Author response: Tenotomy-induced muscle atrophy is sex-specific and independent of NFκB
Year: 2022
Type: peer-review
Abstract: Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Methods Data availability References Decision letter Author response Article and author information Metrics 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. Editor's evaluation This articlce 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. https://doi.org/10.7554/eLife.82016.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Muscle atrophy – the gradual loss of muscle mass – follows injuries to our muscles, tendons, or joints. During atrophy, muscles shrink and become weaker, which can interfere with everyday activities and, ultimately, decrease quality of life. Rotator cuff tears are a common example of such injuries. A rotator cuff is group of four muscles that come together as tendons to form a cuff that normally stabilises our shoulders and allows us to lift and move our arms over our heads. Rotator cuff tears can result from an injury or may be caused by ageing-related wear and tear of the tendon. A signalling protein, called NFκB, is thought to be involved in muscle atrophy. When the NFκB signal is switched on, it interacts with genes that are thought to speed up the loss of muscle mass. However, NFκB's precise role in atrophy and recovery after muscle injury is still poorly understood, particularly following injuries where a tendon is cut or torn. Meyer et al. therefore set out to determine whether or not NFκB played a role in the muscle atrophy following rotator cuff tears. Meyer et al. used genetically engineered mice in which NFκB's signal could be turned off at the time of rotator cuff injury, and specifically in muscle cells (but not other parts of the body). The experiments revealed that stopping NFκβ signalling in these mice did not reduce muscle atrophy after a rotator cuff injury: the levels of atrophy, muscle performance, and muscle composition were the same regardless of whether the NFκβ signal was active. The sex of the mice did, however, affect muscle atrophy, specifically the way in which they lost muscle mass. In male mice, the size of muscle cells decreased, while in female mice, the number of muscle cells decreased. Muscle cells in male mice (but not in females) also accumulated abnormally high amounts of protein, which is an indication of a mechanism of muscle breakdown called autophagy. These results shed new light on the way that we lose muscle mass after injury, and how that could vary depending on the individual. Meyer et al. hope that this study will help guide the development of new, more effective treatments for muscle atrophy, and ultimately contribute to therapies tailored to the characteristics of the patient and the type of injury. Introduction At the most basic level, skeletal muscle atrophy is a shift in the physiological balance between protein synthesis and breakdown. This shift can be initiated by a wide variety of stimuli, from disuse to starvation to cancer, but all eventually converge on a common result – reduced muscle mass. As expected from the diverse stimuli, atrophy can be initiated through a variety of intracellular pathways. However, studies have found surprising commonality downstream, leading to the identification of a number of 'lynchpin' mediators – in particular signaling through nuclear factor kappa beta (NFκB), which has been described as both necessary and sufficient for skeletal muscle atrophy (Jackman et al., 2013; Sandri, 2013). Our current understanding of NFκB's role in adult muscle atrophy has been reviewed in detail (Jackman et al., 2013), where the reader can find illustrations of the pathway. In brief, NFκB is a family of transcription factor subunits that exist as dimers and are localized to the cytoplasm by inhibitory kinases (inhibitor of κB, IκB). Upon stimulation, IκB kinases (IKKα and IKKβ) are phosphorylated and in turn phosphorylate IκB (predominantly IκBα). This leads to cytosolic degradation of Iκβ and nuclear transport of NFκB transcription factor dimers to initiate transcription of genes involved in the atrophy program. Inhibiting IKKβ activity by muscle-specific knockout of its gene (Ikbkb) or electroporating a dominant negative form into skeletal muscle prevented approximately half the loss of muscle mass induced by denervation (Mourkioti et al., 2006), unloading (Van Gammeren et al., 2009), or immobilization (Reed et al., 2011). Similar results were found in the Iκβα superrepressor mouse, which displays dampened NFkB activity, in response to an inflammatory insult (Haegens et al., 2012; Langen et al., 2012) denervation (Cai et al., 2004), unloading (Judge et al., 2007), nutrient deprivation (Lee and Goldberg, 2015), or tumor (Cai et al., 2004). Assessment of NFκB activity by nuclear localization or DNA binding of its subunits confirmed that inhibition of IKKβ activity or IκBα degradation completely (Cai et al., 2004; Senf et al., 2008; Van Gammeren et al., 2009) or partially (Mourkioti et al., 2006) blocked NFκB translocation, demonstrating that NFκB signaling is responsible for a considerable portion of atrophy driven by these diverse stimuli. The best-studied gene targets of NFκB in muscle are the E3 ubiquitin ligases muscle ring finger 1 (MuRF1) and muscle atrophy F-box (MAFbx; a.k.a. Atrogin-1). These genes, termed 'atrogenes,' are thought to control the majority of proteolysis in skeletal muscle across atrophic conditions (Bodine et al., 2001; Gomes et al., 2001). Knockout of MuRF1 results in a similar extent of muscle sparing to NFκB inhibition in denervation (Gomes et al., 2012), unloading (Labeit et al., 2010), glucocorticoid treatment (Baehr et al., 2011), and inflammatory insult (Files et al., 2012), leading it, too, to be labeled 'essential' for skeletal muscle atrophy (Peris-Moreno et al., 2020). While compelling, this explanation remains incomplete. First, because inhibition of NFκB or MuRF1 signaling does not completely prevent atrophy induced by these various stimuli and second because the degree of prevention varies by stimulus and muscle. For example, muscle-specific Ikbkb knockout prevented ~70% of denervation-induced decrease in mass of the soleus muscle, but only ~30% in the gastrocnemius muscle (Mourkioti et al., 2006). Similarly, Iκβα super-repressor prevented only ~25% of the denervation-induced mass loss in the gastrocnemius and tibialis anterior muscles but ~50% of the tumor-induced loss in mass (Cai et al., 2004). Recently, tendon tears (leading to release of muscle tension and muscle retraction) have emerged as potentially employing distinctive mechanisms of muscle atrophy. A comparative study of atrophic signaling between tenotomy and immobilization found that tenotomized gastrocnemius muscle atrophy was primarily characterized by a lysosomal/autophagic signature, in contrast to the classical ubiquitin-mediated proteasomal activity signature of the immobilized gastroc (Bialek et al., 2011). MuRF1 or MAFbx expression did not significantly increase with tenotomy, where dramatic increases were again shown with immobilization, denervation, and glucocorticoid-induced atrophy. This is somewhat surprising given the similarity in signaling induced by other models of unloading that remove either passive loading (e.g., hindlimb suspension and immobilization by casting) or active loading (e.g., denervation). In fact, previous to this study, tenotomy was assumed to employ the ubiquitin-mediated proteasome activity through NFκB and the atrogenes simply because of this similarity (Laron et al., 2012). More recent studies have focused on tenotomy of the rotator cuff (RC) muscles (without suprascapular nerve injury) as a model to investigate the mechanisms of muscle atrophy in human chronic RC tears. These studies have confirmed no increases in MuRF1 or MAFbx following RC tenotomy (Liu et al., 2012) and have uncovered additional evidence for increased autophagic flux (Gumucio et al., 2012; Joshi et al., 2014). Evidence in support of proteasomal activity in the early phase of RC tenotomy, however, also exists (Valencia et al., 2017), leaving open the possibility of coordinated action of multiple pathways. Taken together, these prior studies suggest that although NFκB signaling is a critical component of many disparate atrophy models, it may not be ubiquitous – and in particular may not play a major role in tenotomy-induced atrophy. However, due to the complexity of NFκB signaling, its role in tenotomy-induced atrophy (or lack thereof) cannot be inferred from the lack of atrogene expression or the autophagic state of the muscle alone. First, because evidence suggests that NFκB is capable of driving atrophy independent of MuRF1 or MAFbx (Cai et al., 2004) and second, because there is a complex link between NFκB and autophagy which could cause NFκB to drive the autophagic process (reviewed in Xia et al., 2021). In this study, we sought to determine the role of NFκB signaling in tenotomy-induced atrophy of the RC muscles using muscle-specific gain- and loss-of-function IKKβ transgenic mouse models to inhibit and promote NFκB activity, respectively. We hypothesized that NFκB inhibition would ameliorate tenotomy-induced atrophy through an atrogene-independent mechanism, thus supporting NFκB inhibitors as a novel therapeutic strategy to maintain muscle mass following human chronic RC tears. However, we found that knockdown of IKKβ had no impact on the loss of muscle mass or contractile performance following tenotomy and that overexpression of constitutively active IKKβ was insufficient to drive muscle atrophy. Instead, our data point to an NFκB-independent, sex-specific mechanism driven by autophagy. Results Tenotomy-induced atrophy of the rotator cuff muscles is sex-specific Change in muscle mass was the first outcome assessed following tenotomy of the supraspinatus (SS) and infraspinatus (IS) muscles – the primary abductors in the RC. Although this study was primarily concerned with differences between genotypes (due to changes in NFκB signaling), differences were noted between males and females in the wildtype group. Specifically, at week 8 (W8) post-tenotomy, mass loss in the male SS and IS muscles outpaced that in female mice (Figure 1A). Further investigation in the other outcomes of the study suggests that these sex-specific muscle mass effects arose from sex-specific differences in the morphological mechanisms of atrophy. Atrophy is driven by three primary morphological adaptations: decreases in fiber sizes in the radial dimension (fiber atrophy), decreases in fiber numbers (hypoplasia), and decreases in fiber sizes in the longitudinal dimension (sarcomere subtraction). Only male mice demonstrated decreased fiber cross-sectional area (CSA) at W8 post- tenotomy (Figure 1B). Conversely, only female mice decreased fiber numbers at W8 post-tenotomy (Figure 1C). Female mice also decreased fiber lengths to a greater extent than male mice at both W2 and W8 (Figure 1D). Figure 1 Download asset Open asset WT control mice exhibited sex-specific responses to late-stage tenotomy-induced muscle atrophy. (A) Supraspinatus (SS) and infraspinatus (IS) muscles from male mice lose more mass than female muscles at 8 weeks (W8) post-tenotomy (TEN). Muscle mass is normalized to body mass and the average value from the sham group for each sex. (B) Type 2b fiber cross-sectional area (CSA), assessed in histological sections, is also significantly reduced in males compared with females at W8. (C) The total number of fibers counted per histological section is significantly reduced in the female SS compared with male at W8. (D) Prediction of fiber lengths from measured muscle lengths during physiological testing indicates that female SS muscles lose a greater fraction of their fiber length at both W2 and W8 post-tenotomy. (E) Predictions of the contributions of each morphological change (B–D) to the measured mass deficit (A). Change in (Δ) fiber length (dark gray), fiber number (light gray), and fiber CSA (white) show different trends for males and females at W2 and W8 post-tenotomy. (F) The mass deficit predicted by morphological changes and the mass deficit measured at W2 and W8 are significantly correlated. (A–D) Raw values were normalized to the average of the sham group of the same sex. N = 5–7 per group; *p<0.05, **p<0.01. Figure 1—source data 1 Raw data and statistical analysis results for normalized supraspinatus (SS) mass, normalized infraspinatus (IS) mass, normalized type 2b fiber cross-sectional area (CSA), normalized fiber number, normalized fiber length, % contribution to mass deficit, and predicted vs. measured mass deficit. https://cdn.elifesciences.org/articles/82016/elife-82016-fig1-data1-v2.xlsx Download elife-82016-fig1-data1-v2.xlsx To better understand the relative impact of these morphological changes on muscle mass, a geometrical model was employed to predict the mass deficit from each change. Overall, changes in fiber length accounted for most of the mass deficit with tenotomy in both sexes (Figure 1E, purple). However, there were notable differences in the other sources. While changes in fiber CSA accounted for 25–30% of the mass loss in male mice at 2W and 8W, they accounted for none of the mass loss in female mice. In contrast, changes in fiber number account for 30–40% of the mass loss in females, but only 15–20% in males. To validate that our model captured the major sources of mass loss, we regressed the predicted mass deficit against the measured mass deficit for each muscle across both sexes. The measurements were significantly correlated (r2 = 0.41, p=0.002; Figure 1F). Additionally, the slope of the regression line was close to 1, indicating that the morphological measurements were able to account for the majority of muscle mass loss following tenotomy. Given these differences, all subsequent analyses investigating the effect of IKKβ knockdown and overexpression on tenotomy-induced muscle atrophy consider sex-specific effects. IKKβ gain- and loss-of-function did not affect tenotomy-induced muscle atrophy The gene encoding IKKβ (Ikbkb) was selectively deleted (IKKbMKD) or constitutively activated (IKKbMCA) in mature skeletal muscle fibers of 6–8-month-old transgenic mice. Tamoxifen treatment of IKKbMKD mice caused a 50–60% reduction in the expression of Ikbkb mRNA in supraspinatus muscles that was sustained through W8 (Figure 2A). Conversely, tamoxifen treatment of IKKbMCA mice caused a two- to threefold increase in Ikbkb expression at W8 (Figure 2B). Knockdown of Ikbkb expression decreased IKKβ protein levels by a comparable 50–60% (Figure 1C) while overexpression of constitutively active Ikbkb increased IKKβ protein levels by two- to threefold (Figure 2D). This deletion efficiency measured at the whole muscle level is comparable to other tamoxifen-inducible models in which satellite and interstitial cells also express the gene of interest. While the deletion efficiency in muscle fibers is likely higher than what is measured at the whole muscle level, it is likely incomplete and thus this model is referred to as knockdown rather than knockout. In male WT mice of the IKKbMKD cohort, Ikbkb expression significantly increased by 40–50% in response to tenotomy at both W1 and W8 post-tenotomy (Figure 2A). However, this effect was not seen in the WT mice of the IKKbMCA cohort (Figure 2B), nor was it evident at the protein level (Figure 2C). To ensure that a 50–60% reduction IKKβ protein was sufficient to impact NFκB signaling, we isolated the nuclear fraction from IKKbMKD and WT muscle and quantified NFκB subunits p50 and p65, which reflect the NFκB-proteasomal degradation axis. Nuclear p50 and p65 were reduced by more than 50% in IKKbMKD mice in both sham and tenotomy groups (Figure 2E), confirming inhibition of NFκB nuclear translocation. Furthermore, this assay was validated using positive controls (Figure 2—figure supplement 1). Thus, these genetic manipulations resulted in a consistent two- to three3fold knockdown or overexpression of IKKβ in both sexes. Figure 2 with 1 supplement see all Download asset Open asset Muscle-specific inducible deletion and overexpression of IKKβ induced a twofold decrease and increase in IKKβ expression, respectively. (A) In mice with muscle-specific inducible IKKβ deletion (IKKbMKD), expression of the IKKβ gene (Ikbkb) was reduced 50–60% in male and female sham (SHM) and tenotomized (TEN) supraspinatus (SS) at week 1 (W1) and week 8 (W8) compared with wildtype (WT). (B) In mice with muscle-specific inducible overexpression of constitutively active IKKβ (IKKbMCA), Ikbkb expression was increased two- to threefold in SHM and TEN groups at W8. (C) Protein abundance of IKKβ was 50–60% lower in IKKbMKD compared with WT. (D) Protein abundance of IKKβ was two- to threefold higher in IKKbMCA compared with WT. (E) Protein abundance of NFκB subunits p50 and p65 in the nuclear fraction of SS muscles was reduced 50–60% in IKKbMKD compared with WT. N = 3–6 per group; *p<0.05, **p<0.01, ***p<0.005, ****p<0.001. Figure 2—source data 1 Raw data and statistical analysis results for relative Ikkb expression, IKKβ/actin, and nuclear p50/histone H3. https://cdn.elifesciences.org/articles/82016/elife-82016-fig2-data1-v2.xlsx Download elife-82016-fig2-data1-v2.xlsx Figure 2—source data 2 Western blot raw and uncropped images. https://cdn.elifesciences.org/articles/82016/elife-82016-fig2-data2-v2.zip Download elife-82016-fig2-data2-v2.zip In both sham and tenotomy groups, IKKβ conditional deletion caused a increase in mass of the SS (Figure and IS (Figure muscles normalized to body mass. each treatment group a significant effect of in SS SS tenotomy, and IS sham However, only in the male SS at W1 post-tenotomy was the To determine whether IKKβ conditional deletion the atrophic process post-tenotomy or simply increased muscle mass tenotomized SS and IS muscle were normalized to the mass of the tibialis anterior of the same In this no consistent was observed between genotypes in either muscle, either or Conversely, overexpression of caIKKβ exacerbated tenotomy-induced atrophy to a extent in both SS and IS muscles (Figure and which resulted in a significant treatment effect by to the mass of the these effects (Figure muscles in IKKbMKD mice exhibited the same sex specificity in mass loss (Figure and as noted for their WT (Figure 1). muscle mass from the IKKbMCA cohort did not show this effect normalized to body mass, but there was a significant in body mass between sham and tenotomized male mice that may have the atrophic sex SS and IS normalized to mass significant effects of sex by (Figure Figure Download asset Open asset IKKβ deletion and overexpression muscle mass in sham (SHM) and tenotomy (TEN) groups muscle loss post-tenotomy. (A) Supraspinatus (SS) muscles from IKKbMKD mice had a increase in mass normalized to body mass in both SHM and TEN groups compared with WT. (B) (IS) muscles from IKKbMKD mice had a increase in normalized mass compared with WT. (C) When SS mass was normalized to tibialis anterior mass, there was no between IKKbMKD and WT (D) IS muscles from IKKbMCA mice had a decrease in muscle mass in the TEN group at week 8 (W8) compared with WT. (E) IS muscles from IKKbMCA mice had a decreased in normalized mass compared with WT in the TEN group (F) When SS mass was normalized to mass, there was no between IKKbMCA and WT are the average of all sham values for between genotypes the same sex and compared with week (W1) values the same sex and N = 5–7 per group; compared with sham values the same sex and Figure data 1 Raw data and statistical analysis results for normalized supraspinatus (SS) mass, infraspinatus (IS) mass, SS anterior mass and IS mass. Download IKKβ gain- and loss-of-function did not affect tenotomy-induced muscle loss tenotomy, SS and IS muscles lose of their contractile tension (Figure This loss was not by IKKβ deletion or overexpression of caIKKβ in either muscle or either sex at mass loss, contractile were similar between W2 and W8 in both sexes. This suggests that contractile tension to physiological cross-sectional area for the ongoing mass Figure Download asset Open asset IKKβ deletion and overexpression did not affect the loss of tension following tenotomy (TEN). (A) tension of supraspinatus (SS) muscles normalized to physiological cross-sectional area is not different between WT and IKKbMKD genotypes in sham (SHM) and TEN (B) tension of infraspinatus (IS) muscles is also not different between WT and IKKbMKD genotypes in SHM and TEN (C) tension of SS muscles is not different between WT and IKKbMCA genotypes in SHM and TEN (D) tension of IS muscles is not different between WT and IKKbMCA genotypes in SHM and TEN N = 5–7 per group; compared with sham values the same sex and Figure data 1 Raw data and statistical analysis results for supraspinatus (SS) and infraspinatus (IS) Download following tenotomy were independent of IKKβ knockdown We sought to whether the of the tenotomy-induced muscle mass loss and contractile were by IKKβ For these we focused on the SS muscle SS and IS to tenotomy with to mass and contractile Surprisingly, tenotomy caused changes in fiber CSA compared to other of atrophy for a similar mass At W8 of mass only type 2b fibers (Figure from male muscles exhibited a shift in the of CSA with tenotomy (Figure while type fibers exhibited a shift (Figure of CSA of type and type 2b fibers over time this was a effect of time in analysis of type CSA by to the increase in CSA through W8 (Figure was also a effect of with a significant sex time effect in type 2b CSA (Figure to the sex specificity in Figure The mass loss for by fiber CSA changes was due to decreases in fiber numbers and lengths (Figure and length had an additional effect of sex (Figure as the sex specificity in Figure 1 was evident in the IKKbMKD group as were no significant effects or by in of the indicating that IKKβ knockdown did not shift the mechanisms of mass In contrast to morphological of no consistent in as a of tenotomy or IKKβ knockdown (Figure supplement 1). Figure with 1 supplement see all Download asset Open asset Muscle morphological changes following tenotomy (TEN) were not by IKKβ knockdown and were (A) of histological from sham (SHM) and TEN WT supraspinatus (SS) muscles at week 8 were by against of – type 1 type type and type 2b – and area was quantified by the (B) of cross-sectional (CSA) for type and 2b fibers for male and female mice at W8. of type 2b fiber CSA in male mice are to the following (C) CSA of type fibers and (D) average CSA of type 2b fibers across genotypes and the TEN group. Only type 2b fiber CSA in males decreases following tenotomy. (E) of the total number of fibers in the SS across genotypes and the TEN group. (F) Prediction of fiber length from measurements of muscle length during physiological testing at W2 and W8 the TEN group. data exist at week 1 because physiological testing was not at that The are the average of all sham values for for each sex. N = 5–7 per group; compared with week (W1) values the same sex and Figure data 1 Raw data and statistical analysis results for fiber cross-sectional area (CSA) fiber type fiber number, and predicted fiber Download Tenotomy-induced pathology to sex specificity in autophagy we assessed of pathology that are with tenotomy and thought to impact muscle – fatty and fiber – and and fatty are in tenotomy et al., 2007), and were therefore measured at W8 to the signal (Figure assessed as the area fraction positive for was following tenotomy but a effect of or indicating that tenotomy induced in WT and IKKβ knockdown males and females (Figure assessed as the area fraction positive for was also following tenotomy, an effect that was exacerbated in female mice due to the higher levels of in females (Figure et al., 2012). However, there was no effect of again indicating that IKKβ knockdown did not affect the of fatty was assessed on by a fibers and fibers (Figure there were no fibers in tenotomized muscle, that active was either not or at the for However, there was a gradual increase in fibers over time following tenotomy, which was exacerbated in males (Figure A noted the of in (Figure and These were more in males than females, and this was exacerbated at 8W, where they were increased over W1 levels (Figure These positive for and
Source: eLife
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $2,500
OpenAlex ID: W4320029849