Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBα modulation

  1. Marie Quétin
  2. Audrey Der Vartanian
  3. Christelle Dubois
  4. Juliette Berthier
  5. Marine Ledoux
  6. Stéphanie Michineau
  7. Bernadette Drayton-Libotte
  8. Athanassia Sotiropoulos
  9. Frédéric Relaix
  10. Marianne Gervais

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Abstract

Muscle stem cells (MuSCs) are essential for skeletal muscle repair. Following injury, MuSCs reside in low oxygen environments until muscle fibers and vascularization are restablished. The dynamics of oxygen levels during the regenerative process and its impact on muscle repair has been underappreciated. We confirm that muscle repair is initiated in a low oxygen environment followed by gradual reoxygenation. Strikingly, when muscle reoxygenation is limited by keeping mice under systemic hypoxia, muscle repair is impaired and leads to the formation of hypotrophic myofibers. In vivo , sustained hypoxia decreases the ability of MuSCs to differentiate and fuse independently of HIF-1α. Prolonged hypoxia specifically affects the circadian clock by increasing Rev-erbα expression in MuSCs. Using pharmacological tools, we demonstrate that Rev-ERBα negatively regulates myogenesis by reducing late myogenic cell fusion under prolonged hypoxia. Our results underscore the critical role of progressive muscle reoxygenation after transient hypoxia in coordinating proper myogenesis through Rev-ERBα.

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    Reply to the reviewers

    'The authors do not wish to provide a response at this time.'

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    Referee #3

    Evidence, reproducibility and clarity

    The manuscript by Quétin et al "Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBα modulation" describes the nature of muscle stem cell (MuSC) differentiation within its hypoxic niche using in vivo, ex vivo and in vitro methodologies. Approaches to limit oxygen in a regenerating model of muscle injury showed that muscle oxygenation is necessary for proper muscle repair. They found that the lack of oxygen is associated with the formation of hypotrophic myofibers, due to the inability of MuSCs to differentiate and fuse. Their findings show that the phenotype was independent of HIF-1α. However, RNA-seq of MuSCs 7 day post injury from prolonged hypoxia was shown to have significantly increased circadian clock gene Rev-erbα expression. Pharmacological inhibition of Rev-erbα during hypoxia rescued the myogenic phenotype. Contrarily, the use of Rev-erbα agonist in normoxia impaired the fusion capacity of MuSCs and decreases the number of large mature myofibres. This manuscript is well written and very easy to follow. Though, there are certain shortcomings outlined below. Sometimes the evidence provided does not support the conclusions made. For example, more rigour should be performed to state that there is a self-renewal phenotype.

    Major issues

    1. In Figure 1, why were these timepoints chosen? Is the hypoxia more severe between days 0 and 5 (i.e. when MuSCs begin their activation).
    2. "From 5 to 28 dpi, pimonidazole adduct intensity gradually declined, demonstrating a progressive reoxygenation after transient hypoxia during muscle repair (Fig. 1E and 1F) that correlates with progressive restoration of the vascular network (Fig. 1C) and MuSC return into quiescence (Fig. 1B and 1D)." For this statement, correlating these events to MuSC returning to quiescence might not be appropriate. As Figure 1D shows all the Pax7+ cells, it does not reflect whether they are quiescent. Thus, the timelines might not actually match up with the proportion of self-renewed MuSCs?
    3. The manuscript cites far too many review articles (at least half) and not primary sources. Also, some citations are misrepresented. For example: Reference #13 does not show that HIF-1alpha level increases during muscle injury in rodents, Reference #15 shows fusion is impaired in hypoxic c2c12 cells, not promotion of quiescence, Reference #22 does not support the claim that hypoxia induces myostatin expression, only that myostatin inhibits MyoD expression.
    4. Figure 1E and 1F, does the dye intensity change with it being more accessible to the muscle during early injury as opposed to later recovery. Also, when using the probe for hypoxia determination, the whole tissue is fluorescing intensely suggesting potential non specificity. It would be prudent to use markers of hypoxia on western blots or gene expression to corroborate this data.
    5. a) It is well known that CTX injury does not cause damage to the vasculature but directly to the muscle (Tatsumi et al doi:10.1002/stem.2639; Ramadasan-Nair et al doi:10.1074/jbc.M113.493270; Ohtsubo et al. doi: 10.1016/j.biocel.2017.02.005; Wang et al doi: 10.3390/ijms232113380). How do the authors reconcile their findings that there is vasculature damage with CTX (Fig. 1C).

    b) Moreover, the endothelial cell staining (Fig. 1B) appears to be unchanged in the time course of injury. To prove vascular damage this data should be corroborated, for example with lectin perfusion.

    1. Problems with Figure 3J. There are data points with zero clusters/isolated myofibres suggesting that the hypoxic environment caused MuSCs to not activate from quiescence. There are several outliers for example at 1% there is a zero reading that makes the data significant.
    2. In Figure 1G, Loxl2 after 14 days appears to be significant, as the error bars at 0 and 14 days do not overlap and thus it does not return to normal. An n=3 is not sufficient, as one of the data points at 14 days appears to be an outlier (the data stretching from 1500 to 3000).
    3. In Fig. 2C and 2D, there are no control CSA and myofiber diameter experiments for keeping the mice in hypoxia over 14 and 28 days without injury.
    4. For Figure 3K, how can self-renewing MuSCs be distinguished from MuSCs that never activated? Especially in the 1% O2 condition where few clusters formed. How does hypoxia influence activation? A 4hr or 8hr timepoint is necessary, as well as 24hrs. Also, for Figure 5E and 5F, it is possible that HIFcKO allowed the cells to activate normally, thus explaining the shift from quiescence to activation in the read-outs. This further highlights the importance of analyzing earlier timepoints. One cannot state that these cells are self-renewing or returning to quiescence without performing experiments on earlier timepoints.
    5. The data for Figure 4 does not suggest that transient reoxygenation is required "for proper skeletal muscle repair" as stated by the authors only that reoxygenation has rescued the phenotype in the primary myoblasts. There is no hypoxia in the control (8% O2) for regeneration to occur (Fig. 2B).
    6. One cannot rule out metabolic dysregulation. It's true that glycolytic fibers are generally larger than oxidative, it is likely that that alone does not explain the difference in fiber size. However, the fact that the fibers are more glycolytic does suggest a metabolic shift in the muscle (which was the aim of the experiment), which could also shift MuSC character altering their behaviour. How are MuSCs metabolically responding to hypoxia?
    7. In Figure 2, how can one be sure that reoxygenation is blocked by the hypoxic chamber? Reduced O2 levels will induce hypoxia, but one cannot state that it blocks reoxygenation without further validation such as using pimonidazole as in Fig. 1E. If reoxygenation is blocked, then pimonidazole staining should remain consistent throughout the injury.
    8. For Figure 3G, is a sum appropriate for the graph? Proportions would be more appropriate as cell number is not equal as shown in figure 3E. Can Pax7+/MyoD+ be defined as differentiated? By day 7, many MuSCs will have fused and be expressing MyoG, which is not accounted for by these definitions. Did systemic hypoxia increase self-renewal or impair activation? How can you distinguish these two?
    9. In Figure 6A, while it is interesting that Pax7 levels are elevated in hypoxia and differentiation and fusion markers are down at 7days, it does not necessarily mean that self-renewal is increased. It might suggest that the hypoxic cells might have never activated or might have differentiated precociously. Are any cell cycle genes down regulated? Any other genes involved in quiescence altered?
    10. The use of pimonidazole in Fig. 1E shows the staining within fibers (many with centrally located nuclei). These nuclei are differentiating and not representative of expanding MuSCs. How do the authors reconcile these MuSCs as part of their population.

    Minor Problems

    1. In the introduction, the line "Vascular alterations result in reduced oxygen (O2) levels, disrupting cell homeostasis and contributing to many diseases" is not always true as vascular alterations do not always result in reduced oxygen levels. For example, in angiogenesis there is no reduction of O2. This line should better reflect this.
    2. In the introduction, Paragraph 2, line 9 change "quiescence thought HIF-1α" to "quiescence through HIF-1α".
    3. Paragraph 3, line 8: "lead" instead of "leads"
    4. It is not sure how important the connection between capillary density and Pax7+ cell number is. Both are presumed to occur at the same time in muscle, so both will recover concurrently. To state that it is a coupled response is overstating the evidence presented.
    5. Figure 1B the colour-labels for Pax7 and Dapi over lap with the border.
    6. In the Introduction, the following sentence does not follow the previous sentence: "In vivo, Majmundar and colleagues show that HIF-1a in MuSCs negatively regulates myogenesis by decreasing myogenic differentiation".
    7. In the Introduction, the following statement is not accurate "Hypoxia can also alter myogenic differentiation and myotube formation by inhibiting p21 (as known as p21 and CDKN1A) that leads to an accumulation of the retinoblastoma protein Rb24", for what was found in the reference. The authors should correct this statement.
    8. Paragraph 3, line 5: "as known as p21 and CDKN1A" should perhaps read "also known as CDKN1A"
    9. The following statement is not supported by the results: "Strikingly, the most abundant and intense pimonidazole staining is detected on CTX-injured TAs at 5 dpi, indicating that myogenic cell expansion is initiated in a hypoxic environment in situ (Fig. 1D-1F)." MuSCs are activated and expanding from time zero to 5 days according to Figure 1D.
    10. "....Since glycolytic fibers are larger than oxidative fibers, ...." citation missing
    11. An inconsistent finding is that the authors show that protein synthesis rates are normal between normoxia and hypoxia of regenerating muscle (suppl. Fig. 1E), yet the capacity of protein synthesis is found to be higher in oxidative muscle fibres compared to glycolytic fibers (Van Wessel et al, doi: 10.1007/s00421-010-1545-0), which are formed during regeneration (Fig. 2G and 2H).
    12. Some figure legends that describe graphs do not denote the number of samples or mice used.
    13. In Figure 1C, 1D and 1F what is being compared to obtain statistical significance?
    14. The font size of many figures is too small to follow.
    15. Confusion for the results of figure 3G. Labels in the text do not reflect the labels in figure (which cannot be read anyway because the font is too small). Why is Ki67 used as a marker for activation versus proliferation.
    16. The physiological O2 concentration is 8%, do the authors know what the hypoxic O2 concentration is in the injured environment. Why did they choose hypoxic O2 concentration at 1% for ex vivo and invitro experiments? Why did they choose 10% for the in vivo experiment?
    17. For Figure 2H it is not appropriate to state that type IIA ratio was reduced with hypoxia, as the results show no statistical significance.
    18. For Figure legend 3K, are the cell number/fiber the sums per one mouse or the sum from all mice combined for each condition?
    19. For Figure 3B and 3E "concomitantly with their proliferation peak" seems to imply that hypoxia in Pax7+ cells peaks alongside proliferation, but the evidence doesn't support that conclusion. More timepoints would be needed to show that 5 dpi is truly the peak of hypoxia in Pax7+ cells.
    20. For Figure legend 4E, should read "MHC" not "MCH"
    21. In Figure 4C there is no gap between the significance bar.
    22. In Figure legend 5G, "Experience design" should read "Experimental design"
    23. Representative images Fig 3I and 5E are poor quality.
    24. Confusing statement "In the same way, this presence of smaller myofibers under prolonged hypoxia could not be explain by the glycolytic fiber-type switch from type-IIA to type-IIB, as observed in pathological context of COPD or peripheral arterial disease (PAD), since type-IIB are the largest myofibers in mice."

    Referees cross-commenting

    I agree with the thoughtful reviews and issues raised by Reviewers 1 and 2. I do not have anything more to add.

    Significance

    General Assessment: This manuscript is well written and easy to follow. It rigorously investigates the influence of oxygenation on MuSC behaviour. The authors utilize in vivo, ex vivo, and in vitro models to support their study and executed their work to a high degree. A limitation is that all experiments are only performed in mice and might not be applicable in humans. In addition, some claims made by the authors were over-reaching. The study can be improved by further validating some of the authors' claims, as has been suggested in the review.

    Advance: This study is the first to report the effect of hypoxia on MuSCs in an ex vivo culture and in vivo injury model using a hypoxia chamber. This study helps clarify the role of HIF-1α on MuSC behaviour by suggesting that it does have a role in MuSC fate decisions. Finally, the authors make a novel link between circadian rhythm and MuSC behaviour in hypoxia.

    Audience: A specialized audience that is interested in myogenesis, muscle stem cells, and/or hypoxia will be interested in this study. It highlights the important role of oxygen in muscle regeneration and may help researchers understand the role of oxygen in MuSC fate decisions.

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    Referee #2

    Evidence, reproducibility and clarity

    The manuscript, Transient hypoxia followed by progressive reoxygenation is required for efficient skeletal muscle repair through Rev-ERBa modulation, revisits the role of hypoxia in skeletal muscle regeneration after acute injury. They first nicely demonstrate, using the pimonidazole hypoxia probe, that during regeneration skeletal muscle is transiently hypoxic at 5 days post injury (DPI). Then they show skeletal muscle regeneration is impaired in mice housed in a hypoxic (10% 02) chamber; the regenerated muscle mass is smaller, due to smaller regenerated myofibers and there is a shift in myofiber type so that there are more IIB myofibers. In addition, at 7 DPI when mice are raised in a hypoxic environment there is a shift in muscle stem cells so that they are more proliferative and fewer have differentiated. Ex vivo experiments culturing muscle stem cells in association with EDL myofibers in 1% 02, as compared with 8% 02, also led to fewer differentiated Pax7-MyoD+ cells, but could be restored if 02 was subsequently increased to 8%. They also found that low oxygen inhibited myoblast fusion in vitro. They then tested, via Pax7CreERT2/+;HIF-1afl/fl, whether HIF-1a signaling mediated the response of muscle stem cells to hypoxia in vivo. Surprisingly, they found that loss of HIF-1 did not impair myofiber regeneration in normoxic or hypoxic conditions, but they do provide some data suggesting that HIF-1a is required for the hypoxic-induced increase in Pax7+MyoD- muscle stem cells. Bulk RNA-seq analysis of 7 DPI muscle from mice housed in normoxic versus hypoxic conditions uncovered the interesting mis-regulation of circadian rhythm associated genes - in particular, the circadian clock repressor Rev-ERBa. Using a pharmacological antagonist of Rev-ERBa they show in culture that blocking Rev-ERBa (in contrast to loss of HIF-1a) rescues the fusion defect of muscle stem cells cultured in 1% 02. Conversely, they show that a Rev-ERBa agonist inhibits fusion in 8% 02. Altogether, the paper provides interesting new data on the controversial role of hypoxia and HIF-1a as well as data suggesting a connection between hypoxia and circadian rhythm genes. The data is logical and well presented, and the paper will be of strong interest to the regeneration and skeletal muscle research communities. I have two major comments and a list of smaller suggestions to improve the manuscript.

    Major comments:

    1. In vivo experiments (presented in Figures 2, 3, 5, 6, 7) house mice in hypoxic (10% oxygen) chambers, and the authors suggest that this blocks the progressive reoxygenation of skeletal muscle during regeneration. Surprisingly, the authors do not test when the mice are in hypoxic chambers whether, in fact, skeletal muscle is hypoxic at homeostasis and whether during regeneration muscle experiences prolonged hypoxia. The obvious experiment would be to use the pimonidazole probe on skeletal muscle sections of muscle at homeostasis and at 0, 5, 6, 14, and 28 DPI CTX injury in mice housed in hypoxic chambers. Without some demonstration that skeletal muscle oxygenation is changed when the mice are housed in hypoxic chambers, it is impossible to interpret these experiments.

    2. The authors claim that reducing reoxygenation by maintaining the mice under systemic hypoxia impairs skeletal muscle repair by limiting the differentiation and fusion capacity of MuSCs in HIF-1a-independent manner, while it favors their return into quiescence through HIF-1a activation. They provide some in vitro evidence that Hif1ais required for the high levels Pax7+MyoD- muscle stem cells in 1% O2. They should also show that the elevated levels of Pax7+ muscle stem cells at 7 DPI (seen in Fig. 3D-G) requires HIF1a via analysis of Pax7CreERT2/+;HIF-1afl/fl mice.

    Minor comments:

    1. Please provide a reference for the pimonidazole probe. Reference 26, Hardy et al., is not the right one.

    2. Please provide references that Loxl-2, Pdgfb, and Ang2 are HIF-inducible target genes.

    3. Fig. 2C shows changes in average myofiber diameter. How was this calculated? Is this the largest diameter? Is there a reason that cross-sectional area was not measured (the more standard measurement)? Also, generally this type of data is shown as bar graphs - which is how these data are shown in Fig. 5C. Please also show the data in Fig. 2C as bar graphs.

    4. Please provide reference for 8% 02 being physioxia in culture.

    5. Fig.5 should also quantify the number of centronuclei/myofiber (as in Fig. 2I) for Pax7CreERT2/+;HIF-1afl/fl mice 14 and 28 DPI - to further demonstrate that differentiation defects in hypoxia are HIF-1a independent.

    6. Please provide a graphical model of your research findings.

    7. There are many typos and verb tense issues. Please fix these. The most amusing is Stinkingly in the Discussion.

    Referees cross-commenting

    I think several important issues are raised by myself and reviewer 3. First, the authors need to explain and support their use of 10% O2 hypoxia in vivo chambers and 1% O2 for hypoxic in vitro experiments. Second, the authors have not demonstrated that reoxygenation of muscle is prevented in mice raised in hypoxic chamber. There are questions about how well the pimonidazole probe is working (the widespread expression at 5 dpi in Fig. 1E suggests there may be specificity issues) and this probe is also not shown for muscle from mice living in hypoxic chambers. Another method of demonstrating hypoxia in muscle tissue would be useful.

    Significance

    The paper provides interesting new data on the controversial role of hypoxia and HIF-1a as well as data suggesting a connection between hypoxia and circadian rhythm genes.

    This paper will be of interest to researchers studying the role of hypoxia on regeneration and also to researchers studying muscle regeneration.

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    Referee #1

    Evidence, reproducibility and clarity

    SUMMARY

    Quétin et al investigated the dynamics of oxygen levels during the skeletal muscle regeneration following sterile damage and its impact on muscle repair. They combined in vivo and ex-vivo model systems, together with genetic and pharmacological manipulations. They found results consistent with the fact that a dynamic oxygeneation process, hypoxia during the early phase followed by reoxygenation, are involved in muscle repair. Prolonged hypoxia leads to defective myogenesis and muscle repair. These activities apper to be meadiated by modulation of Rev-ERBα levels. Collectively, the study provide intriguing insight regarding the role of oxygen in muscle repair.

    MAJOR COMMENTS

    1. In Figure 1, the 5 days post CTX injury is too late to claim that "myogenic cell expansion is initiated in a hypoxic environment". Indeed, at day 5 myofibers are already regenerated, although immature. To support their claim, the authors should perform analyses and quantification of Pax7+, Pax7+Ki67+ and hypoxia at earlier timepoints.

    2. In Figure 2B, a larger number of mononuclear cells is present in hypoxia mice. Is hypoxia affecting the number/activity of extra-muscular cells important for muscle regeneration like for example FAPs, macrophages, etc?

    3. In Figure 5H, the myotubes formed by HIF-1α cKO appear thinner than control myotubes. Is myotube size affected by lack of HIF1 α?

    4. The choice of the 7 days post CTX for the RNA-seq is odd. Indeed, at that timepoint there are obvious histological abnormalities in hypoxia mice. Hence, it is highly likely that many DEGs are simply secondary to the defect in regeneration and not directly linked to hypoxia exposure. This is probably the reason why the authors found so many (close to 4K) DEGs. To focus on the genes closely-associated to the primary defect, the authors should have performed the RNA-seq at an earlier timepoint, in which minimal histological defects were present. While repeating the RNA-seq would be costly and time consuming, the authors could at least address this issue by RT-qPCR. Are muscle stem cell fate, repair, and circadian clock genes significantly altered 3 and 5 days after CTX injury in hypoxia vs normoxia?

    5. Given that compounds have frequently off-target effects, the authors must independently support their Rev-ERBα findings by performing genetic manipulations, at least ex-vivo.

    6. A recent study (PMID: 38333911), which was not cited by the authors, reports muscle atrophy and weakness, impaired muscle regeneration, and increased fibrosis in hypoxia exposed mice. Intriguingly, this was due to impaired MuSC proliferation and differentiation following HIF-2α stabilization under hypoxia. Hence, the authors should investigate if HIF-2α plays any role in the phenotypes they describe. For example, is HIF-2α a regulator of circadian clock genes expression?

    Referees cross-commenting

    The other reviewers raised very relevant issues and I fully agree with their comments. In particular, I concur with Reviewer #3 that in several instances the evidence provided by the authors does not support the conclusions made.

    Significance

    SIGNIFICANCE

    There is a limited knowledge regarding the role of oxygen supply during tissue differentiation and repair. In the muscle field, there are conflicting reports in the literature. This study combines genetic, pharmacological and oxygen manipulations both in vivo and ex-vivo to investigate the role of oxygen during regeneration following sterile skeletal muscle injury. The results are very intriguing and potentially relevant both for muscle, but possibly also for other tissue repair. Aspects of the study that must be improved concern the role of HIF-1a and HIF-2α in the process, and the characterization of the molecular mechanism through which Rev-ERBα is regulated by oxygen and regulates muscle repair.

    • AUDIENCE: specialized, basic research, translational research; results could potentially extend beyond the muscle field.

    • FIELD OF EXPERTISE: muscle differentiation, muscular dystrophy, gene expression regulation.

  5. Version published to 10.1101/2024.05.02.592180 on bioRxiv