Myoglobin-derived iron causes wound enlargement and impaired regeneration in pressure injuries of muscle

  1. Nurul Jannah Mohamed Nasir
  2. Hans Heemskerk
  3. Julia Jenkins
  4. Nur Hidayah Hamadee
  5. Ralph Bunte
  6. Lisa Tucker-Kellogg

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Abstract

The reasons for poor healing of pressure injuries are poorly understood. Vascular ulcers are worsened by extracellular release of hemoglobin, so we examined the impact of myoglobin (Mb) iron in murine muscle pressure injuries (mPI). Tests used Mb-knockout or treatment with deferoxamine iron chelator (DFO). Unlike acute injuries from cardiotoxin, mPI regenerated poorly with a lack of viable immune cells, persistence of dead tissue (necro-slough), and abnormal deposition of iron. However, Mb-knockout or DFO-treated mPI displayed a reversal of the pathology: decreased tissue death, decreased iron deposition, decrease in markers of oxidative damage, and higher numbers of intact immune cells. Subsequently, DFO treatment improved myofiber regeneration and morphology. We conclude that myoglobin iron contributes to tissue death in mPI. Remarkably, a large fraction of muscle death in untreated mPI occurred later than, and was preventable by, DFO treatment, even though treatment started 12 hr after pressure was removed. This demonstrates an opportunity for post-pressure prevention to salvage tissue viability.

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  1. Version published to 10.7554/elife.85633 on eLife
  2. Review Commons

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    Reply to the reviewers

    Manuscript number: RC-2022-01668

    1. General Statements

    We are grateful to reviewers for their thorough, insightful, and highly constructive feedback.

    GENERAL REPLY #1.

    We clarify a major misunderstanding. All instances of the phrase “failure of phagocytosis” or__ “phagocytic dysfunction” should be re-worded as “persistence of dead tissue”__ or simply “necro-slough.” The word “phagocytic” contains an implication of “cells” or micro-scale issues, which was not our thinking. We apologize for the ambiguity. Our claim is strictly about the millimeter-scale tissue outcome, not about cell activities to cause the tissue outcome. Please consider how strongly this miscommunication may have affected reviewers’ requests.

    The relevant hypothesis statement now reads as follows: “Given that pressure ulcers often have slough or eschar, we hypothesize that mPI will have persistence of dead tissue in the wound bed, and that sterile mPI will have slough, despite the absence of bacterial biofilm.” This is a clinically-oriented claim about the relationship between bacterial infection and sloughing, not a cell biology claim about the relationship between macrophages and efferocytosis. We do not believe (and we do not wish to hypothesize) that mPI phagocytes are present and healthy while refusing to perform efferocytosis. To the contrary, such cells appear to be dead or absent at day 3 of mPI. Cells cannot clear debris when they are dead/absent. To satisfy the requests of peer review, we will perform some measurements looking for altered efferocytosis activity in monocytic cells, but we caution that the results might be negative.

    GENERAL REPLY #2.

    Reviewers asked us to characterize specific details of the immune system, especially for monocytes/macrophages. We did already measure many general immune-related factors at different timepoints, and found that surprisingly few of the general immunology analytes had any statistical significance (Supplementary Tables 3, 5 and 6), despite the large fold-changes seen in damage-related epitopes of oxidative stress.

    We wish to avoid making any claims about the immune system in mPI, other than the absence of intact immune cells in untreated day-3 mPI wounds, and the DFO-induced increase in the presence/influx of immune cells at days 7 & 10. These serendipitous findings about immune cells are not required for any of the five chief hypotheses listed in our introduction. To characterize which cell types “should have been” present, and why they are absent, cannot possibly be established by the Reviewer’s request for greater rigor in the CTX-versus-mPI comparison, because CTX is the wrong comparison for that purpose.

    To address reviewers’ requests, we provide multiple additional experiments characterizing limited aspects of the immune system. We are interested in how mPI wounds diverge from the dominant theory of what causes non-healing wounds. The dominant theory is that non-healing wounds are caused by excessive inflammation due to pre-existing morbidities (e.g., diabetes) and/or pro-inflammatory disruption (e.g., infection) that extend the duration of the inflammation phase. According to this theory, prolonged inflammation is what causes damage and blocks the granulation phase from progressing. Our mPI model violates expectations in two ways. Firstly, the dominant theory expects a non-healing wound to have elevated presence/infiltration of immune cells, but we found absence of intact immune cells at the earliest timepoint. The second is that mPI levels of oxidative damage were inversely correlated with immune cell abundance, suggesting that immune cells were not the largest source of oxidative damage. As expected, oxidative damage was highly correlated with poor healing (and was downstream of myoglobin iron). In summary, we will perform multiple additional studies toward “*better describing the immune response ensuing the injury” *which will promote the shared goal of understanding mPI and elucidating what the DFO drug is accomplishing.

    We have one minor question about editorial policy for re-displaying the same control images for multiple experiments.

    Reviewer Two initial comments (minor)

    “- Figure 5 C, E, G: please provide illustrations for control treatment.

    - Figure 6 K, L, M: please provide illustrations for control treatment."

    The controls for Figure 5C, 5E, and 5G were already shown in Figure 2 (2I, 2L and 2B), and we hesitate to show them again without permission from the editor to duplicate figures. Similarly, controls for Figure 6K, 6L and 6M are already shown in Figure 2F-G.

    2. Description of the planned revisions

    __PLANNED ADDITION #1. __Measuring the iron system via immuno-staining.

    *Reviewer Two initial comments: *

    “2. Is myoglobin also released in the injured tissue after CTX and how does it compare to mPI (Mb+ surface area, quantity)?

    • What about expression levels of proteins involved in heme/iron detoxifying proteins haptoglobin and hemopexin? Are they present in the injured tissue and are they differentially expressed between types of injury and mouse genotypes? Same goes for their receptors (CD163 and CD91, respectively): are they differentially expressed on macrophages found in the injured tissue?*

    - Is hemoglobin found mPI and CTX wounds from WT or Mb-/- mice?”

    Reviewer Two cross-comments:

    “The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area, __myoglobin deposition/accumulation in the wound, __better describing the immune response ensuing the injury. Hence my major comments 1 to 7.”

    Authors' reply: We interpret this feedback to mean that measurements of myoglobin and iron detoxification factors would be help explain why mPI has higher iron and what consequences the iron may have. However, we believe the collapse or destruction of vessels in mPI (described below in the section for Completed Revision 2) might suffice to explain why iron-containing waste accumulates in mPI while the same waste gets removed in CTX. Furthermore, iron detoxification factors might be unable to enter the wound without the blood vessels.

    For Planned Addition #1, we will perform the following five measurements, which should provide data for two core issues: globin protein presence in the wound, and iron detoxification factors in the wound. The methods we will perform are immunostaining for the following factors, comparing mPI against CTX at day 3 (each treated with saline and no other drugs, each with n=3 replicates).

    1. Myoglobin
    2. Hemoglobin
    3. Hemopexin
    4. Haptoglobin
    5. Haptoglobin receptor CD163 Expected outcomes, regarding myoglobin abundance at day 3 post-injury in mPI vs CTX:

    • If Myoglobin is elevated in mPI vs CTX, then this finding would corroborate the increased ferric iron in mPI (measured by Prussian blue staining). It would also support the interpretation that myoglobin is the source of the excess iron that decreases upon myoglobin knockout.

    • If Myoglobin is not elevated in mPI vs CTX, then our myoglobin hypothesis could be in question and/or the myoglobin might have degraded to a state that remains redox active without binding the anti-myoglobin antibody (as occurs for hemoglobin [6]) and/or the day 3 timepoint could be too early/too late to observe the phenomenon. Expected outcomes, regarding iron detoxification factors (hemopexin, haptoglobin and CD163) at day 3 post-injury in mPI vs CTX:

    • If hemopexin and haptoglobin and/or haptoglobin receptor CD163 are elevated in mPI vs CTX, some readers will interpret this to mean that mPI has increased heme and/or increased scavenging of extracellular myoglobin/hemoglobin. Elevated hemopexin would corroborate our finding that Prussian blue staining is increased in mPI. One serious problem for interpretation of haptoglobin is that the haptoglobin-myoglobin complex has low affinity, while the haptoglobin-hemoglobin complex has high affinity, and some hemoglobin is probably present. Therefore, we also perform hemoglobin staining. Note also that CD163 is often used as a biomarker for “M2” macrophages, in addition to being the receptor for haptoglobin.

    • If hemopexin, haptoglobin, and/or haptoglobin receptor CD163 are not elevated in mPI vs CTX, some readers might interpret the measures to be irrelevant because the loss of blood vessels in mPI might prevent involvement of circulating factors. Other readers might interpret it to mean that mPI did not need, or did not utilize increased levels of the circulating factors. Other considerations are that the globin/heme source could degrade and the day 3 timepoint might be too early or too late to observe the phenomena of interest.

    • We cannot guarantee the primary antibodies will pass quality control and provide desired results. PLANNED ADDITION #2. Describing the immune response in vivo and in vitro.

    Reviewer One initial comments:

    “It would be interesting to see if myoglobin prevents monocyte or macrophage migration/chemotaxis. Another aspect is how cells reach the injured area… Given that the study is already quite huge with numerous experiments, the reviewer is reluctant to ask for additional experiments…

    Also the points mentioned above, about the role of myoglobin in immune cell infiltration and the role of myoglobin in the vessel properties should be at least discussed, if they are not experimentally addressed.”

    *Reviewer Two initial comments: *

    “8. The in vitro experiments with macrophages could be further supported by in vivo experiments, where types of injury (mPI vs. CTX) and mouse genotypes (Mb-/- vs. WT) could be evaluated for the ability of macrophages to perform efferocytosis: coupling apoptotic cell detection (Tunel staining) to macrophage immunostaining. Quantification of overlapping signal would give some information (albeit indirect) regarding the macrophages' ability to clear the tissue from dead cells. From my perspective, this would be the minimal set of data required to highlight a potential "efferocytic failure" in mPI.”

    Reviewer Two cross-comments:

    “The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area, myoglobin deposition/accumulation in the wound, better describing the immune response ensuing the injury. Hence my major comments 1 to 7.

    Beyond these points, the authors can then re-assess whether or not to include the role of myoglobin on monocyte/macrophage infiltration on the site of injury and the phagocytic activity of these recruited macrophages as part of this manuscript.”

    Reviewer One cross-comments:

    “Point 8 should be investigated if the authors wish to claim about efferocytosis.”

    Authors' reply: We interpret this to mean that most readers will want to see more cell-specific and macrophage-specific data to complement the tissue experiments and molecular experiments. The specific choice of experiments is left up to us, but reviewers both agree something more is needed.

    For Planned Addition #2, we will perform the following five measurements, which should provide quality data for at least three core issues: macrophages ex vivo, neutrophils ex vivo, and in vitro response of macrophages to myoglobin treatment. The methods we will perform are the following:

    1. Perform Tunel staining alongside macrophage (F4/80) immuno-staining, to look for whether macrophages have engulfed apoptotic debris. We will compare mPI+Saline versus mPI+DFO at day 7, to see whether iron depletion affects the amount of engulfed debris inside macrophages.
    2. Perform quadruple staining of pan-macrophage marker F4/80, pro-inflammatory macrophage marker iNOS, pro-regenerative macrophage marker Arginase-1, and DAPI nuclear stain.
    3. Perform immuno-staining for Ly6G, a marker of neutrophils, and myeloperoxidase, a marker of neutrophil extracellular traps (NETs/NETosis).
    4. Perform immuno-staining for CD38 and CD86. CD38 is a marker of CD4+, CD8+, B and Natural Killer cells. CD86 is a marker of dendritic cells, macrophages, B cells and other antigen-presenting cells. For greater information content, this staining might be multiplexed with the neutrophil staining, if antibody optimization is successful.
    5. Measure the impact of Myoglobin on monocytic cell functions in vitro. We will test naïve and M1-differentiated RAW264.7 monocytes/macrophages, with or without treatment with myoglobin. The highest priority is to measure efferocytosis activity, but we will consider three functional assays: phagocytosis, efferocytosis, and transwell migration.

    For the ex vivo studies (items 1-4 above), we will compare mPI+Saline versus mPI+DFO at day 7, using CTX+saline at day 3 as the positive control (n=3 per group). The in vitro treatment groups are naïve and M1-differentiated RAW264.7 monocytes/macrophages treated with or without myoglobin. The positive control is H2O2 treated RAW264.7 cells. The experiments will be carried out in quadruplicates.

    Expected results for co-localization of Tunel+ F4/80 in mPI vs CTX.

    • If Tunel+viable F4/80 co-localization is decreased in mPI vs CTX, then some readers might interpret a decrease in phagocytic activity by macrophages, which might help explain the persistence of dead tissue in mPI.

    • If Tunel+viable F4/80 co-localization is not decreased in mPI vs CTX, then some readers may interpret that iron and its scavenger DFO cause no difference in the phagocytic function of macrophages, but some might question the timepoint or methods.

    • Note that if we cannot see Tunel+F4/80 co-localization in day 3 samples of CTX injury (the positive control condition), then we consider that the assay has failed. Expected results for immuno-staining of Ly6G, myeloperoxidase, CD38 and CD86 in mPI vs CTX.

    • If Ly6G, CD38, and CD86 are observed inside cells, they can indicate categories of immune cells present in the wounds. If observed extracellularly, they will be interpreted as debris from cells previously present.

    • Myeloperoxidase is expected to be extracellular in the case of extracellular traps.

    • Any observations will reflect only the timepoint measured, which may be before or after the peaok for that analyte. Note that we cannot guarantee that these antibodies will pass quality control and provide useful results, and we only have enough tissue samples to measure each analyte in triplicate. Expected results for cell-based assays of RAW264.7 cells when treated with myoglobin.

    • If myoglobin-loaded macrophages exhibit decreased cell functions of efferocytosis, phagocytosis, and/or migration in vitro, then some readers might see this as the cellular mechanism for persistence of dead tissue and sloughing. However, such a finding would not rule out other causes of necro-slough. For example, the relative primacy of macrophages and fibroblasts in efferocytosis is subject to debate.

    • If no change is detected, many readers will interpret this to mean mPI macrophages have no change in cell function after myoglobin loading.

    • Our claims are unaffected either way, because what we see are dead immune cells and delayed presence/influx into the wound, and cells cannot clear debris when they are dead/absent.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Completed Revision #1. Major text amendments

    Reviewer One initial comments:

    “Given that the study is already quite huge with numerous experiments, the reviewer is reluctant to ask for additional experiments. Rather, the reviewer suggests to reshape the text, remove unnecessary details to get straight to the points and to emphasize the important result. …Discussion sections about oxidative stress, endogenous iron, prevention studies, antiDAMPs strategies, slough and debridement are poorly informative and poorly referenced and should be either removed or shortened.”

    Reviewer Two cross-comments:

    “I also agree with Rev#1's assessment that some claims should be toned down…* __Beyond these points, the authors can then re-assess whether or not to include __the role of myoglobin on monocyte/macrophage infiltration on the site of injury and the phagocytic activity of these recruited macrophages as part of this manuscript.”*

    Authors' reply: We have amended the main text as follows:

    • Changed the title to omit the term “phagocytic dysfunction”.
    • Changed the text to emphasize tissue physiology and not evoke concepts of cell biology. Changed terminology so that all “failure of phagocytosis” will be written as “persistence of dead tissue.”
    • Shortened our discussion and conclusion by 33%, especially the sections entitled “the context of oxidative stress”, “anti-DAMP strategies”, and “debridement of slough.” Our abstract now says, “Unlike acute injuries (from cardiotoxin), mPI regenerated poorly with a lack of viable immune cells, persistence of dead tissue (necro-slough), and abnormal deposition of iron.” The old version had said, “mPI regenerated poorly with a lack of viable immune cells, failure of phagocytosis….”

    Completed Revision #2. Surface area and vascular comparisons between CTX and mPI injuries.

    *Reviewer Two initial comments: *

    “I find the direct comparison made between the two types of injury, CTX and mPI, difficult to interpret. From my perspective, a more rigorous and systematic comparison between the two models of injury would be key to convincingly convey the findings of this work, especially regarding key features impacting repair.

      • Time for tissue repair not only depends on the type of injury, but also on the extent of the injury. In other words__, how the mPI and CTX models compare in terms of surface of injured tissue__ (and resulting ischemia)?”* Reviewer Two cross-comments:

    “The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area,….”

    Authors' reply: We interpret the feedback to mean that the surface area of injured muscle should be comparable between CTX and mPI in order to claim that the tissue repair is different between the two injuries. We will provide the requested measurements showing similarity between the wounds, but we disagree with the premise that one should seek similarity. To that end, we will provide additional data (not requested), showing other forms of dissimilarity. Our scientific claims don’t rely on comparisons between CTX and mPI, and we urge readers to refrain from direct comparisons between dissimilar wounds.

    We have revised the transferred manuscript as follows:

    1. Added requested data (Suppl. Table 1) showing that both CTX and mPI have comparable size of dead muscle at the initial timepoint. To avoid making claims about CTX, we will delete the word “normal,” we will delete our phrase saying CTX lacks the dysfunctions seen in mPI, and we will explain that the purpose of CTX is to show a dissimilar example of muscle regeneration after an acute injury.
    2. Added supplementary images (Suppl. Fig 2) showing dramatic differences in vasculature between CTX injury and mPI. Add accompanying text will explain that the goal of examining different types of wounds is model-description and hypothesis-generation, not hypothesis-testing Completed Revision #3: “Minor” edits suggested

    Minor Edit #1

    Suppl. Fig 4 is added to show intact immune cells at the wound margin and the absence of intact immune cells in the compressed region 3 days after mPI. This is as per Reviewer One’s suggestion to change Suppl. Fig 6 and call it in the results section that, “In the discussion section, there is reference to a SupplFig6, which seems to be not the good one in the document. In the FigS6 described in the text, it is mentioned that cells are kind of "stopped" at the boundaries of the damage… mPI Discussion end of page 10. Unfortunately, suplFig6 is missing (and is not called in the result section).”

    Minor Edit #2

    Main Fig 2L and Main Fig 5E have been changed to more representative images of HO-1 fluorescence in Day 3 saline- and DFO-treated mPI respectively, as per Reviewer Two’s minor comment, “Figure 2: HO-1 staining seem decreased in mPI compared to CTX and thus doesn't support the quantifications. Please 2x-check quantifications and images to provide consistent quantifications-illustrations pairing.”

    Minor Edit #3

    Suppl. Fig 5 (previously Suppl. Fig 3) has concentration and treatment times added to the figure caption as per Reviewer Two’s minor comment,* “Provide concentrations and treatment times in figure legends (sup Fig3).”*

    Minor Edit #4

    Suppl. Fig 6 (previously Suppl. Fig 4) has DNA gel electrophoresis results (Suppl. Fig 6D) added as requested by Reviewer Two in minor comment, “Show all the data mentioned in the manuscript (DNA gel electrophoresis supp fig 4)”

    Minor Edit #5

    Suppl. Fig 8, Suppl. Fig 10 and Suppl. Fig 12 have labels added to the image sets as per Reviewer Two’s minor comment, *“Missing information in supp Fig 5 A-D: which images from WT or Myb-KO?” *

    Minor Edit #6

    Suppl. Fig 11A-D have a different, more representative image set to show F4/80, CitH3 and DAPI triple-stain in saline-treated mPI (day 3). DAPI staining was not shown previously (only F4/80 and CitH3.)

    Minor Edit #7

    Suppl. Fig 12E has a blue dashed line added to the graph for the level of MerTK fluorescence in uninjured skinfold.

    Minor Edit #8

    Clarifying text and citation on the BODIPY 581/591 fluorescent probe that we used has been added in the Results section, as per Reviewer Two’s minor suggestion, “Bodipy is not a probe for lipid peroxidation. Due to its lipophilic nature, this dye can be used as a generic lipid satin to image intracellular lipid depots. Therefor the experiments using bodipy as a proxy for lipid peroxidation is incorrect and derived conclusions erroneous. Modulation in bodipy signals probably reflects modulation of intracellular lipid deposition.”

    Minor Edit #9

    The section, “Data availability”, which discloses the link to the Zenodo database containing the mice numbers and primary data has been moved from Suppl. Methods (Suppl. Text) to Methods in the main text.

    Minor Edit #10

    The acknowledgements section has been updated.

    4. Description of analyses that authors prefer not to carry out

    Reviewer Two initial comments:

    *“4. *Does in situ Mb supplementation in Mb-/- mice worsens mPI repair to an extent that is comparable to WT mice?”

    Authors' reply: Further study of knockout mice (Mb-/-) was mentioned by Reviewer Two, but the reviewer did not prioritize this experiment. We will not carry this out because we have already spent many years breeding descendants of our Mb-/- mice, trying to generate more Mb-/- pups, but the later years of breeding have had zero live births of homozygous knockouts. Because we have reached the ethical limit of wasted animals, any further study of myoglobin knockout would require an entirely new conditional knockout system, which is a long-term future investment.

    Citations:

    [1] Wang Y, Lu J, Liu Y. Skeletal Muscle Regeneration in Cardiotoxin-Induced Muscle Injury Models. Int J Mol Sci. 2022 Nov 2;23(21):13380.

    [2] Averin AS, Utkin YN. Cardiovascular Effects of Snake Toxins: Cardiotoxicity and Cardioprotection. *Acta Naturae. *2021 Jul-Sep;13(3):4-14.

    [3] Naldaiz-Gastesi N, Goicoechea M, Alonso-Martín S, Aiastui A, López-Mayorga M, et al. Identification and Characterization of the Dermal Panniculus Carnosus Muscle Stem Cells. Stem Cell Reports. 2016 Sep 13;7(3):411-424.

    [4] Ahmed AK, Goodwin CR, Sarabia-Estrada R, Lay F, Ansari AM, et al. (2016). A non-invasive method to produce pressure ulcers of varying severity in a spinal cord-injured rat model. Spinal Cord, 54(12), 1096–1104.

    [5] Turner CT, Pawluk M, Bolsoni J, Zeglinski MR, Shen Y, et al. (2022). Sulfaphenazole reduces thermal and pressure injury severity through rapid restoration of tissue perfusion. Scientific Reports, 12(1), 12622.

    [6] Bahl N, Du R, Winarsih I, Ho B, Tucker-Kellogg L, Tidor B, et al. (2011) Delineation of lipopolysaccharide (LPS)-binding sites on hemoglobin: from in silico predictions to biophysical characterization. J Biol Chem. 286(43), 37793-803.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Jannah and colleagues highlight a pathophysiological mechanism involving myoglobin for the poor repair capacity of ulcerative pressure wounds. The authors modeled muscle pressure injury (mPI) using a magnet and compared it to cardiotoxin (CTX)-induced muscle injury. They show a significant delay in repair kinetics for mPI compared to CTX, recapitulating the notoriously poor repair process associated with ulcerative pressure injuries. Using mice genetically invalidated for myoglobin (Mb), they show an improved mPI recovery, with improved tissue repair quality over a shorter period of time. Mechanistically, the authors link the poor repair of mPI to the oxidative and pro-inflammatory effect of Mb released from injured skeletal muscle fibers.

    Major comments:

    The authors hypothesis regarding the role of Mb in the pathophysiology of ulcerative pressure injuries is interesting. However, the work here seems quite preliminary with major points remaining to be clarified before considering reaching the author's conclusion. I find the direct comparison made between the two types of injury, CTX and mPI, difficult to interpret. From my perspective, a more rigorous and systematic comparison between the two models of injury would be key to convincingly convey the findings of this work, especially regarding key features impacting repair. My major comments are listed below.

    1. Time for tissue repair not only depends on the type of injury, but also on the extent of the injury. In other words, how the mPI and CTX models compare in terms of surface of injured tissue (and resulting ischemia)?
    2. Is myoglobin also released in the injured tissue after CTX and how does it compare to mPI (Mb+ surface area, quantity)?
    3. Does Mb co-injection with CTX mimics mPI injury in terms of inflammation and repair kinetics?
    4. Does in situ Mb supplementation in Mb-/- mice worsens mPI repair to an extent that is comparable to WT mice?
    5. A better characterization of the inflammatory between types of injury and mice (Mb-/- vs. WT) before and after 3- and 10-days post-injury would be very informative. Comparing the relative proportions of leukocyte populations would provide valuable information regarding the kinetics of the repair process.
    6. Macrophages in particular play a central role in the orchestration of tissue repair, through their immunomodulation abilities. On the same token, characterizing macrophage infiltrates (number per surface area of injured tissue) and phenotype would potentially provide valuable information to link observed differences between types of injuries to Mb. Ideally, assessment of leukocyte and macrophages infiltration and populations would be analyzed by flow cytometry after injured (vs. uninjured) tissue dissociation (enzymatic or mechanical). Otherwise, although less quantitative, this can also be done by cell infiltration using specific immunostaining and quantification (cell number/injured tissue surface area).
    7. Macrophage phenotype (inflammatory vs. anti-inflammatory/reparative) can be achieved by RTqPCR, using well-define combination of mRNA encoding proteins associated with inflammatory (e.g. iNos, Cox-2, Cd86) or anti-inflammatory (Ym1, Arg-1, RELMa, Cd206).
    8. The in vitro experiments with macrophages could be further supported by in vivo experiments, where types of injury (mPI vs. CTX) and mouse genotypes (Mb-/- vs. WT) could be evaluated for the ability of macrophages to perform efferocytosis: coupling apoptotic cell detection (Tunel staining) to macrophage immunostaining. Quantification of overlapping signal would give some information (albeit indirect) regarding the macrophages' ability to clear the tissue from dead cells. From my perspective, this would be the minimal set of data required to highlight a potential "efferocytic failure" in mPI.
    9. What about expression levels of proteins involved in heme/iron detoxifying proteins haptoglobin and hemopexin? Are they present in the injured tissue and are they differentially expressed between types of injury and mouse genotypes? Same goes for their receptors (CD163 and CD91, respectively): are they differentially expressed on macrophages found in the injured tissue?

    Minor comments:

    • Figure 2: HO-1 staining seem decreased in mPI compared to CTX and thus doesn't support the quantifications. Please 2x-check quantifications and images to provide consistent quantifications-illustrations pairing.
    • Figure 5 C, E, G: please provide illustrations for control treatment.
    • Figure 5J: it would have been nice to add Mb-/- mice to the comparison.
    • Figure 6 K, L, M: please provide illustrations for control treatment.
    • Figure 8: please maintain consistency in the way you convey data between timepoints: area of regenerated (E, F) or unregenerated (G) tissue.
    • Bodipy is not a probe for lipid peroxidation. Due to its lipophilic nature, this dye can be used as a generic lipid satin to image intracellular lipid depots. Therefor the experiments using bodipy as a proxy for lipid peroxidation is incorrect and derived conclusions erroneous. Modulation in bodipy signals probably reflects modulation of intracellular lipid deposition.
    • Provide concentrations and treatment times in figure legends (sup Fig3).
    • Show all the data mentioned in the manuscript (DNA gel electrophoresis supp fig 4)
    • Indicate the number of experimental repeats and the statistical tests used in the figure legends.
    • Missing information in supp Fig 5 A-D: which images from WT or Myb-KO?
    • Is hemoglobin found mPI and CTX wounds from WT or Mb-/- mice?

    Referees cross-commenting

    I have read the report from Reviewer#1 and below are my cross-comments.

    I agree with Rev#1's minor comments. I also agree with Rev#1's assessment that some claims should be toned down as data don't support them. The phagocytic dysfunction is certainly one of them, for the reasons mentioned, but not the only one. Indeed, I believe that virtually all the claims could use some dampening to various extent because the level of evidence is not very high throughout. A more rigorous description of the injury model would address this point in my opinion (narrower scope with a demonstration).

    The way I understand the authors' work, instead of broadening the scope of the work with a cellular mechanism (phagocytic dysfunction), I would rather suggest the authors to focus on strengthening the description of their model of injury (mPI) versus CTX, on key points that are known to influence tissue repair/regeneration as well as their main findings: evaluation of the injury's surface area, myoglobin deposition/accumulation in the wound, better describing the immune response ensuing the injury. Hence my major comments 1 to 7.

    Beyond these points, the authors can then re-assess whether or not to include the role of myoglobin on monocyte/macrophage infiltration on the site of injury and the phagocytic activity of these recruited macrophages as part of this manuscript.

    Significance

    The field of tissue repair and regeneration is an exciting field and improving our understanding of the molecular mechanisms involved in muscle tissue injury has clear and impactful clinical applications.

    The pathophysiological mechanism involving Mb that the author address in this work has the potential to interest both basic science and clinical researchers and can potentially benefit not only the field of skeletal muscle regeneration but also the field of cardiac remodeling.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary: The study by Nasir et al. investigates the healing properties of skeletal muscle after a pressure injury and the impact of myoglobin in this process. First, they compared cardiotoxin versus pressure injuries and showed that the latter heals slowly and badly (this is shown through a large series of parameters). They then used myoglobin deficient mice or WT mice treated with the iron chelator DFO to show that in the absence of myoglobin, there is an improvement of the regeneration process.

    Major comments:

    The study is well written, clear, and the experiments are carefully presented and conducted. Although the text is usually very detailed and nicely referenced, some of the claims should be dampened. Notably the title since the phagocytic dysfunction is not evidenced by the results, not the wound enlargement (since DFO has no impact on it).
    Also the text is very long, notably the discussion, which contains 18 sections (!) and several sections are not very informative and poorly referenced, looking more as a thesis dissertation than as an article discussion.

    Concerning immune cells, the conclusion cannot be that myoglobin impedes their phagocytic function. All the data concur that in the absence of myoglobin there are more immune cells in the regenerating muscle at day 3. Consequently, more macrophages will lead to a better cleansing of debris. Thus, the difference would not rely on phagocytic properties per se, but more on the number of macrophages that arrive at the site of injury. It would be interesting to see if myoglobin prevents monocyte or macrophage migration/chemotaxis. Another aspect is how cells reach the injured area. In the discussion section, there is reference to a SupplFig6, which seems to be not the good one in the document. In the FigS6 described in the text, it is mentioned that cells are kind of "stopped" at the boundaries of the damage. This is very interesting. If the vessels are physically flattened or squashed by the injury, extravasation can not occur properly, in comparison with cardiotoxin. Then the role of myoglobin in extravasation, or in the "reshaping" of the vessels after the removal of the magnet would be interesting to investigate. Of note, in the myoglobin deficient mice, the vascular network is increased, favoring immune cell infiltration.

    Given that the study is already quite huge with numerous experiments, the reviewer is reluctant to ask for additional experiments. Rather, the reviewer suggests to reshape the text, remove unnecessary details to get straight to the points and to emphasize the important results. Also the points mentioned above, about the role of myoglobin in immune cell infiltration and the role of myoglobin in the vessel properties should be at least discussed, if they are not experimentally addressed.

    Minor comments:

    • Page 5: "The panniculus layer of mPI was nearly devoid of intact immune cells." It is not clear here if the authors refer to the absence of immune cells or to damage of immune cells present in the injured area.
    • Page 6: "In summary, measures of innate immune response became less abnormal after Mb knockout". Cardiotoxin injury is not the "normal" situation since this kind of injury is not physiological and is highly inflammatory as compared with others (Hardy et al., Plos One 2016).
    • Discussion sections about oxidative stress, endogenous iron, prevention studies, antiDAMPs strategies, slough and debridement are poorly informative and poorly referenced and should be either removed or shortened.
    • Discussion end of page 10. Unfortunately, suplFig6 is missing (and is not called in the result section).

    Referees cross-commenting

    I agree with the rev#2's review and later comments. His comments are quite complementary to those I raised. If the author have the capacity to make the experiments that are proposed by Rev#2, it would be super nice. Point 8 should be investigated if the authors wish to claim about efferocytosis.

    Significance

    This study is of interest because it provides insights on muscle regeneration after an injury that may occur in daily life just as contusion, or crush, to the contrary of the whole muscle necrosis induced by cardiotoxin (the main model used in the field). In that aspect, it is more physiological and of interest for the readers in the fields of in muscle regeneration and tissue trauma. The study is descriptive, but is very well conducted and well discussed. Additional experiments investigating the impact of myoglobin on vessel properties/extravasation of immune cells would raise the impact of the study but the study is publishable as it is (with editing as suggest above).

    The field of expertise of the reviewer is muscle regeneration and inflammation.

  5. Version published to 10.1101/2022.03.07.483146v3 on bioRxiv
  6. Version published to 10.1101/2022.03.07.483146v2 on bioRxiv
  7. Version published to 10.1101/2022.03.07.483146v1 on bioRxiv