Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration
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Abstract
Skeletal muscle regeneration requires extracellular matrix (ECM) remodeling, including an acute and transient breakdown of collagen that produces gelatin. Although the physiological function of this process is unclear, it has inspired the application of gelatin to injured skeletal muscle for a potential pro-regenerative effect. Here, we investigated a bi-phasic effect of gelatin in skeletal muscle regeneration, mediated by the hormetic effects of reactive oxygen species (ROS). Low-dose gelatin stimulated ROS production from NADPH oxidase 2 (NOX2) and simultaneously upregulated the antioxidant system for cellular defense, reminiscent of the adaptive compensatory process during mild stress. This response triggered the release of the myokine IL-6, which stimulates myogenesis and facilitates muscle regeneration. By contrast, high-dose gelatin stimulated ROS overproduction from NOX2 and the mitochondrial chain complex, and ROS accumulation by suppressing the antioxidant system, triggering the release of TNFα, which inhibits myogenesis and regeneration. Our results have revealed a bi-phasic role of gelatin in regulating skeletal muscle repair mediated by intracellular ROS, the antioxidant system and cytokine (IL-6 and TNFα) signaling.
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Reply to the reviewers
Reply to the Reviewers
We would like to thank the reviewers for their thoughtful comments and efforts towards improving our manuscript. Based on the reports, we have revised the manuscript entitled “Bi-phasic effect of gelatin in myogenesis and skeletal muscle r____egeneration (RC-2021-00854 )”. We have addressed all the concerns, revised the text and figures. Modified parts are in red, and line numbers are tracked in this response letter. Our detailed point-by-point responses to the reviewers’ comments are listed below. We believe that these changes strengthen the manuscript and are grateful to the reviewers for all of their suggestions.
Point-by-point …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
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Reply to the reviewers
Reply to the Reviewers
We would like to thank the reviewers for their thoughtful comments and efforts towards improving our manuscript. Based on the reports, we have revised the manuscript entitled “Bi-phasic effect of gelatin in myogenesis and skeletal muscle r____egeneration (RC-2021-00854 )”. We have addressed all the concerns, revised the text and figures. Modified parts are in red, and line numbers are tracked in this response letter. Our detailed point-by-point responses to the reviewers’ comments are listed below. We believe that these changes strengthen the manuscript and are grateful to the reviewers for all of their suggestions.
Point-by-point description of the revisions
R____eviewer #2 (Evidence, reproducibility and clarity (Required)):
**Summary:**
The manuscript by Xiao Ling Liu and colleagues titled "Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration" deals with the effect of gelatin on differentiation of myoblast cell line, in vitro, and on skeletal muscle regeneration upon muscle injury, in vivo. In vivo, the gelatin is a product of collagen breakdown associated with skeletal muscle regeneration upon acute or chronic muscle damage.
Specifically, the authors define a dose-dependent effect of gelatin, beneficial at low dose and detrimental at high dose. This effect is mediated by the level of ROS accumulation leading to the induction of different cytokines with opposite effects on skeletal muscle regeneration.
**Major comments:**
*The experimental purpose is well tackled from both biochemical and functional point of view, and the proposed experiments are quite exhaustive. *
Response: We are grateful for the encouraging comments from this reviewer.
However, I would suggest some additional experimental analyses to improve the robustness and quality of the study, as well as text and figure editing, as reported below.
Regarding the additional experiments/analyses/images:
- Figure 5: I would suggest to add an image of C2C12 cells in GM (growth medium), as* representative images of proliferation analysis upon LCG/HCG/NAC treatment. Response: We appreciate this suggestion by the reviewer. New images of C2C12 cells upon LCG/HCG/NAC treatment have now been added as in Supplementary Figure 4B. The new results are described in main text Lines 237-239.
- Figure 5: I would suggest to repeat the main si-NOX2 experiments with an alternative siRNA to rule out off target effects.*
Response: We thank the reviewer for this suggestion. We have now added a new siRNA targeting NOX2 with an independent sequence and shown the results in Figure 5F-J and Supplementary Figure 4H-J. The sequences of si-NOX2 and si-NC are shown in Materials and Methods Lines 681-687. The newly added siRNAs confirmed results with the previous siRNA, suggesting unlikely off-target effects. This result is described in the main text Lines 246-256.
- In vivo experiments could be improved by adding DHE or DCFH staining on muscle TA cryosections to quantify the level of oxidative stress.*
Response: We appreciate this suggestion by the reviewer. We have now stained DCFH-DA in TA cryosections to quantify oxidative status in situ and show the results in Supplementary Figure 8A-B. Indeed, low- and high-dose gelatin injections both triggered ROS production, and high-dose injection resulted in a high accumulation of ROS. The new result is described in the main text Lines 318-321.
- The proposed model could be better tackled by additional in vivo treatment with Ab anti IL-6 or anti TNFalpha in combination with CTX and LCG or HCG, followed by H/E staining at 14 dpi.*
Response: We appreciate this suggestion by the reviewer. We have now added in vivo treatment with IL-6 or TNFα neutralizing antibody (Ab) in combination with CTX and LCG or HCG. The procedure is illustrated in Figure 8J and described in main text Lines 522-525. H&E staining showed that anti-IL-6 Ab injection significantly reduced the beneficial effect of LCG, but had no effect on HCG-treated mice. By contrast, anti-TNFα Ab injection significantly suppressed infiltration of macrophages into the injury site upon HCG, reversed the deleterious effect of HCG on muscle repair, resulting in myofibers with higher CSA and more myofibers with central nuclei. The new results are described in Figure 8J-K and main text Lines 331-346.
**Minor comments:**
- Each acronym should be indicated in full in the main text at the first mention (for instance BHP, NAC and others). Moreover, I would suggest to add an acronym list for reagents and factors Response: We thank the reviewer for this suggestion and have now added an acronym list in Material and Methods, Lines 466-504.
Experimental methods should be better detailed; for instance I would suggest:
- Add a detailed description of the quantification of differentiation indexes* Response: We thank the reviewer for this suggestion. We have now added a detailed description on the quantification method of myogenesis and differentiation to Material and Methods, Lines 577-582.
- Explain how cell growth (OD 450nm) and optical density (570nm) assays have been performed*.
Response: We thank the reviewer for pointing this out. The cell growth was examined by measuring dehydrogenase activities that generate a soluble formazan dye, whose OD 450nm value was measured as a proportional value to the number of viable cells in the sample (CCK-8 kits according to manufacturer’s instructions).
The transwell cell migration ratio was determined by measuring the optical density of crystal violet staining (570 nm) of migrated cells on the bottom of the transwell filters (transwell filters have 8 μm pores to allow cells to pass through). The non-migrated cells on the top of the transwell filter were scraped away.
Detailed descriptions have now been added to Material and Methods, Lines 593-609.
- Explain how ROS species and antioxidant enzymes have been measured (Fig 4C and 4D)*
Response: We thank the reviewer for pointing this out. The levels of ROS species (O2.- , OH· and H2O2) and antioxidant enzymes in Figure 4C and 4D were examined using commercial kits according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, cells were lysed in RIPA lysis buffer and protein concentrations were determined using BCA method. The O2.- level and SOD activity were measured by adding electron transfer substances that reduce azo blue tetrazole to blue methionine. The activity of SOD was evaluated by the absorption of methionine. GSH-PX facilitates the reaction between H2O2 and GSH to produce H2O and oxidized glutathione (GSSG). The activity of GSH-PX can thus be obtained by measuring the consumption of GSH in this enzymatic reaction. OH· was measured using Fenton reaction. The level of H2O2 was determined according to the reaction with molybdic acid. We have now added the corresponding information to Figure. 4 legend and main text Lines 1066-1067. Detailed protocols can be found in Material and Methods, Lines 704-714.
Figures and figure legends:
- Please add in the figures the figure number in order to facilitate the reading of the pdf file Response: We thank the reviewer for this suggestion and have added figure numbers to the PDF file.
The sequence of the panels should be coherent with the alphabet and reading left to right and up to down
Response: Yes, we have rearranged the figure layouts to be coherent.
- In the Figure 1, I would suggest to add the whole TA sections for H/E staining in order to appreciate the overall beneficial or detrimental effect of LCG and HCG, respectively.*
Response: We thank the reviewer for this suggestion and have added the whole-section view of TA with H&E staining as in Supplementary Figure 1C. The results are described in the main text Lines 132-136.
- I would suggest to show Supplementary figures 1C and 1D in the Figure 1*.
Response: We thank the reviewer for this suggestion and have now moved Supplementary Figures 1C and 1D to Figure 1F and 1G.
In the Supplementary Fig 2A, I would suggest to the authors to show and comment only the data about proliferation: the earlier orientation, fusion and differentiation of C2C12 exposed to LCG are a consequence of the positive effect of LCG on proliferation
Response: We thank the reviewer for this suggestion and have removed data and comments except for proliferation of C2C12 in Supplementary Figure 2A.
- The quality of representative images of western blot is not always high: the bands are fuse and, consequently, the quantification is not reliable. For instance Fig S4 B, S4 G; in Fig 2 the representative image does not really represent the reported quantification.*
Response: We thank the reviewer for this suggestion and have replaced western blot images in Supplementary Figure 4B, 4G and Figure 2.
- Please specify in the figure legends the meaning of the acronym in the axis title (for instance RFU, MFI or DCF) and in the axis title the unit of measure (for instance Count (?)).*
Response: We thank the reviewer for this suggestion and have spelt out “RFU” as “Relative fluorescence intensity”, changed “MFI” to “Relative fluorescence intensity of NOX2” in Figure 4H and Figure 8H. The unit of measurement is the fluorescence intensity. The related modifications have been described in the legend of Figure 4H and Figure 8H, Lines 1071, 1125-1126. We have now included “DCF: 2',7'-dichlorofluorescein” in the acronym list in Material and Methods, Line 473. DCF positive ratio is now used to reflect ROS level in the population. We have explained “Count” in the legend as “Cell counts”.
The authors always wrote "filed" in place of "field".
Response: We apologize for this typo and have corrected it with others throughout the text.
- In the figures 7 and 8 the letters for densitometry panel are missed.*
Response: We could not identify those missing labels and letters in the densitometry panel. We suspect this issue could be from the soft wares opening the documents.
- In the figure legend 8 the panel letters do not match the panels in the figure*.
Response: We thank the reviewer for pointing this mistake out and have corrected Figure legend 8.
- Figure 3I: replace "nucleis" with "nuclei"*.
Response: We thank the reviewer for pointing out this mistake and have modified Figure 3I.
Text editing:
- Line 64: Satellite cells (SCs) would be more appropriate than myoblasts* Response: We thank the reviewer’s suggestion and have replaced myoblasts with satellite cells in the main text Line 60.
- Line 64: Please define more carefully the location of SCs*
Response: We thank the reviewer for this suggestion. SCs are underneath the basal laminin and myofiber plasma membrane in the resting skeletal muscle. The results are described in the main text Lines 60-65.
- Line 67: MyoD+/Myog+ would be more appropriate than Pax7+/Myog+*
__Respons__e: We thank the reviewer’s suggestion and have changed pax7+/MyoG+ into MyoD+/MyoG+ in the main text Line 64.
- Line 67: (Pax7+)/Myog+ "myocytes" in place of myotubes ... and fuse with each other to generate myotubes*
Response: We thank the reviewer for pointing this out and have modified the sentence in Lines 64-65.
- Line 69: Please add Myf6/MRF4 to MRFs list*
Response: We thank the reviewer for pointing out and have added Myf6/MRF4 to MRFs list in the main text Line 66.
- Line 112: replace "its" with "their"*
Response: We thank the reviewer for pointing out this mistake and have replaced “its” with “their”.
- Lines 330-331: "promoted IL-6" "enhanced TNF", please insert secretion/production*
Response: We thank the reviewer’s suggestion and have inserted “production and secretion” behind IL-6 and TNFa in the main text Lines 310-312. .
- Line 352-353: this sentence is not necessary*
Response: We have deleted this sentence.
Reviewer #2 (Significance (Required)):
The study reports robust and interesting data applicable to both basic research and translational research, such as tissue engineering applications.
Response: We thank the reviewer for sharing this positive and important opinion on our work. We are delineating the biological pathways of gelatin treatments, motivated by the application of this biocompatible and industrial material for treating disease and aging-related skeletal muscular dystrophies
Keywords for field of expertise of this reviewer:
Skeletal muscle regeneration
Duchenne Muscular Dystrophy
Inflammation
Macrophages
Oxidative stress
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
**Summary**
The Review Commons submission by Liu and colleagues entitled "Biphasic effect of gelatin in myogenesis and skeletal muscle regeneration" provide a systematic in vitro and in vivo evaluation of the effects of myogenic cell exposure to low or high dose gelatin. Through these analyses they uncover pro- and anti-regenerative effects of gelatin that are dose dependent and through a series of cell and molecular studies, they attribute these dual effects to a ROS-IL6/TNFa signaling axis. The study is well designed and executed. For the most part the figures and experimental details are clear and transparent, and most of the conclusions are supported by the data. Specific comments follow.
Response: We are grateful for the reviewer’s encouraging comments.
**Major Comments**
- Study framing in the Introduction is mis-aligned with the research conducted. Currently the Introduction sets the stage for an in vivo exploration of the effects of endogenous gelatin produced in the course of muscle regeneration. However, there are no experiments in this paper investigating the presence or effects of endogenous gelatin.* Response: We thank the reviewer for pointing this out and apologize for the misleading parts in our abstract and introduction. The reported phenomenon of a temporary breakdown of collagen after skeletal muscle injury has inspired us to apply gelatin for achieving a pro-regenerative effect. Although it will be a very interesting biological pathway to study, no adequate research tools are available for measuring endogenous gelatin in vivo. We have re-written parts in abstract and introduction to avoid confusion, see Lines 34-35, 78-93.
1.a. The impact of the study would indeed be increased by including a systematic characterization of endogenous gelatin levels during muscle regeneration in healthy mice as compared to in those where fibrosis is prevalent. A demonstration that ROS-IL6/TNFa levels align with patterns seen in the in vitro studies, and pharmacological manipulations to 'rescue' would all provide a demonstration of a hormesis gelatin response in vivo. Meaning, is this process something that naturally occurs in the physiological context, or is it one that is possible, but only be supraphyiological gelatin injections?
Response: We resonate with the reviewer and would have loved to investigate whether what we have observed is a fundamental mechanism of the natural healing process. But we are currently limited by the lacking of adequate tools for endogenous gelatin quantification. We appreciate the reviewer’s suggestion to compare normal and aberrant repairing processes such as fibrosis and would like to explore the possibility in a separate future study.
1.b. Alternatively to 1.a., the authors should reframe the Introduction to focus on understanding the effects of gelatin as a biomaterial that is being used in regenerative medicine applications. In this case, the authors should delete/edit/reframe lines 74-102 and instead use lines 103 on to motivate the study so as to be consistent.
Response: We have rephrased sections of Abstract and Introduction to focus on gelatin as a biomaterial. Please find the changes in the main text Lines 34-35, 78-93.
- Satellite cell conclusions in Figures 2C-D that are based upon representative images provided in 2A, are questionable. Pax7 staining in mouse tissue sections is notoriously difficult and the antibodies can have dramatic lot to lot variability. The immunostaining provided in the representative images is not convincing, and hence, draws into question the conclusions based upon them.*
Response: We thank the reviewer for the suggestion and have optimized Pax7 staining based on the published protocol by Feng et al., 2018 in JoVE. The new images can be found in Figure 2A.
2.a. If the authors wish to leave the satellite cell conclusions in their study, they will need to optimize their Pax7 staining and repeat this study. They should focus on Pax7+ objects that contain a nucleus and are located below the basal lamina. Also, the word 'activation' in line 180 should be edited to 'expansion' as the histological analysis and study design preclude an evaluation of satellite cell activation.
Response: Our new results after optimizing staining protocol support that low-dose gelatin injection causes more Pax7+ cells (green) underneath the basal lamina (red) in injured TA muscle and high-dose gelatin injection suppressed the number of Pax7+ SCs at 7 D.P.I. The new results were shown in Figure 2A and described in the main text Lines 152-155.
We have changed the word “activation” into “expansion” according to reviewer’s suggestion in the main text Line 161.
2.b. Alternatively to 2.a., it would not diminish the impact of this study to remove the 'satellite cell' findings in their entirety from the manuscript.
Response: Please see above. We hope the new images are convincing to this reviewer.
- It is surprising that the molecular hallmarks of low vs high gelatin injection shown in Fig. 8 would still be present at a time point 2-weeks after the initial injection.*
Response: Please see below 3.b.
3.a. It would increase the impact of the study to better understand the basis of this surprising observation. This point links to point 1.a. as one would ideally need to quantify baseline gelatin levels pre-injury and post-injury. For example, is the injected gelatin still present 14-days after injection? Or is the MMP profile altered in a way that sustains these levels one direction or the other? Etc.
Response: Please see below 3.b.
3.b. Alternatively to 3.a., the authors should use the Discussion to note this point and speculate on the significance.
Response: We thank the reviewer for making this important point. The sustained effect of gelatin materials has been reported in several previous studies, and we now have discussed possible mechanisms such as MMP expression profiles and lasting interplays between SCs, macrophages, ECM, and myoblasts. See the main text Lines 437-459.
**Minor Comments**
- The authors should conduct a careful review of the manuscript to address minor typos and grammatical errors.* Response: We thank the reviewer for pointing this out and have carefully reviewed the manuscript and corrected minor typos and grammatical errors.
- It is unclear from the Figure Legends, Results, or Methods what the 'PBS' condition in Figure 1 refers to. Is this the uninjured control? If so, consider using 'Cntrl' as the label and then defining it in the figure legend for clarity.*
Response: We thank the reviewer for pointing this out. Yes, PBS/phosphate-buffered saline is the vehicle for delivering CTX thus representing the uninjured model. In the revised manuscript, we have replaced “PBS: phosphate-buffered saline” with “Ctrl” in Figures, Figure Legends, Results and Methods.
- It is unclear from the Figure Legends, Results, or Methods what is quantified to read-out the 'myogenesis index' and 'fusion index' that is reported in Fig. 5D & E. Please reconcile.*
Response: This point has been addressed in the previous section. Myotube formation was quantified using myogenesis and fusion index. Myogenesis index (% nuclei within MyHC-stained myocytes/total nuclei) and fusion index (% nuclei in myotubes with >5 nuclei/total nuclei in MyHC-stained cells) are now explained both in legends and in Materials and Methods. Please see the main text Lines 577-582.
Reviewer #3 (Significance (Required)):
The manuscript constitutes a technical advance, and offers a molecular mechanism, in support of the notion that intramuscular injection of low dose gelatin serves to expedite the process of skeletal muscle regeneration. This study has translational implications for a regenerative medicine application of this knowledge. It is my opinion that this aspect of the study is well supported by the results and requires Response: We thank the reviewer for sharing this positive and important comment. Indeed, the motivation of this work was to delineate biological pathways of gelatin treatments in myogenesis and muscle regeneration for potential therapeutic applications.
The manuscript would constitute both a technical and a conceptual advance by addressing Major Point 1a as the authors would show, for the first time, that low and high dose gelatin levels naturally exist in vivo to mediate the process of muscle endogenous repair. This would be highly significant, because as the authors rightly point out in Lines 74-76 of their manuscript "...by what mechanisms ECM regulates the functional, morphological, and molecular events of skeletal muscle regeneration remain poorly understood". It is my opinion that making this latter point would require 1-3 months of additional studies.
Response: We thank the reviewer for pointing to this important scientific direction. But regrettably, missing adequate tools for quantifying endogenous gelatin in vivo is currently prohibitive.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary
The Review Commons submission by Liu and colleagues entitled "Biphasic effect of gelatin in myogenesis and skeletal muscle regeneration" provide a systematic in vitro and in vivo evaluation of the effects of myogenic cell exposure to low or high dose gelatin. Through these analyses they uncover pro- and anti-regenerative effects of gelatin that are dose dependent and through a series of cell and molecular studies, they attribute these dual effects to a ROS-IL6/TNFa signaling axis. The study is well designed and executed. For the most part the figures and experimental details are clear and transparent, and most of the …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Summary
The Review Commons submission by Liu and colleagues entitled "Biphasic effect of gelatin in myogenesis and skeletal muscle regeneration" provide a systematic in vitro and in vivo evaluation of the effects of myogenic cell exposure to low or high dose gelatin. Through these analyses they uncover pro- and anti-regenerative effects of gelatin that are dose dependent and through a series of cell and molecular studies, they attribute these dual effects to a ROS-IL6/TNFa signaling axis. The study is well designed and executed. For the most part the figures and experimental details are clear and transparent, and most of the conclusions are supported by the data. Specific comments follow.
Major Comments
- Study framing in the Introduction is mis-aligned with the research conducted. Currently the Introduction sets the stage for an in vivo exploration of the effects of endogenous gelatin produced in the course of muscle regeneration. However, there are no experiments in this paper investigating the presence or effects of endogenous gelatin.
1.a. The impact of the study would indeed be increased by including a systematic characterization of endogenous gelatin levels during muscle regeneration in healthy mice as compared to in those where fibrosis is prevalent. A demonstration that ROS-IL6/TNFa levels align with patterns seen in the in vitro studies, and pharmacological manipulations to 'rescue' would all provide a demonstration of a hormesis gelatin response in vivo. Meaning, is this process something that naturally occurs in the physiological context, or is it one that is possible, but only be supraphyiological gelatin injections?
1.b. Alternatively to 1.a., the authors should reframe the Introduction to focus on understanding the effects of gelatin as a biomaterial that is being used in regenerative medicine applications. In this case, the authors should delete/edit/reframe lines 74-102 and instead use lines 103 on to motivate the study so as to be consistent.
- Satellite cell conclusions in Figures 2C-D that are based upon representative images provided in 2A, are questionable. Pax7 staining in mouse tissue sections is notoriously difficult and the antibodies can have dramatic lot to lot variability. The immunostaining provided in the representative images is not convincing, and hence, draws into question the conclusions based upon them.
2.a. If the authors wish to leave the satellite cell conclusions in their study, they will need to optimize their Pax7 staining and repeat this study. They should focus on Pax7+ objects that contain a nucleus and are located below the basal lamina. Also, the word 'activation' in line 180 should be edited to 'expansion' as the histological analysis and study design preclude an evaluation of satellite cell activation.
2.b. Alternatively to 2.a., it would not diminish the impact of this study to remove the 'satellite cell' findings in their entirety from the manuscript.
- It is surprising that the molecular hallmarks of low vs high gelatin injection shown in Fig. 8 would still be present at a time point 2-weeks after the initial injection.
3.a. It would increase the impact of the study to better understand the basis of this surprising observation. This point links to point 1.a. as one would ideally need to quantify baseline gelatin levels pre-injury and post-injury. For example, is the injected gelatin still present 14-days after injection? Or is the MMP profile altered in a way that sustains these levels one direction or the other? Etc.
3.b. Alternatively to 3.a., the authors should use the Discussion to note this point and speculate on the significance.
Minor Comments
- The authors should conduct a careful review of the manuscript to address minor typos and grammatical errors.
- It is unclear from the Figure Legends, Results, or Methods what the 'PBS' condition in Figure 1 refers to. Is this the uninjured control? If so, consider using 'Cntrl' as the label and then defining it in the figure legend for clarity.
- It is unclear from the Figure Legends, Results, or Methods what is quantified to read-out the 'myogenesis index' and 'fusion index' that is reported in Fig. 5D & E. Please reconcile.
Significance
The manuscript constitutes a technical advance, and offers a molecular mechanism, in support of the notion that intramuscular injection of low dose gelatin serves to expedite the process of skeletal muscle regeneration. This study has translational implications for a regenerative medicine application of this knowledge. It is my opinion that this aspect of the study is well supported by the results and requires <1month of additional edits to finalize the manuscript.
The manuscript would constitute both a technical and a conceptual advance by addressing Major Point 1a as the authors would show, for the first time, that low and high dose gelatin levels naturally exist in vivo to mediate the process of muscle endogenous repair. This would be highly significant, because as the authors rightly point out in Lines 74-76 of their manuscript "...by what mechanisms ECM regulates the functional, morphological, and molecular events of skeletal muscle regeneration remain poorly understood". It is my opinion that making this latter point would require 1-3 months of additional studies.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
The manuscript by Xiao Ling Liu and colleagues titled "Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration" deals with the effect of gelatin on differentiation of myoblast cell line, in vitro, and on skeletal muscle regeneration upon muscle injury, in vivo. In vivo, the gelatin is a product of collagen breakdown associated with skeletal muscle regeneration upon acute or chronic muscle damage.
Specifically, the authors define a dose-dependent effect of gelatin, beneficial at low dose and detrimental at high dose. This effect is mediated by the level of ROS accumulation leading to the induction of …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
Summary:
The manuscript by Xiao Ling Liu and colleagues titled "Bi-phasic effect of gelatin in myogenesis and skeletal muscle regeneration" deals with the effect of gelatin on differentiation of myoblast cell line, in vitro, and on skeletal muscle regeneration upon muscle injury, in vivo. In vivo, the gelatin is a product of collagen breakdown associated with skeletal muscle regeneration upon acute or chronic muscle damage.
Specifically, the authors define a dose-dependent effect of gelatin, beneficial at low dose and detrimental at high dose. This effect is mediated by the level of ROS accumulation leading to the induction of different cytokines with opposite effects on skeletal muscle regeneration.
Major comments:
The experimental purpose is well tackled from both biochemical and functional point of view, and the proposed experiments are quite exhaustive. However, I would suggest some additional experimental analyses to improve the robustness and quality of the study, as well as text and figure editing, as reported below.
Regarding the additional experiments/analyses/images:
- Figure 5: I would suggest to add an image of C2C12 cells in GM (growth medium), as representative images of proliferation analysis upon LCG/HCG/NAC treatment.
- Figure 5: I would suggest to repeat the main si-NOX2 experiments with an alternative siRNA to rule out off target effects.
- In vivo experiments could be improved by adding DHE or DCFH staining on muscle TA cryosections to quantify the level of oxidative stress.
- The proposed model could be better tackled by additional in vivo treatment with Ab anti IL-6 or anti TNFalpha in combination with CTX and LCG or HCG, followed by H/E staining at 14 dpi.
Minor comments:
Each acronym should be indicated in full in the main text at the first mention (for instance BHP, NAC and others). Moreover, I would suggest to add an acronym list for reagents and factors
Experimental methods should be better detailed; for instance I would suggest:
- Add a detailed description of the quantification of differentiation indexes
- Explain how cell growth (OD 450nm) and optical density (570nm) assays have been performed
- Explain how ROS species and antioxidant enzymes have been measured (Fig 4C and 4D)
Figures and figure legends:
- Please add in the figures the figure number in order to facilitate the reading of the pdf file
- The sequence of the panels should be coherent with the alphabet and reading left to right and up to down
- In the Figure 1, I would suggest to add the whole TA sections for H/E staining in order to appreciate the overall beneficial or detrimental effect of LCG and HCG, respectively.
- I would suggest to show Supplementary figures 1C and 1D in the Figure 1
- In the Supplementary Fig 2A, I would suggest to the authors to show and comment only the data about proliferation: the earlier orientation, fusion and differentiation of C2C12 exposed to LCG are a consequence of the positive effect of LCG on proliferation
- The quality of representative images of western blot is not always high: the bands are fuse and, consequently, the quantification is not reliable. For instance Fig S4 B, S4 G; in Fig 2 the representative image does not really represent the reported quantification.
- Please specify in the figure legends the meaning of the acronym in the axis title (for instance RFU, MFI or DCF) and in the axis title the unit of measure (for instance Count (?)).
- The authors always wrote "filed" in place of "field"
- In the figures 7 and 8 the letters for densitometry panel are missed.
- In the figure legend 8 the panel letters do not match the panels in the figure
- Figure 3I: replace "nucleis" with "nuclei"
Text editing:
- Line 64: Satellite cells (SCs) would be more appropriate than myoblasts
- Line 64: Please define more carefully the location of SCs
- Line 67: MyoD+/Myog+ would be more appropriate than Pax7+/Myog+
- Line 67: (Pax7+)/Myog+ "myocytes" in place of myotubes ... and fuse with each other to generate myotubes
- Line 69: Please add Myf6/MRF4 to MRFs list
- Line 112: replace "its" with "their"
- Lines 330-331: "promoted IL-6" "enhanced TNF", please insert secretion/ production
- Line 352-353: this sentence is not necessary
Significance
The study reports robust and interesting data applicable to both basic research and translational research, such as tissue engineering applications.
Keywords for field of expertise of this reviewer:
Skeletal muscle regeneration
Duchenne Muscular Dystrophy
Inflammation
Macrophages
Oxidative stress
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