exclusively in neonatally denervated muscles of female mice, suggesting a sex-dependent role for the MSTN pathway in modulating longitudinal muscle growth and contracture formation. Curiously, MSTN inhibition improves muscle proteostasis in denervated female muscles by circumventing known downstream signaling pathways to directly target the 20S proteasome. These discoveries establish a critical link between denervation and impaired longitudinal muscle growth, which helps guide translation of safer strategies for preventing contractures. Furthermore, they highlight the importance of sex as a biological variable in disease pathology and treatment strategies, as well as in future studies dissecting the molecular regulation of longitudinal muscle growth.

Secondary muscle contractures in pediatric neuromuscular disorders such as NBPI and cerebral palsy are major drivers of joint immobility, limb deformity, and physical dysfunction and disability (Hale et al., 2010). As existing treatments for contractures have focused on palliative mechanical solutions and do not address the underlying pathophysiology of contracture formation, the effectiveness of these strategies in restoring physical function is limited (Newman et al., 2006; Pedowitz et al., 2007; van der Sluijs et al., 2004). Specifically, the paucity in our understanding of contracture pathophysiology impedes clinical efforts to prevent contractures. To overcome these limitations, we have previously identified the dysregulation of muscle proteostasis, as characterized by an increase in proteasome-mediated protein degradation, as a causative mechanism of impaired longitudinal muscle growth and contracture formation following NBPI (Nikolaou et al., 2019). This discovery therefore highlights the possibility of pharmacologic strategies to prevent contractures through targeting of a biological mechanism. As our recent findings revealed a need for chronic pharmacologic proteasome inhibition with the potential for cumulative off-target toxicity (Goh et al., 2021), we must continue to identify safe and efficacious strategies for preventing pediatric muscle contractures by targeting muscle-specific regulators of proteostasis.

In our present study, we pharmacologically targeted MSTN, a prominent muscle-specific negative regulator of proteostasis, and identified several key findings regarding MSTN signaling in muscle growth and contracture pathophysiology. First, we discovered that pharmacologic MSTN inhibition with ACVR2B-Fc enhanced the size and protein content of normally innervated forelimb muscles in 1-month-old mice at P33, which is equivalent to the completion of neonatal muscle development in human infants (Dutta and Sengupta, 2016). In comparison, the youngest age that prior studies on MSTN inhibition have reported gains in lean body mass was in 2-month-old mice (McPherron and Lee, 2002). Our current finding therefore indicates a key regulatory role for MSTN signaling in governing muscle growth during the neonatal window, an often overlooked period in developmental muscle biology. This discovery not only reveals greater insights in skeletal muscle development, but also offers potential therapeutic strategies for overcoming low muscle mass arising from a host of genetic, metabolic, and hormonal disorders in the pediatric population (Orsso et al., 2019). In addition, MSTN inhibition also protected against atrophy and sarcomere overstretch in neonatally denervated forelimb muscles, as well as prevented contractures in female but not male mice. These findings therefore establish a sex-specific role for MSTN as a driver of impaired muscle growth and contracture formation exclusively in female mice. Critically, this sex dimorphism lies in the pathophysiology of denervation-induced atrophy and contractures rather than drug pharmacokinetics or pharmacodynamics, given that MSTN inhibition promotes robust growth in normally innervated muscles of both female and male mice. Indeed, MSTN inhibition restored proteostasis only through the attenuation of 20S proteasomal subunit activity in denervated muscles of female mice, without associated changes in overall levels of protein synthesis or K48-linked polyubiquitin. This postulates an intriguing mechanism for MSTN-dependent impairment of longitudinal muscle growth, whereby MSTN putatively circumvents canonical signaling pathways of proteostasis to directly regulate the 20S proteasome after neonatal muscle denervation. Beyond offering potential translational opportunities for female NBPI patients, these seminal findings highlight the underappreciated role of sex as a biological variable in the pathophysiology and treatment of acquired neuromuscular disorders. Our results also establish that longitudinal muscle growth is governed through sex-divergent and non-canonical pathways, which offers valuable directions for future investigations to undertake.

Skeletal muscle exhibits high levels of sex dimorphisms. Female and male muscles typically display heterogeneity in body composition and protein turnover (Griffin and Goldspink, 1973; Smith and Mittendorfer, 2016), regenerative and hypertrophic processes (Kitajima and Ono, 2016; Seko et al., 2020), metabolism and substrate utilization (Rosa-Caldwell and Greene, 2019; Maher et al., 2009), drug pharmacodynamics (Salamone et al., 2022), and even the development of disease-induced pathologies (De Jonghe et al., 2002). While the role of sex in neonatal muscle denervation is largely unknown, we speculate that sex mediates divergent pathways that ultimately lead to contracture formation. Our current discovery of a sex-specific role for MSTN signaling in mediating contractures thus establishes a critical link between neonatal muscle denervation and impaired longitudinal muscle growth. In neonatally denervated female muscles, the improvement in functional muscle length following MSTN inhibition is associated with reduced proteasome subunit activity. Our seemingly paradoxical finding of reduced proteasomal catalytic activity in the absence of any alterations in K48 polyubiquitin is not without precedent. Indeed, the phenomenon of ubiquitin-independent proteasomal degradation has been supported by several prior studies in a subset of proteins, which share common features of belonging to the Intrinsic Disordered Protein (IDP) family (Erales and Coffino, 2014; Jariel-Encontre et al., 2008), such as p21 and p53 (Asher et al., 2005; Chen et al., 2004; Tsvetkov et al., 2010). Since these proteins and their associated intrinsically disordered regions critically regulate various cellular processes, including transcriptional regulation, post-translational modification, and molecular signaling (Wright and Dyson, 2015), it is possible that neonatal muscle denervation induces a direct degradation of these substrates. If so, the sex-specific reduction in proteasome catalytic activity with MSTN inhibition may be due to the preservation of these IDPs, which would not lead to an accumulation of polyubiquitinated substrates. It remains unclear what specific protein substrates are polyubiquitinated with NBPI, and future studies are needed to address this question. Additionally, since we observe discrepancies in the temporal expression of upstream ubiquitin ligases in our current and prior studies (Nikolaou et al., 2019), we wonder if there is a transient temporal window for the upregulation of muscle-specific ligases after NBPI. Thus, a more extensive investigation of ubiquitin ligase expression and function in future studies will provide a better understanding of their role in neonatal denervation and muscle growth.

Our finding of sex-specific reductions in proteasome activity with MSTN inhibition compelled us to thoroughly scrutinize our prior results on proteasome inhibition with bortezomib through the lens of sex differences (Goh et al., 2021). Here, we discovered that bortezomib treatment preferentially prevented elbow and shoulder contractures in male mice after the neonatal stage of muscle development and into skeletal maturity. Though the sex-associated phenotypic differences are more subtle in this context, they further illustrate sex-divergent pathways in the modulation of NBPI-induced contractures. Intriguingly, the effects of MSTN inhibition on proteasome activity in female mice parallel our earlier results of proteasome inhibition with bortezomib (Goh et al., 2021). Specifically, we observed that a 30–45% attenuation in either β1 or β5 subunit activity at 4 weeks post-NBPI is associated with a 50–55% and a 45–60% reduction in elbow and shoulder contractures, respectively. Furthermore, this magnitude of reduction in either subunit activity is associated with an improvement of normalized sarcomere length by 14–15%. The absence of subunit specificity in preventing contractures thus points to a less prominent role for the different proteolytic sites of the 20S proteasome in modulating sarcomerogenesis and muscle length than previously proposed (Goh et al., 2021). Alternatively, the independent alterations in caspase- and chymotrypsin-like activities could potentially reflect different affinities of the two enzymatic processes for specific sarcomeric, cytoskeletal, or regulatory proteins involved in muscle growth and sarcomere addition. This notion is supported by Kisselev et al., 2006, who showed that the unique amino acid sequence of a protein substrate differentially determines the activities of the 20S proteolytic sites during protein degradation, thereby leading to variability in efficacy of the different inhibitors (Kisselev et al., 2006). Importantly, mutations and crystallography of yeast 20S proteasome by Groll et al., 1999 revealed that the three active β subunits are processed autocatalytically and independently of each other, with the processing of one subunit unaffected by the inactivation of another subunit (Groll et al., 1999). The independent processing function of the individual subunits, along with their unique affinities for specific protein substrates, may explain the independent changes in enzymatic activity observed in our current and prior studies. Although, we acknowledge that our findings contradict observations of allosteric interactions among the active subunits observed by Kisselev et al., 1999, specifically between the caspase- and chymotrypsin-like sites and vice versa, in eukaryotic proteasomes (Kisselev et al., 1999). Additional studies are required to reconcile the independent changes of the subunits with total changes in proteasome content, as we continue to develop insights on proteasome function in contracture pathophysiology.

Nevertheless, our collective findings indicate that different upstream pathways in contracture formation between sexes ultimately converge at the 20S proteasome, thereby validating the role of the proteasome as a key mediator of NBPI-induced contractures. Despite these insights, the precise mechanism(s) by which MSTN signaling modulates proteasome activity in denervated female muscles remains to be determined. While we observed altered Akt/mTOR and Smad2/3 signaling with NBPI, which are corroborated by studies from other groups using different mouse denervation models (Castets et al., 2019; MacDonald et al., 2014; Tando et al., 2016). MSTN inhibition failed to impact these canonical pathways in female mice. These findings thus suggest a non-canonical signaling mechanism through which MSTN regulates the 20S proteasome in female muscles after NBPI. Future studies are needed to elucidate this prospective alternative pathway. Further investigations into the molecular interactions between MSTN and proteasome activity are also critical for unraveling the complex regulatory machinery in longitudinal muscle growth, a poorly understood aspect of skeletal muscle biology (Jorgenson and Hornberger, 2019; Jorgenson et al., 2020).

Additionally, it is unclear how proteostasis dysregulation and contractures are mediated in denervated male muscles. To this end, it is conceivable to postulate a role for sex hormones in contracture pathophysiology post-NBPI. An earlier study on juvenile frogs reported a loss of laryngeal muscle fibers only in male frogs following surgical denervation, whereas androgen treatment resulted in an additive effect on total fiber numbers only in innervated female laryngeal muscles (Tobias et al., 1993). These results not only indicate a sexually dimorphic interaction between innervation and the endocrine system in regulating muscle fiber number, but they also suggest a protective effect of the female sex hormones against muscle fiber loss with denervation. Additional studies further verified the role of the female endocrine system in regulating muscle homeostasis. Ovariectomy in adult female rats prevented muscle mass recovery after hindlimb unloading (Sitnick et al., 2006), whereas genetic deletion of estrogen receptor β in muscle stem cells compromised muscle regeneration in juvenile female mice after local injections of barium chloride (Seko et al., 2020). Thus, to gain mechanistic insights on sexual dimorphisms in MSTN-mediated contracture formation, future studies must rigorously interrogate the contributions of sex in contracture pathophysiology by exploring the relationships between the different sex hormones and MSTN signaling in NBPI.

The finding of sex dimorphism in MSTN signaling itself is not without precedent. Targeted deletion of MSTN during skeletal maturity increased masseter mass and bite force in male mice only (Williams et al., 2015). In contrast, long-term MSTN deletion in skeletal muscles increased lean muscle mass only in aged female mice (Tavoian et al., 2019). These discrepancies might be attributable to sex-related differences of MSTN expression itself in skeletal muscles. Indeed, while men are prone to having higher expression of genes encoding ribosomal and mitochondrial proteins, women tend to have higher gene expression of the ACVR2B receptor (Welle et al., 2008). Moreover, there is an increased transcriptional and translational expression of latent MSTN in hindlimb muscles of adult female mice compared to their male counterparts (McMahon et al., 2003). As the increased expression of MSTN and its receptor ligands facilitate higher MSTN activity in females, they conceivably predispose female muscles to more prominent responses from MSTN inhibition. These sex differences in MSTN and receptor gene expression may further account for the mixed outcomes of MSTN inhibition therapy for treating Duchenne Muscular Disease and other muscle disorders (Lee, 2021; Rybalka et al., 2020). In our current study, these intrinsic sex differences could potentially elucidate how pharmacologic MSTN inhibition confers greater neonatal growth in normally innervated female muscles and protects against atrophy in denervated muscles of female mice. Future studies should perform a comprehensive analysis of the expression of MSTN and other TGF-β superfamily members (such as Activin A), as well as the associated receptors (ACVR2 and ACVR2B) across sexes and muscle groups to account for potential sex and/or muscle-dependent regulation.

Our discovery of sex dimorphisms in denervated muscle growth also calls into question prior conclusions on the role of MSTN signaling in denervation-induced atrophy from studies analyzing only male mice. In a juvenile mouse model of sciatic nerve resection, MacDonald et al. reported an inability of both rapamycin and ACVR2B-Fc treatment to prevent atrophy in male mice, and surmised that denervation atrophy is not Akt/mTOR or MSTN-dependent (MacDonald et al., 2014). These findings are similar to our current observations of male mice in a neonatal mouse model of NBPI. In our present study, we extend MacDonald et al.’s earlier findings by revealing a novel role for MSTN in mediating atrophy solely in denervated muscles of female mice. Importantly, the sex dimorphisms in muscle atrophy are not limited to neonatal denervation, as sex differences in the pathophysiology of muscle atrophy have also been documented by an independent group using an adult mouse model of tenotomy (Meyer et al., 2022). Overall, these latest discoveries underscore the need to account for sex as a biological variable in future studies investigating in different models of muscle atrophy. Our study is not without limitations. 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. Furthermore, we cannot be certain whether the lack of normal distribution observed for elbow extension in female NBPI mice with MSTN inhibition (Figure 3B) is attributed to different treatment response or an inherent variability in contracture formation with NBPI. Indeed, we have consistently observed varying levels of elbow joint mobility and contracture severity in our mouse model of NBPI in prior studies (Nikolaou et al., 2011; Weekley et al., 2012; Nikolaou et al., 2014; Nikolaou et al., 2015; Nikolaou et al., 2019; Goh et al., 2021; Ho et al., 2021). At this point, we do not have sufficient mechanistic insights to explain this variability. However, despite this variability, the effects of MSTN inhibition seen in this study remain apparent and statistically significant.

Additionally, we observed that the ACVR2B-Fc decoy receptor attenuated growth of other non-skeletal muscle tissues, including the heart, spleen, and bone. Indeed, the broad ligand specificity of ACVR2B-Fc increases the risk of toxicity to non-skeletal muscle tissues and cell types, ranging from epistaxis to telangiectasias (Campbell et al., 2017). Our observations are thus consistent with prior reports of off-target effects with pharmacologic MSTN inhibition (Lee, 2021). The off-target effects we observed in our study could have been minimized through genetic models to manipulate MSTN signaling in the skeletal muscle. While such genetic models could reveal additional insights in contracture formation, the off-target effects do not fully explain the sex specificity of our outcomes. As MSTN plays a key role in cardiac energy homeostasis (Biesemann et al., 2014), the attenuation in heart size of male mice could impair cardiac function and lead to reduced muscle activity. We are unable to ascertain this possibility as we did not perform any metabolic measurements in the current study, though there were no observable differences in spontaneous cage activity with ACVR2B-Fc treatment. Of particular note is the effect of MSTN inhibition on bone growth, which could potentially confound our measurements of muscle parameters that are normalized to humerus length. However, the reduced bone length in male denervated limbs following MSTN inhibition would, if anything, cause an apparent increase in muscle size and/or rescue of contractures. The absence of such muscle findings despite this effect on bone growth reinforces the lack of a beneficial effect of MSTN inhibition on male denervated muscle, further underscoring the sex dimorphisms seen in muscle parameters. Prior studies have failed to find an effect of embryonic MSTN deletion on humerus length in adult mice, although the use of a mixed-sex approach may have masked sex-related differences (Hamrick et al., 2002). Finally, although we identified a link between MSTN inhibition and proteasome activity without perturbations in canonical MSTN signaling pathways, the molecular mechanisms by which MSTN could be regulating proteasome activity remain unclear. Potential non-canonical pathways could include p21-activated kinase 1 (PAK1) signaling, which has recently been shown to be permissive to gains in skeletal muscle mass with MSTN inhibition (Barbé et al., 2021). Additional non-canonical alternatives that should be further explored include TAK1 and its downstream targets JNK and p38, as well as Ras and its downstream targets MEK1 and ERK1/2. Alternatively, MSTN might target contractures through the autophagy–lysosome pathway instead (Wang et al., 2015). Future studies are needed to fully elucidate mechanistic links and complete our understanding of the role of MSTN signaling in contracture pathophysiology.

For a more nuanced interpretation of our findings, we must also consider the specificity of the ACVR2B-Fc decoy receptor as a ligand trap, as mentioned above. Previous studies have reported that many members of the TGF-β superfamily, such as Activin A and GDF-11, are capable of binding Activin Type IIB receptors (Oh et al., 2002; Olsen et al., 2015). This broad array of targeted ligands makes the decoy receptor a potent inhibitor of not only MSTN, but also an inhibitor of various TGF-β family members that signal downstream of Activin binding. Of note, simultaneous inhibition of MSTN and Activin A leads to more effective muscle hypertrophy and force production in rodents and primates (Latres et al., 2017). Consequently, this ligand trap putatively elicits more robust increases in skeletal muscle growth than MSTN-specific agents (Lee, 2021). In our current study, it is possible that Activin A is differentially expressed and/or blocked with ACVR2B-Fc between sexes, which may account for the sex dimorphisms observed in neonatally denervated muscles. Indeed, a sex-specific role for Activin A in pancreatic ductal adenocarcinoma (PDAC)-induced cachexia has recently been reported by Zhong et al., 2022. The authors observed that sex-specific differences in endogenous levels of Activin A contribute to sex dimorphisms in PDAC-induced cachexia, as well as the differential outcomes of ACVR2B-Fc treatment in attenuating tumor-induced Activin (Zhong et al., 2022). Alternatively, we also wonder whether Activin A and/or other TGF-β family members are more acutely involved in the regulation of neonatal longitudinal muscle growth and contractures than MSTN. Future work with genetic models and expression profiles for the various TGF-β superfamily members are necessary to address these limitations in our current study. Finally, we cannot yet comment on the long-term efficacy of MSTN inhibition beyond 4 weeks following denervation. However, the focus of this study was to establish a proof of concept that targeting a muscle-specific regulator of proteostasis (MSTN) can effectively prevent contractures during the critical window of neonatal muscle growth (4 weeks). Furthermore, we have previously established that contracture formation plateaus at 4 weeks post-NBPI, making this time point ideal for such a proof of concept investigation. As we have established that MSTN inhibition does prevent contractures in a sex-specific manner during this stage of development, ongoing studies are currently planned to build on our findings here by interrogating the efficacy of this pharmacological approach in preventing contractures at skeletal maturity and beyond.

All data generated or analyzed during this study are included in the manuscript and supporting file.

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Author details

1. Marianne E Emmert

   Department of Medical Sciences, University of Cincinnati College of Medicine, Cincinnati, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing – review and editing

   Competing interests

   No competing interests declared

2. Parul Aggarwal

   Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States

   Contribution

   Data curation, Formal analysis, Investigation

   Competing interests

   No competing interests declared

3. Kritton Shay-Winkler

   Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States

   Contribution

   Data curation, Formal analysis, Investigation, Project administration

   Competing interests

   No competing interests declared

4. Se-Jin Lee

   1. The Jackson Laboratory, Farmington, United States
   2. Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, United States

   Contribution

   Resources, Methodology, Writing – review and editing

   Competing interests

   consulting fees from Biohaven Pharmaceuticals, Inc, and Alnylam Pharmaceuticals, Inc; payment/honoraria: Regeneron Pharmaceuticals, Inc; patent: "Therapeutics targeting transforming growth factor beta family signaling"

5. Qingnian Goh

   1. Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
   2. Department of Orthopaedic Surgery, University of Cincinnati College of Medicine, Cincinnati, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Investigation, Visualization, Writing - original draft, Project administration, Writing – review and editing

   For correspondence

   Qingnian.Goh@cchmc.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4108-2957

6. Roger Cornwall

   1. Division of Orthopaedic Surgery, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
   2. Department of Orthopaedic Surgery, University of Cincinnati College of Medicine, Cincinnati, United States
   3. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, United States
   4. Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, United States

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing – review and editing

   For correspondence

   Roger.Cornwall@cchmc.org

   Competing interests

   No competing interests declared

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