Tenotomy-induced muscle atrophy is sex-specific and independent of NFκB
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eLife assessment
The purpose of the study was to evaluate the transcription factor NF-kB, a common transcription factor that is thought to mediate muscle atrophy, in the setting of a rotator cuff injury. The authors used gain of function and loss of function NF-kB inhibitors to show that, surprisingly, NF-kB does not seem to be a major mediator of muscle atrophy in this model (as compared to other atrophy models), but there are sex-related differences. They found that male mice were more likely to have atrophy regulated by autophagy, both of which are interesting, novel findings.
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Abstract
The nuclear factor-κB (NFκB) pathway is a major thoroughfare for skeletal muscle atrophy and is driven by diverse stimuli. Targeted inhibition of NFκB through its canonical mediator IKKβ effectively mitigates loss of muscle mass across many conditions, from denervation to unloading to cancer. In this study, we used gain- and loss-of-function mouse models to examine the role of NFκB in muscle atrophy following rotator cuff tenotomy – a model of chronic rotator cuff tear. IKKβ was knocked down or constitutively activated in muscle-specific inducible transgenic mice to elicit a twofold gain or loss of NFκB signaling. Surprisingly, neither knockdown of IKKβ nor overexpression of caIKKβ significantly altered the loss of muscle mass following tenotomy. This finding was consistent across measures of morphological adaptation (fiber cross-sectional area, fiber length, fiber number), tissue pathology (fibrosis and fatty infiltration), and intracellular signaling (ubiquitin-proteasome, autophagy). Intriguingly, late-stage tenotomy-induced atrophy was exacerbated in male mice compared with female mice. This sex specificity was driven by ongoing decreases in fiber cross-sectional area, which paralleled the accumulation of large autophagic vesicles in male, but not female muscle. These findings suggest that tenotomy-induced atrophy is not dependent on NFκB and instead may be regulated by autophagy in a sex-specific manner.
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Author Response
Reviewer #1 (Public Review):
This excellent manuscript challenged the premise that NF-kappaB and its upstream kinase IKKbeta play a role in muscle atrophy following tenotomy. Two animal models were used - one leading to enhanced muscle-specific NF-kappaB activation and the other a muscle-specific deletion. In both models, there was no significant relationship to observed muscle changes following tenotomy. Overall this work is significant in that it challenges the existing dogma that NF-kappaB plays a crucial role in muscle atrophy.
Surprisingly the authors noted that there were basal differences observed in the phenotypes of their models that were sex-dependent. They note that male mice lose more muscle mass after tenotomy and specifically type 2b fiber loss.
Overall this is an outstanding study that challenges …
Author Response
Reviewer #1 (Public Review):
This excellent manuscript challenged the premise that NF-kappaB and its upstream kinase IKKbeta play a role in muscle atrophy following tenotomy. Two animal models were used - one leading to enhanced muscle-specific NF-kappaB activation and the other a muscle-specific deletion. In both models, there was no significant relationship to observed muscle changes following tenotomy. Overall this work is significant in that it challenges the existing dogma that NF-kappaB plays a crucial role in muscle atrophy.
Surprisingly the authors noted that there were basal differences observed in the phenotypes of their models that were sex-dependent. They note that male mice lose more muscle mass after tenotomy and specifically type 2b fiber loss.
Overall this is an outstanding study that challenges the notion that NF-kappaB inhibitors are likely to improve muscle outcomes following injuries such as rotator cuff tears. Its main weakness is that there were no pharmacological arms of investigation; this fails to definitively exclude the hypothesis that inhibition may exert some effect in healing, perhaps in surrounding non-muscle matrix tissue that in turn may assist in healing.
Thank you for your careful and thoughtful review. We agree that the finding that NFkb is not driving tenotomy-induced atrophy is both surprising and interesting. We look forward to further uncovering the atrophic mechanisms responsible. We also agree that an investigation using pharmacological NFkb inhibitors will improve our understanding of the full scope of the role of NFkb in the tenotomy pathology. As you and another reviewer note, this work has only blocked NFkb signaling in the mature muscle fiber and thus cannot assess the role of NFkb in satellite cell, fibroblast, immune cell activation etc in the healing response. However, we avoided using these inhibitors in this study due to the potential for these systemic effects to obscure the role of NFkb in the muscle fiber. While we believe that a pharmacological investigation is beyond the scope of this study, it will make an excellent follow on investigation.
Reviewer #2 (Public Review):
The primary strength of this paper is a rigorous approach to 'negative' data. Did the authors definitively prove that NF-kB has no role in the tenotomy-induced atrophy? Probably not entirely, since there are limitations of the mouse model and the knockdown mice. There cannot be complete elimination of load since mice heal with some scar tissue, and the knockdown is not complete elimination. However, even with these limitations, this presents important findings that tenotomy, which induces mechanical unloading of the muscle-tendon unit, provides a unique biomechanical environment for the muscle to undergo atrophy, which warrants a more in-depth look given that these injuries are unique and extremely common. It must be mentioned that the results are entirely supported by their data and that even though the model is not 'perfect' it truly supports that NF-kB has a limited role in atrophy. The sex-mediated differences based on autophagy are a secondary hypothesis and are interesting but possibly less clinically relevant based on the differences shown.
We appreciate your thoughts on the “negative” data in this study. A manuscript in which the data refute your hypothesis and that of the field is difficult to write. There is a higher burden of validation and closer scrutiny of limitations. We agree that the model does have some limitations, but overall strongly supports a limited role for NBkb in tenotomy-induced muscle atrophy.
The important next step for this group and others is to evaluate the 'how and why' of tenotomy atrophy if not through NF-kB. Is it that there are many redundant processes that the muscle may have to circumnavigate the NF-kB pathway given that it is so ubiquitous that the authors didn't see a difference? Could it be differences in axial vs appendicular muscle? Or should there be a closer look at the mechanosensors in the muscle cells to determine if there are other key drivers of atrophy? Regardless, this paper shows that tenotomy-induced muscle atrophy is unique and supports the conclusion that muscle has many ways to atrophy based on the injury it undergoes.
We agree that the major next step for this work is to investigate the mechanism(s) responsible for tenotomy-induced atrophy. Autophagy in particular needs a more thorough investigation using autophagic inhibitors in naive wildtype mice to investigate its role in the sex-specificity of tenotomy-induced atrophy. The question of axial vs. appendicular muscle is intriguing. There could also be an upper vs. lower body difference that is worth exploring in future work.
Reviewer #3Public Review):
The authors provided thorough analyses of muscle morphology, biochemistry, and function, which is a major strength of the study. However, there are some key confounding variables authors failed to address. For example, the difference in the estrous cycle in female animals was not controlled. The study could have been significantly improved by controlling sex hormone levels or at least testing differences in response to injury.
We appreciate your careful and insightful review of our work. We designed this study to assess the role of myofiber NFkb in tenotomy-induced atrophy, which led us to a rigorous assessment of morphology, biochemistry and function, which we agree is the strength of the study. We also agree that a major limitation of this study is that the secondary observations of sex-specificity and autophagic signaling are not as well controlled or supported. This is because these observations were made at the end of the study when the histological analyses were completed by the blinded rater. The sex-specificity in the basophilic puncta that the rater observed sparked us to reconsider the sex-specificity in our other data and to stain for autophagic vesicles. As you suggest, to rigorously assess sex-specificity it would be good to control of estrous cycle and analysis of sex hormones which would require initiation of another study, planning for these variables in advance. We think this is beyond the scope of the current question of the role of NFkb in tenotomy-induced atrophy but think it should be undertaken as a follow on to eliminate confounding variables of genetic manipulation and tamoxifen treatment.
However, since we still need to report the sex specificity we observed while ensuring that our findings are not misconstrued, we reviewed the language in the manuscript to emphasize that these are retrospective observations that require further investigation. We have also added discussion of these variables and their potential influence on the results to the Discussion.
Discussion: “Additionally, it is important to note that estrous cycle was not controlled in these mice and sex hormone levels weren’t measured in this study. These preliminary observations, though intriguing, will require more rigorous follow up evaluations to define the interaction between sex, tenotomy, and autophagy in naïve wildtype mice.”
Furthermore, more data are needed to link NFkB signaling and autophagy to make any kind of conclusions. Overall, in the current form of the manuscript, the presented data seem underdeveloped, and the addition of more supporting data could significantly improve the quality of the manuscript and enhance our understanding of NFkB signaling and muscle wasting in rotator cuff injury.
We agree that more data are needed to complete the picture of autophagy in tenotomy-induced muscle atrophy. The p62 and LC3 positive intracellular puncta in male tenotomized muscle are distinctive, but only limited conclusions can be drawn physiologically because 1) they are only present in a fraction of fibers and 2) it is impossible to tell whether they result from increased autophagic flux or altered vesicle processing. Western blot for LC3 (and now p62) indicates only small changes in total protein, but since these proteins are synthesized and degraded during active autophagy, it is possible for their levels to remain constant while flux increases. Direct measures of autophagic flux would require treating mice with an autophagosome block which would require initiation of another study. However, we agree with the reviewer that we can add some additional measures to better characterize the instantaneous state.
We have added analysis of p62 protein expression to LC3 since p62 protein content in muscle can be decoupled from LC3 (PMID: 27493873). We also added expression data for genes involved in autophagy (Lc3b, Gabarapl1, Becn1, Bnip3, and Atg5). Finally, we have commented on the limitations of our data in the Discussion.
Discussion: “Evidence for autophagy regulating tenotomy-induced atrophy has been mounting over recent years (Bialek et al., 2011; Gumucio et al., 2012; Joshi et al., 2014; Ning et al., 2015; Hirunsai & Srikuea, 2021). The evidence presented here supports this contention, but we find surprisingly small effect sizes for all markers investigated. This could be because we are not directly assessing autophagic flux and so are missing some temporal dynamics since synthesis and degradation are ongoing simultaneously.”
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eLife assessment
The purpose of the study was to evaluate the transcription factor NF-kB, a common transcription factor that is thought to mediate muscle atrophy, in the setting of a rotator cuff injury. The authors used gain of function and loss of function NF-kB inhibitors to show that, surprisingly, NF-kB does not seem to be a major mediator of muscle atrophy in this model (as compared to other atrophy models), but there are sex-related differences. They found that male mice were more likely to have atrophy regulated by autophagy, both of which are interesting, novel findings.
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Reviewer #1 (Public Review):
This excellent manuscript challenged the premise that NF-kappaB and its upstream kinase IKKbeta play a role in muscle atrophy following tenotomy. Two animal models were used - one leading to enhanced muscle-specific NF-kappaB activation and the other a muscle-specific deletion. In both models, there was no significant relationship to observed muscle changes following tenotomy. Overall this work is significant in that it challenges the existing dogma that NF-kappaB plays a crucial role in muscle atrophy.
Surprisingly the authors noted that there were basal differences observed in the phenotypes of their models that were sex-dependent. They note that male mice lose more muscle mass after tenotomy and specifically type 2b fiber loss.
Overall this is an outstanding study that challenges the notion that NF-kappaB …
Reviewer #1 (Public Review):
This excellent manuscript challenged the premise that NF-kappaB and its upstream kinase IKKbeta play a role in muscle atrophy following tenotomy. Two animal models were used - one leading to enhanced muscle-specific NF-kappaB activation and the other a muscle-specific deletion. In both models, there was no significant relationship to observed muscle changes following tenotomy. Overall this work is significant in that it challenges the existing dogma that NF-kappaB plays a crucial role in muscle atrophy.
Surprisingly the authors noted that there were basal differences observed in the phenotypes of their models that were sex-dependent. They note that male mice lose more muscle mass after tenotomy and specifically type 2b fiber loss.
Overall this is an outstanding study that challenges the notion that NF-kappaB inhibitors are likely to improve muscle outcomes following injuries such as rotator cuff tears. Its main weakness is that there were no pharmacological arms of investigation; this fails to definitively exclude the hypothesis that inhibition may exert some effect in healing, perhaps in surrounding non-muscle matrix tissue that in turn may assist in healing.
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Reviewer #2 (Public Review):
The purpose of this study was to evaluate the transcription factor NF-KB, a common transcription factor that is thought to mediate muscle atrophy, in the setting of a rotator cuff injury. Unlike many other models of atrophy such as hind limb suspension, aging, and neurologic injury, the tenotomy model represents a unique mechanical change where the muscle is acutely unloaded from the bone, which is relevant for rotator cuff injuries as well as achilles tendon ruptures.
The premise of the study was that NF-kB, a known central regulator of muscle atrophy, would be a central mediator of this process in a tenotomy model as well. The study hypothesized that NF-kB inhibition would reduce atrophy in a rotator cuff model through atrogene-independent mechanisms, a hypothesis that is well supported by literature in …
Reviewer #2 (Public Review):
The purpose of this study was to evaluate the transcription factor NF-KB, a common transcription factor that is thought to mediate muscle atrophy, in the setting of a rotator cuff injury. Unlike many other models of atrophy such as hind limb suspension, aging, and neurologic injury, the tenotomy model represents a unique mechanical change where the muscle is acutely unloaded from the bone, which is relevant for rotator cuff injuries as well as achilles tendon ruptures.
The premise of the study was that NF-kB, a known central regulator of muscle atrophy, would be a central mediator of this process in a tenotomy model as well. The study hypothesized that NF-kB inhibition would reduce atrophy in a rotator cuff model through atrogene-independent mechanisms, a hypothesis that is well supported by literature in other models.
Using gain of function and loss of function NF-kB inhibitors, the IKKB family, to evaluate this pathway after a tenotomy model. The results were rigorously approached with appropriate timelines and controls, and the analyses were well done. Surprisingly, the study found that NF-kB did not appear to be an important regulator of tenotomy-induced atrophy, which they did an excellent job of exploring in detail with their gain/loss of function mice, and by looking at cellular changes, protein changes, and architectural changes after rotator cuff injury. They did find that autophagy, which was more pronounced in male mice, was a sex-dependent mechanism that seemed to regulate atrophy.
The primary strength of this paper is a rigorous approach to 'negative' data. Did the authors definitively prove that NF-kB has no role in the tenotomy-induced atrophy? Probably not entirely, since there are limitations of the mouse model and the knockdown mice. There cannot be complete elimination of load since mice heal with some scar tissue, and the knockdown is not complete elimination. However, even with these limitations, this presents important findings that tenotomy, which induces mechanical unloading of the muscle-tendon unit, provides a unique biomechanical environment for the muscle to undergo atrophy, which warrants a more in-depth look given that these injuries are unique and extremely common. It must be mentioned that the results are entirely supported by their data and that even though the model is not 'perfect' it truly supports that NF-kB has a limited role in atrophy. The sex-mediated differences based on autophagy are a secondary hypothesis and are interesting but possibly less clinically relevant based on the differences shown.
The important next step for this group and others is to evaluate the 'how and why' of tenotomy atrophy if not through NF-kB. Is it that there are many redundant processes that the muscle may have to circumnavigate the NF-kB pathway given that it is so ubiquitous that the authors didn't see a difference? Could it be differences in axial vs appendicular muscle? Or should there be a closer look at the mechanosensors in the muscle cells to determine if there are other key drivers of atrophy? Regardless, this paper shows that tenotomy-induced muscle atrophy is unique, and supports the conclusion that muscle has many ways to atrophy based on the injury it undergoes.
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Reviewer #3 (Public Review):
This study is designed to test the mechanistic role of NF-kB signaling in muscle atrophy following rotator cuff injury. The authors utilized a genetic gain-of-function and loss-of-function model to manipulate NF-kB activation and how this alters muscle plasticity following rotator cuff tendon transection.
The authors provided thorough analyses of muscle morphology, biochemistry, and function, which is a major strength of the study. However, there are some key confounding variables authors failed to address. For example, the difference in the estrous cycle in female animals was not controlled. The study could have been significantly improved by controlling sex hormone levels or at least testing differences in response to injury. Furthermore, more data are needed to link NFkB signaling and autophagy to make …
Reviewer #3 (Public Review):
This study is designed to test the mechanistic role of NF-kB signaling in muscle atrophy following rotator cuff injury. The authors utilized a genetic gain-of-function and loss-of-function model to manipulate NF-kB activation and how this alters muscle plasticity following rotator cuff tendon transection.
The authors provided thorough analyses of muscle morphology, biochemistry, and function, which is a major strength of the study. However, there are some key confounding variables authors failed to address. For example, the difference in the estrous cycle in female animals was not controlled. The study could have been significantly improved by controlling sex hormone levels or at least testing differences in response to injury. Furthermore, more data are needed to link NFkB signaling and autophagy to make any kind of conclusions.
Overall, in the current form of the manuscript, the presented data seem underdeveloped, and the addition of more supporting data could significantly improve the quality of the manuscript and enhance our understanding of NFkB signaling and muscle wasting in rotator cuff injury.
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