BETs inhibition attenuates oxidative stress and preserves muscle integrity in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) affects 1 in 3500 live male births. To date, there is no effective cure for DMD, and the identification of novel molecular targets involved in disease progression is important to design more effective treatments and therapies to alleviate DMD symptoms. Here, we show that protein levels of the Bromodomain and extra-terminal domain (BET) protein BRD4 are significantly increased in the muscle of the mouse model of DMD, the mdx mouse, and that pharmacological inhibition of the BET proteins has a beneficial outcome, tempering oxidative stress and muscle damage. Alterations in reactive oxygen species (ROS) metabolism are an early event in DMD onset and they are tightly linked to inflammation, fibrosis, and necrosis in skeletal muscle. By restoring ROS metabolism, BET inhibition ameliorates these hallmarks of the dystrophic muscle, translating to a beneficial effect on muscle function. BRD4 direct association to chromatin regulatory regions of the NADPH oxidase subunits increases in the mdx muscle and JQ1 administration reduces BRD4 and BRD2 recruitment at these regions. JQ1 treatment reduces NADPH subunit transcript levels in mdx muscles, isolated myofibers and DMD immortalized myoblasts. Our data highlight novel functions of the BET proteins in dystrophic skeletal muscle and suggest that BET inhibitors may ameliorate the pathophysiology of DMD.

mechanisms. I think this is one of the major downsides of this report. The authors do not show any proof of this concept and do not provide any information about the specificity of the antibody used. The specificity of the anti-BRD4 must be evaluated in BRD4 KO mice or at least in BRD4 silenced C2C12 cells. 2-Another major downside of this report is the fully lack of information about JQ1. Is this compound selective for BRD4 or it has affinity also for the other BRD proteins? In this latter case, there is the risk that results shown in this report have been misinterpreted. In addition, there is no information about time (2 weeks) and dosage. 3-In some representative blots, the anti-vinculin or GADPH antibody produces only one band while in other figures I see two bands. Can the authors explain why? 4-The authors do not provide any information about the bioinformatics analysis. Please provide all the necessary information about the consensus sequences, score, type of software used etc. 5-The authors state that BET proteins promote the transcriptional activity of NADPDH oxidase sub genes. However, Chip assay is a useful tool to demonstrate the binding of transcription factors to DNA. It would be more straightforward to perform also luciferase assays. 6-The effect of JQ1 on muscle regeneration is very confusing. It would be a good idea to evaluate the effect of JQ1 on satellite cell proliferation and differentiation. 7-Since in mdx mice the onset is before 5 weeks and these mice do not show a decline in their regeneration capacity at early age, can the authors explain why they used 10 week-old mdx mice? What about the effect of JQ1 when the disease phenotype is more aggravated? 8-I noticed a complete lack of information about DMD donors. This needs to be clearly stated in the manuscript. 9-I have noted irregularities in the stats analysis. Authors stated that for experimental groups >2 they used ANOVA followed by Tukey's test. However, it is not mentioned in the manuscript if the biological replicates are normally distributed or not. If are not normally distributed they would need to be analysed via Kruskal-Wallis test with Dunn's post hoc (not Tukey) and be presented as box & whisker.
Reviewer #3: Remarks to the Author: This work from Segatto et al provides compelling evidence that BET inhibition with JQ1 ameliorates a number of the important pathological features of the mdx mouse model of Duchenne Muscular Dystrophy. The rationale and study design is well justified, the paper is clearly written and enjoyable to read, and the data overall appear robust and appropriately analyzed (with minor exceptions detailed below). In sum, the work reflects an impressive biochemical characterization, validated with functional rescue, and the overall result is both novel and significant. I have a few suggestions aimed at increasing the overall scope and relevance of the work, and some small text edits. Major: 1. The relevance of this work would be significantly strengthened by a demonstration of BRD4 upregulation, or increased BRD4 occupancy of key targets such as Nox2, in human DMD tissue. The upregulation of BRD4 targets (such as Nox2) may be able to be simply mined from existing RNA sequencing data sets from DMD boys (such as in Khairallah et al., Science Signaling 2013). While the authors do show that JQ1 can reduce Nox2 transcript in immortalized DMD cells, a demonstration that BRD4 upregulation is a relevant feature beyond the mdx mouse would significantly strengthen the impact of this work.
Minor: 2. The authors conduct a fairly comprehensive biochemical characterization of many key dystrophic features ameliorated by JQ1 (oxidative stress, autophagy, inflammation, fibrosis). Given the relatively large number of recent reports linking Nox2-mediated ROS production to microtubule abnormalities that contribute to DMD pathology (e.g. Randazzo et al., Hum Mol Genet 2019, Prosser et al., Science 2011, Loehr et al., Elife 2018, Nelson et al., Hum Mol Genet 2018, Kerr et al., Nature Communications 2015, Khairallah et al., Science Signaling 2013, it seems a missed opportunity to determine whether JQ1 also corrects microtubule abnormalities that contribute to disease pathology. This could be accomplished by a simple western blot of key markers of microtubule misregulation, such as expression of detyrosinated-tubulin and TUBB6, and would provide further evidence that proximal changes in Nox2 contribute to the microtubule dysfunction well characterized in DMD. 3. For histological characterizations, for example of EBD and SHD imaging, there is no mention of the imaging or analysis being performed blinded to the treatment groups. These techniques are particularly prone to user bias, and positive results have historically been difficult to replicate when subjected to blinded analysis (there is a considerable history of this in the DMD field). These samples should be imaged and analyzed blindly, and if this is not possible, it should be stated as such in the methods. 4. Regarding the histology, the presence of centrally nucleated fibers is a classic hallmark of DMD pathology, and their correction a benchmark of pathological rescue. Thus it is odd that the authors do not quantify CNFs. This seems particularly important given the links to BET inhibition and differentiation/regeneration. 5. What is the definition of, or inclusion criteria for, an "intact" fiber as quantified in figure 1? Again, was this analysis performed blind? 6. The word "remarkably" is used in considerable excess (>10 times) to describe JQ1 effects. This degree of hyperbole throughout the manuscript is not needed, the robust results speak for themselves! 7. The authors state that the protection from oxidative stress "was not ascribed to the transcriptional activation of anti-oxidant genes", yet do not seem to probe for Nrf2, which regulates broad antioxidant proteins implicated in DMD pathology. This seems important to include, or at least explain, if the authors wish to make this claim. 8. Page 9 top, reference should be included for "as previously observed in mdx muscles" 9. Please edit page 12, "JQ1-treated mdx mice exhibited significantly increased resistance to fatigue in the treadmill test, and we observed a significant improvement in endurance." 10. Why not include the full time course of S.

Authors response to reviewers' comments.
The authors thank the three reviewers for their efforts in reviewing our manuscript, for recognizing the novelty of our experimental work and the useful comments that we used to clarify and improve the manuscript. We performed a substantial body of new experiments and below we present our point-topoint response, outlying modifications in the text, as well as in main or supplemental figures. Novel data or changes in the text are shown in red.
Reviewer #1 (Remarks to the Author): The authors address the role of bromodomain and extraterminal (BET) proteins, which are acetyl-chromatin binding factors, in the pathogenesis of muscular dystrophy. In muscle from the mdx mouse model of muscular dystrophy, expression of the BRD4 BET family member is elevated at the protein level. Treatment of mdx mice with the pan-BET inhibitor, JQ1, has various salutary effects, including increasing the number of intact skeletal muscle fibers and reducing the number of damaged fibers. This correlated with various effects that are predicted to be beneficial, such as reduced oxidative stress. In this regard, in vitro and in vivo studies revealed that JQ1 suppresses expression of NAPDH oxidase subunits.
The ability of JQ1 to improve the muscular dystrophy phenotype is impressive and important. However, the manuscript lacks the detailed mechanistic insight and investigational rigor that is required for publication in Nature Communications.
Specific points 1. All in vivo studies employed three animals per group, which is unacceptable. We employed more than 3 animals per group in our in vivo experiments, as described in figure legends. We usually used between 6-8 animals in immunoblots of Mdx-Veh and Mdx-JQ1 treated animals. We used an average of 7 animals per group for RNA qPCR analysis and used 3 animals per group in morphological evaluation of skeletal muscles, where multiple sections of each muscle were analyzed. We employed 3 animals only for WT animals, which behaved in a very homogenous way.
2. Additional studies should be performed to understand which BET family member(s) regulate the phenotype. This could be accomplished by knocking down expression of BRD2, BRD3 and BRD4 in the authors' cell culture models. We agreed with the reviewer and performed knockdown experiments with siRNAs against BRD2/BRD3/BRD4 and evaluated transcript levels of the NAPDH oxidase subunits in the different conditions. These data are presented in Fig.5H. Page 11-12: "To define which BET protein plays a major role in NADPH oxidase subunits modulation, we employed a siRNA approach in C2C12 myoblasts. BRD4 knockdown reduced the transcript levels of Nox2, Nox4, p47-phox and p67-phox, while BRD2 depletion didn't affect Nox4 mRNA but decreased Nox2, p47-phox and p67-phox expression. BRD3 did not influence NADPH subunits expression levels (Fig. 5H and Fig. S9A)." 3. The ChIP-PCR findings in Fig. 5 are mislabeled on the y-axis. ChIP-PCR is not used to quantify mRNA expression. We thank you the reviewer for spotting this mistake. We corrected the y-axis in Fig. 5I, which represents the percentage of Input.
4. The regulatory regions that were amplified in the ChIP-PCR study should be defined. We introduced a scheme indicating the position of the amplified regions in the genes and explained in the text that we derived these regulatory regions by our previously published ChIP-seq data and from previous reports (Page 12). "Because of BRD2 and BRD4 ability to modulate NADPH oxidase subunits, we performed ChIP assays for these two BET proteins in skeletal muscles of control, vehicleand JQ1-treated mdx mice. We amplified chromatin regulatory regions that we previously found to be occupied by BRD4 in TA of control mice by ChIP-seq assays, 32 which were shown to include previously described regulatory regions 55-60 . " 5. For immunoblotting, full-length gels with molecular weight markers should be shown. Representative gels for main and supplemental figures, with molecular weight markers, are reported in the Data Source file.
6. In Fig. 4, C2C12 cells were treated with H2O2 and JQ1 for 24 hours, but in Fig. 5E, cells were pretreated with JQ1, then co-treated with H2O2 for 8 hours. The experimental conditions should be kept constant. Some piece of information was missing in figure legend 4. In the experiments shown in Fig. 4, we also pre-treated cells with JQ1 for 24 hr, as in Fig.5, and then we co-treated with H 2 O 2 for 8 hours. While the time of JQ1 pre-treatment was the same in the two figures (24hr), we employed different H 2 O 2 treatment time (8 versus 24 hours). The rationale for this difference in H 2 O 2 treatment was that in figure 5E we were looking at the RNA and in Fig. 4 at the protein levels: we reasoned that RNA changes precede protein alterations. We have changed figure legend 4B-D accordingly, adding the information regarding pre-treatment. Page 27: "Cells were pretreated with JQ1 (200nM) for 24 hr and then stimulated with H 2 O 2 for 24 hr". 7. Since the idea is that JQ1 decreases oxidative stress in mdx mice, it would be better to pre-treat cells with H2O2, then co-treat with JQ1. Additionally, treatment with JQ1 alone should be included. We performed these experiments and reported these data in Supplementary Fig. S5. We have found that our initial data were confirmed in this setting. These figures are now described on page 9-10: Page 9-10. "To test whether JQ1 is effective when oxidative stress is already established, we administered JQ1 after treating C2C12 myotubes with H 2 O 2 for 2 hours, a sufficient time to To test whether JQ1 is effective when oxidative stress is already established, we administered JQ1 after treating C2C12 myotubes with H 2 O 2 for 2 hours, a sufficient time to induce oxidative stress in C2C12 cells (Fig. S5A). The modulation of Sirt1, p62, LC3 as well as of phosphorylated AKT, AMPK and Ulk1 were similar to the one obtained when cells were pretreated with JQ1, followed by H 2 O 2 stimulation (Fig. S5B-C). In addition, JQ1 treatment alone did not affect p-AKT and p62 levels, but it increased Sirt1, lipidated LC3 levels, and AMPK and Ulk1 phosphorylation (Fig. S5B-D). In this experimental setting, we confirmed that NAM treatment prevented AMPK activation and ULK phosphorylation (Fig. S5C)." 8. The authors suggest that SIRT1 accumulation and SRC phosphorylation are the links between oxidative stress and autophagy, although they did not dig deeper into this possibility. Additional immunoblots of LC3 and p62 for Fig. S3B would enhance the manuscript. The link between SRC phosphorylation, oxidative stress and autophagy in the mdx mouse was previously reported (Pal et al., 2014). We performed the suggested experiments and included these results in Supplemental Fig.  5D. We discussed these results on page 10. "Moreover, Sirt1 pharmacological blockade hindered LC3 accumulation and p62 downregulation in cells in which oxidative stress was induced by H 2 O 2 followed by JQ1 treatment (Fig. S5D), further supporting the idea that Sirt1 plays a key role in AMPK activation and autophagy regulation." Reviewer #2 (Remarks to the Author): In this report Segatto et al. demonstrate that the pharmacological inhibition of BET proteins has important structural and functional benefits on skeletal muscle quality of dystrophic mice as well as in primary myoblasts isolated from DMD donors. Authors use a variety of experimental methods however, there are important weaknesses that should be addressed.
1-There is a strong discrepancy between the mRNA and protein expression level of BRD24 in skeletal muscles of mdx mice. The authors suggest that this is possibly due to post-transcriptional mechanisms. I think this is one of the major downsides of this report. The authors do not show any proof of this concept and do not provide any information about the specificity of the antibody used. The specificity of the anti-BRD4 must be evaluated in BRD4 KO mice or at least in BRD4 silenced C2C12 cells. We have tested the specificity of the anti-BRD4 antibody in BRD4 silenced cells and reported this data in Supplemental Fig.S1 (Material and Methods section). Page 23: "BRD4 antibody specificity was tested in C2C12 myoblasts silenced for BRD4 (Fig. S1A)." We agree that the difference between mRNA and protein level is an interesting but open question. Data found on DMD muscles and included in Fig. 1C,D are pointing to a relevant role for this stabilization also in the human pathology, where BRD4 protein levels are higher in DMD muscles but transcript levels are comparable to healthy donors, in independently published RNA-Seq datasets. We are working on the mechanisms underlying BRD4 stabilization, which will require several efforts to define possible posttranslational modifications affecting protein degradation and/or to establish which E2/E3 complex is involved in BRD4 degradation and its modulation in the mdx muscle. Since the available tools to dissect these molecular mechanisms are still limited (for instance antibodies for BRD4 post-translational modifications), we will include these studies in a future manuscript.
2-Another major downside of this report is the fully lack of information about JQ1. Is this compound selective for BRD4 or it has affinity also for the other BRD proteins? In this latter case, there is the risk that results shown in this report have been misinterpreted. In addition, there is no information about time (2 weeks) and dosage. JQ1 binds to both BRD3 and BRD4 with a similar Kd and with higher Kd to BRD2 (Filippakopoulos et al., 2010). Interestingly, BRD2 and BRD4 were shown to regulate transcription in concert in several contexts (Cheung K. et al, 2017;Frnandez-Alonso R. et al., 2017). More targeted loss of function experiments, through siRNA transfection, were used to address the ability of BET proteins to regulate specific targets. These data were included in Fig. 5H and S9A and were described on pages 11-12. "JQ1 binds with different affinity the BET proteins, thus being capable to displace BRD2/BRD3/BRD4 from chromatin 53 . To define which BET proteins play a major role in NADPH oxidase subunits modulation, we employed a siRNA approach in C2C12 myoblasts. BRD4 knockdown reduced the transcript levels of Nox2, Nox4, p47-phox and p67-phox, while BRD2 depletion didn't affect Nox4 mRNA but decreased Nox2, p47-phox and p67-phox expression (Fig. 5H and Fig. S9A)." We also introduced more information about JQ1 chronic treatment. Page 6: "We daily treated 10-week-old mdx mice with JQ1 (20 mg/kg per day) by intraperitoneal injection" 3-In some representative blots, the anti-vinculin or GADPH antibody produces only one band while in other figures I see two bands. Can the authors explain why? Vinculin can display a main band and a fainter upper band (metavinculin), while GAPDH can display a fainter lower band in certain cell types (photo 4, https://www.abcam.com/gapdh-antibody-6c5-loading-control-ab8245.html). In some of our representative gels the second fainter band was not present because of the thicker gel concentration, running conditions or exposure settings. Representative western blot are now included in Data source.

4-The authors do not provide any information about the bioinformatics analysis.
Please provide all the necessary information about the consensus sequences, score, type of software used etc. We did not show ChIP-Seq data and thus we did not provide this information.
5-The authors state that BET proteins promote the transcriptional activity of NADPH oxidase sub genes. However, Chip assay is a useful tool to demonstrate the binding of transcription factors to DNA. It would be more straightforward to perform also luciferase assays. BRD4 doesn't directly bind to DNA and it requires histones for its anchoring to the chromatin. Luciferase assays are useful means to show the ability of transcription factors to promote transcription, once bound to their consensus site; their relevance is less straightforward with chromatin factors. In particular BRD4 requires acetylated histones for recruitment to DNA, which may not be properly assembled on plasmid DNA in a transient transfection assay. ChIP is a more direct and less artificial mean to prove BRD4 chromatin occupancy to genomic regions. We do agree with the reviewer that chromatin association doesn't necessarily imply transcription, but the siRNA assays we introduced in fig. 5H show that BRD2 and BRD4 depletion do influence NADPH subunit levels. Therefore, we changed the text on page 12 accordingly: "Overall, these findings revealed that BRD4 and BRD2 occupy the NADPH oxidase subunits regulatory genes in the mdx muscle and modulate their transcription." 6-The effect of JQ1 on muscle regeneration is very confusing. It would be a good idea to evaluate the effect of JQ1 on satellite cell proliferation and differentiation. We isolated satellite cells and evaluated JQ1 effects on proliferation and differentiation. These data are now included in Supplemental figure S10 and are described on page 14: "In vitro, JQ1 (200nM) treatment of satellite cells did not prevent their ability to differentiation, nor significantly decreased their proliferation rate (Fig. S12D and E). " 7-Since in mdx mice the onset is before 5 weeks and these mice do not show a decline in their regeneration capacity at early age, can the authors explain why they used 10 weekold mdx mice? What about the effect of JQ1 when the disease phenotype is more aggravated? We employed 10 weeks old animals to depict a disease stage in which the muscle is still plastic, but it also displays several concurrent hallmarks of the pathology. For example, we were able to study fibrosis at this stage, since it starts to manifest around 12 weeks. Importantly, we were also interested in autophagy, which is still active at 6 weeks and get less functional later on (Fiacco et al., 2016). In addition, Nox2 protein is upregulated at 5-9 weeks of age in the mdx mouse (Whitehead et al., 2010), but not in younger animals. Another aspect of interest was inflammation, which peaks around 4-8 weeks but it is still present at 10-12 weeks. Therefore, we selected a timepoint in which we were able to dissect different aspects of the disease pathophysiology, of the adult mouse. We do agree that it is interesting to further characterize JQ1 effects with a more aggravated phenotype. We treated mdx mice at 11 month of age for 4 weeks and focused out attention on overall morphology, inflammation, fibrosis and NADPH oxidase subunits transcription level. The longer treatment time was employed because we reasoned that certain pathological features, such as fibrosis, would be more difficult to challenge in older mice. These data are presented in Supplemental Fig. S11 and described on page 13-14: "To evaluate the impact of JQ1 treatment in older animals, we daily treated 11 months old mdx mice with JQ1 (20mg/ml/day) by intraperitoneal injection, for 4 weeks. The longer treatment time was employed because we reasoned that certain pathological features, such as fibrosis, would be more difficult to challenge in older mice. At this stage of the disease progression, JQ1 administration led to a reduction in the transcript levels of inflammatory markers TNFα and IL6 (Fig. S11A), which was paralleled by a decrease in the levels of CD45 and F4/80 proteins (Fig. S11B), as well as of inflammatory infiltrate (Fig. S11C). BET blockade led to a trend towards increasing the number of peripheral nucleated fibers and reducing the centrally nucleated fibers, although not significantly (Fig. S11D). Fibrosis was reduced in 12 months old JQ1-treated mdx TAs, as shown by Sirius red staining (Fig. S11E). Transcript levels of NADPH oxidase subunits and collagen 1α1 were also reduced following JQ1 administration (Fig. S11F  and G). Overall, these results show that, in the mdx mouse model, JQ1 treatment has a beneficial impact also when the disease phenotype is aggravated." 8-I noticed a complete lack of information about DMD donors. This needs to be clearly stated in the manuscript. We included this information in Supplemental materials, in supplemental Methods, page 1. This work from Segatto et al provides compelling evidence that BET inhibition with JQ1 ameliorates a number of the important pathological features of the mdx mouse model of Duchenne Muscular Dystrophy. The rationale and study design is well justified, the paper is clearly written and enjoyable to read, and the data overall appear robust and appropriately analyzed (with minor exceptions detailed below). In sum, the work reflects an impressive biochemical characterization, validated with functional rescue, and the overall result is both novel and significant. I have a few suggestions aimed at increasing the overall scope and relevance of the work, and some small text edits.

Donor myoblasts for immortalized cell lines
Major: 1. The relevance of this work would be significantly strengthened by a demonstration of BRD4 upregulation, or increased BRD4 occupancy of key targets such as Nox2, in human DMD tissue. The upregulation of BRD4 targets (such as Nox2) may be able to be simply mined from existing RNA sequencing data sets from DMD boys (such as in Khairallah et al., Science Signaling 2013). While the authors do show that JQ1 can reduce Nox2 transcript in immortalized DMD cells, a demonstration that BRD4 upregulation is a relevant feature beyond the mdx mouse would significantly strengthen the impact of this work. We agreed with the reviewer that showing BRD4 levels in DMD patients is a relevant point, and we made several attempts to obtain DMD tissues from European and US biobanks. We were able to obtain 4 DMD and 2 control samples from the AFM-Myobank (Paris) and data show that BRD4 levels increase in DMD muscles, despite the limited number of control age-matched muscles. These data are reported in fig. 1C. We also confirmed a significant decrease of Nox2, p47-phox and p67-phox transcripts in RNA-seq data of DMD vs control muscles, published in Khairallah et al. (Khairallah et al., Science Signaling 2012). These results are reported in Fig. 5E. From the same dataset we also found that BRD4 transcript levels are not significantly different in healthy and DMD donors (Fig 1D).
Page 5: " We next analyzed BRD4 levels in DMD muscle samples, and found that BRD4 protein was higher in muscles of DMD patients than in aged matched controls (Fig. 1C). We therefore interrogated RNA-Seq results published by Khairallah et al. 36 and found that BET transcript levels do not significantly change in DMD muscles (Fig.1D)." Minor: 2. The authors conduct a fairly comprehensive biochemical characterization of many key dystrophic features ameliorated by JQ1 (oxidative stress, autophagy, inflammation, fibrosis). Given the relatively large number of recent reports linking Nox2-mediated ROS production to microtubule abnormalities that contribute to DMD pathology (e.g. Randazzo et al., Hum Mol Genet 2019, Prosser et al., Science 2011, Loehr et al., Elife 2018, Nelson et al., Hum Mol Genet 2018, Kerr et al., Nature Communications 2015, Khairallah et al., Science Signaling 2013, it seems a missed opportunity to determine whether JQ1 also corrects microtubule abnormalities that contribute to disease pathology. This could be accomplished by a simple western blot of key markers of microtubule misregulation, such as expression of detyrosinated-tubulin and TUBB6, and would provide further evidence that proximal changes in Nox2 contribute to the microtubule dysfunction well characterized in DMD. We thank the reviewer for this interesting suggestion. We performed immunoblots with detyrosinated-tubulin, alpha tubulin and Tubulin 6. Tubulin 6 results are particularly striking, because of its strong decrease following JQ1 treatment. These data are now reported in fig. 6H, and described on page 14 in the text. "In DMD muscles, dystrophin absence alters the cytoskeleton, which results as a disorganized net of denser microtubules. Since the microtubules network conveys mechanotransduction signals to Nox2-dependent enhancement of ROS 17,36,[62][63][64] in adult mdx muscles, we asked whether JQ1 treatment was able to correct microtubules anomalies that contribute to the DMD pathology. We confirmed that total and detyrosinated alpha-tubulin is increased in adult mdx muscles, and we found that JQ1 treatment decreased both alpha-tubulin and de-tyrosinated tubulin (Fig. 6H). Tubulin6 protein significantly increased in adult mdx TAs when compared to control animals 63,64 , and JQ1 reduced its levels to the ones of control mice (Fig. 6H)." 3. For histological characterizations, for example of EBD and SHD imaging, there is no mention of the imaging or analysis being performed blinded to the treatment groups. These techniques are particularly prone to user bias, and positive results have historically been difficult to replicate when subjected to blinded analysis (there is a considerable history of this in the DMD field). These samples should be imaged and analyzed blindly, and if this is not possible, it should be stated as such in the methods. The imaging or analysis wasn't performed blinded, we added this statement in the methods, page 20.
4. Regarding the histology, the presence of centrally nucleated fibers is a classic hallmark of DMD pathology, and their correction a benchmark of pathological rescue. Thus it is odd that the authors do not quantify CNFs. This seems particularly important given the links to BET inhibition and differentiation/regeneration. We performed this analysis and introduced a graph in figure 2B and in figure S11D, in which 12 months old animals were used. These data are described on page 6 and 14.
Page 6: "JQ1 administration significantly increased the number of peripherally nucleated fibers and decreased the centrally nucleated fibers (Fig. 2B)" Page 13-14: "BET blockade led to a trend towards increasing the number of peripheral nucleated fibers and reducing the centrally nucleated fibers, although not significantly (Fig. S11D)."

5.
What is the definition of, or inclusion criteria for, an "intact" fiber as quantified in figure 1? Again, was this analysis performed blind? Since it was not very clear, we changed the label of intact fiber to "peripheral nucleated fibers". This definition is more intuitive also in comparison with the closely related centrally nucleated fibers. This count was also not blinded and it stated in the methods section (page 19). "Peripheral and centrally nucleated fibers imaging and analysis wasn't performed blinded to the treatment group." 6. The word "remarkably" is used in considerable excess (>10 times) to describe JQ1 effects. This degree of hyperbole throughout the manuscript is not needed, the robust results speak for themselves! We deleted the word "remarkably" in the text, except once.
7. The authors state that the protection from oxidative stress "was not ascribed to the transcriptional activation of anti-oxidant genes", yet do not seem to probe for Nrf2, which regulates broad antioxidant proteins implicated in DMD pathology. This seems important to include, or at least explain, if the authors wish to make this claim. We did analyze Nrf2 targets in Fig. S6, and now introduced an immunoblot showing that Nrf2 doesn't significant change following JQ1 treatment (S7B). Nrf2 transcript is also unaffected by JQ1 administration (S7A). These data are described on page 11. "In the mdx muscle, JQ1 administration did not affect mRNA and protein levels of Nrf2, a transcription factor that plays a key role in the antioxidant response pathway (Fig.  S7A, B). Likewise, JQ1 treatment did not alter the transcriptional regulation of Nrf2 targets, Hmox1, Gclm and Gclc (Fig. S7C), suggesting that restoration of ROS metabolism was not ascribed to the transcriptional activation of anti-oxidant genes. " 8. Page 9 top, reference should be included for "as previously observed in mdx muscles" This wasn't a previously published result, but we refer at our data presented in Fig.4 of this manuscript. We rephrased the sentence as: Page 9: "Nevertheless, JQ1/H 2 O 2 co-treatment restored LC3II abundance and reduced p62 levels, as observed for mdx muscles in Fig. 4C." 9. Please edit page 12, "JQ1-treated mdx mice exhibited significantly increased resistance to fatigue in the treadmill test, and we observed a significant improvement in endurance." This sentence is now on pages 15. We did edit this phrase as follow: "In agreement with the overall reduced muscle damage, JQ1-treated mdx mice significantly increased resistance to fatigue in the treadmill test, and we observed a substantial amelioration in endurance." 10. Why not include the full time course of S. Fig 7 in Fig 6H? It seems odd to have two separate time courses, and the time course of relapse after JQ1 withdrawal is meaningful and warrants inclusion in the primary figures. We agreed with the reviewer to reunite time courses. Since Fig. 6 was very crowded, particularly after adding the tubulin immunoblots, we decided to move time courses in a separate figure, Fig 7.