PGAM2 Regulates Sepsis-Induced Diaphragmatic Atrophy via the JAK2/STAT3 Pathway

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

PGAM2, a glycolytic enzyme that catalyzes the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, is involved in skeletal muscle function through glycolytic regulation. The authors previously shown that anisodamine alleviates sepsis-induced diaphragm atrophy, and the mechanism may be related to inhibiting the JAK2/STAT3 signaling pathway (International Immunopharmacology, 133, 2024, 112133). In this study, they investigated the role of PGAM2 in sepsis-induced diaphragmatic atrophy and its underlying mechanisms.

They show that

• Sepsis-induced diaphragmatic atrophy is associated with upregulation of PGAM2 (figure 1).
• TNF-α increases PGAM2 expression and promotes myotube atrophy in C2C12 myotubes (figure 2).
• PGAM2 knockdown attenuates TNF-α–induced activation of muscle atrophy–related E3 ligases and myotube atrophy (figure 3).
• PGAM2 overexpression enhances TNF-α–induced activation of atrophy-related E3 ligases and aggravates myotube atrophy (figure 4).
• PGAM2 knockdown significantly exacerbated the cecal ligation and puncture (CLP)-induced phosphorylation of JAK2 and STAT3 (figure 5).

They propose that PGAM2 is a novel mediator of sepsis-induced diaphragmatic atrophy and suggest that targeting PGAM2 may provide therapeutic benefits. Although the precise molecular mechanism by which PGAM2 regulates the JAK2/STAT3 signaling pathway remains unclear, the study is well organized and demonstrates the therapeutic potential of targeting PGAM2 for sepsis-induced diaphragm atrophy in humans.

I have one comment that should be addressed before a decision can be made. In reference to the authors’ previous paper (International Immunopharmacology, Volume 133, 2024, 112133), I am wondering whether treatment with anisodamine in the CLP mouse model of sepsis and in the TNF-α–stimulated C2C12 myotube model affects (i.e., suppresses) the expression of PGAM2.

Minor:

CLP does not stand for Directory of open access journals (lane 378).

Author Response

好的，这是根据您提供的格式排版好的回复内容：

**Comments 1:** CLP does not stand for Directory of open access journals (lane 378).

**Response 1:** Thank you for your careful review. We have corrected the definition of CLP . CLP now refers to “cecal ligation and puncture” rather than “Directory of Open Access Journals.”

Reviewer 2 Report

Comments and Suggestions for Authors

The manuscript is written in clear and accessible language and presents a well-defined objective: to investigate the role of phosphoglycerate mutase 2 (PGAM2) in sepsis-induced diaphragmatic atrophy. The findings enhance our understanding of the mechanisms underlying sepsis-associated diaphragmatic dysfunction, demonstrating that PGAM2 mediates muscle atrophy by activating the JAK2/STAT3 signaling pathway and regulating the atrogenes MAFbx and MuRF1. Despite the limitations acknowledged by the authors, the study is original, methodologically sound, and provides meaningful contributions to the field, with translational potential by identifying PGAM2 as both a biomarker and a therapeutic target in sepsis-associated respiratory muscle dysfunction.I have a few comments and suggestions for the authors:

1. Manuscript Formatting: The manuscript should be carefully revised to correct formatting issues, particularly the absence of spaces between sentences (e.g., lines 41, 168, 170, and 179).
2. Sample Size Clarification: The number of animals used in the study, as well as in each experimental analysis, is not clearly stated in the main text and appears only in the figure legends. This information should be explicitly included in the Methods section.
3. Experimental Design Schematic: The inclusion of a detailed schematic illustrating the experimental design is recommended. This could be similar to the schematic representation of the cecal ligation and puncture (CLP) procedure.
4. Immunohistochemistry Details: The Methods section (Section 2.3) should clearly specify the primary and secondary antibodies used in the immunohistochemical analyses. Although primary antibodies are mentioned earlier (lines 78–89), this information should be better organized. Additionally, the type of myosin used (e.g., embryonic myosin) should be specified.
5. Muscle Fiber Analysis:The authors should provide a more detailed description of the methodology used to measure muscle fiber cross-sectional area, including the number of fibers analyzed and the specific region of the diaphragm examined.
6. Discussion of Early Atrophy: The results indicate a significant loss of body weight and muscle atrophy within 3 days of sepsis induction. This is a noteworthy finding and warrants a more comprehensive discussion in the manuscript.

 

Author Response

Comments 1: Manuscript Formatting: The manuscript should be carefully revised to correct formatting issues, particularly the absence of spaces between sentences (e.g., lines 41, 168, 170, and 179).

Response 1: Thank you for pointing this out. We have carefully checked the entire manuscript and corrected the formatting issues, particularly the missing spaces between sentences. We have also revised the manuscript for consistency in punctuation, spacing, and overall formatting.

Comments 2: Sample Size Clarification: The number of animals used in the study, as well as in each experimental analysis, is not clearly stated in the main text and appears only in the figure legends. This information should be explicitly included in the Methods section.

Response 2: Thank you for this important comment. We agree that the number of animals used in the study and in each experimental analysis should be clearly stated in the Methods section. In the revised manuscript, we have added detailed information regarding the total number of animals, the number of animals assigned to each group, and the number of biological replicates included in each analysis. The exact n values are now described in the Methods section and are consistent with those shown in the figure legends.

Comments 3: Experimental Design Schematic: The inclusion of a detailed schematic illustrating the experimental design is recommended. This could be similar to the schematic representation of the cecal ligation and puncture (CLP) procedure.

Response 3: Thank you for this valuable suggestion. We have added a detailed schematic illustration of the overall experimental design in the revised manuscript. The schematic summarizes the animal grouping, CLP/sham procedure, postoperative observation, sample collection, and subsequent analyses. This figure has been included in Figure 1-B to improve the clarity and readability of the experimental workflow.

Comments 4: Immunohistochemistry Details: The Methods section (Section 2.3) should clearly specify the primary and secondary antibodies used in the immunohistochemical analyses. Although primary antibodies are mentioned earlier (lines 78–89), this information should be better organized. Additionally, the type of myosin used (e.g., embryonic myosin) should be specified.

Response 4: Thank you for this helpful comment. We have reorganized the antibody information in the Methods section to make it clearer and more complete. Regarding myosin staining, we used an antibody against myosin heavy chain (MyHC) without distinguishing specific isoforms. Therefore, the revised manuscript now specifies that the staining represents total MyHC/pan-MyHC rather than a specific MyHC subtype such as embryonic MyHC, MyHC I, or MyHC II.

Comments 5: Muscle Fiber Analysis: The authors should provide a more detailed description of the methodology used to measure muscle fiber cross-sectional area, including the number of fibers analyzed and the specific region of the diaphragm examined.

Response 5: Thank you for this helpful comment. We have revised the Methods section to provide a more detailed description of the muscle fiber cross-sectional area analysis, including the specific diaphragm region examined, the number of sections and fields analyzed, the approximate number of fibers measured, and the quantification method. In the revised manuscript, we now specify that muscle fiber cross-sectional area was measured on transverse cryosections from the middle portion of the costal diaphragm adjacent to the intercostal region. For each mouse, at least four immunofluorescence-stained sections were analyzed, and three fields of view were captured from this region in each section at the same magnification. Approximately 200 muscle fibers were measured per section using ImageJ software. Obliquely sectioned, damaged, or poorly defined fibers were excluded from the analysis, and the mean value from each mouse was used as one independent biological replicate for statistical analysis.

Reviewer 3 Report

Comments and Suggestions for Authors

The study investigates the involvement of PGAM2 in sepsis-related diaphragmatic wasting. Utilizing a cecal ligation and puncture (CLP) mouse model and TNF-$\alpha$-stimulated C2C12 myotubes, the authors demonstrate that:

1-PGAM2 expression is significantly upregulated in the diaphragm during sepsis.

2-Silencing PGAM2 attenuates myotube atrophy and the expression of E3 ubiquitin ligases (MuRF1 and MAFbx).

3-PGAM2 promotes these effects by facilitating the activation of the JAK2/STAT3 pathway

The primary contribution of this work is identifying a metabolic enzyme (PGAM2) as a potential regulatory node in inflammatory muscle degradation, offering a new perspective on the intersection of metabolism and signaling in sepsis. However, to meet the high standards of Biomedicines, several critical methodological and interpretive weaknesses must be addressed. Below are specific points for your consideration:

1-The transition from PGAM2’s role in glycolysis to its role in the JAK2/STAT3 pathway feels abrupt. The Introduction cites that PGAM1 interacts with STAT3, but more justification is needed for why PGAM2-specifically in the muscle-would adopt this non-canonical signaling role under septic stress

2-The abstract lacks specific quantitative results (e.g., percentage changes in CSA or protein levels), making it purely descriptive

3-For several critical experiments, the sample size is limited to n=3 or n=4. In biological systems with inherent variability like the CLP model, such small numbers may lack sufficient statistical power.

4-The authors list primer sequences but omit the sequences for the siRNAs used to knock down PGAM2. This hinders the reproducibility of the study.

5-The methodology focuses entirely on histological (CSA) and molecular markers . Including physiological assessments, such as ex vivo diaphragmatic force production, would significantly strengthen the clinical relevance of the findings.

6-In the Results text, the authors state that PGAM2 overexpression exacerbated atrophy and E3 ligase expression. However, the legend for Figure 4 (Lines 246-248) explicitly states that PGAM2 overexpression suppressed the elevation of MAFbx and MuRF1. This is a fundamental error that confuses the entire narrative of the paper

7-Similarly, the legend for Figure 5 (Lines 278-279) claims that PGAM2 knockdown exacerbated CLP-induced phosphorylation of JAK2/STAT3, whereas the actual data and Results text claim the opposite (reduction). Such inconsistencies must be addressed to ensure data integrity

8-Figure 1G cites n=3, which appears to be a typo. Additionally, Figure 1 legend mentions "SD" while the methods section specifies "SEM".

9-The authors speculate that PGAM2 might directly bind to JAK2 or STAT3, but provide no experimental proof (e.g., Co-IP or molecular docking). Without this, the link remains correlative rather than causative.

10-As PGAM2 is a glycolytic enzyme, the Discussion should more thoroughly address how sepsis-induced metabolic shifts (like the Warburg-like effect in muscle) relate to PGAM2 upregulation. It is counter-intuitive that a "growth-promoting" glycolytic enzyme would be upregulated in an atrophic state .

Author Response

Comments 1: The transition from PGAM2's role in glycolysis to its role in the JAK2/STAT3 pathway feels abrupt. The Introduction cites that PGAM1 interacts with STAT3, but more justification is needed for why PGAM2—specifically in the muscle—would adopt this non-canonical signaling role under septic stress.

Response 1: Thank you for this insightful comment. We agree that the transition from the canonical glycolytic role of PGAM2 to its potential involvement in JAK2/STAT3 signaling required further clarification. In the revised manuscript, we have added additional rationale to better explain why PGAM2, a muscle-enriched glycolytic enzyme, may acquire a non-canonical signaling-related role under septic stress. Specifically, we now emphasize that septic stress is associated with profound metabolic remodeling and inflammatory signaling activation in skeletal muscle, and that metabolic enzymes may participate in signaling regulation beyond their classical catalytic functions. In addition, to further support this possibility, we have included exploratory experimental evidence in the supplementary document showing an interaction/association between PGAM2 and phosphorylated STAT3. We have treated these data as preliminary mechanistic evidence rather than definitive proof. Accordingly, we have revised the relevant statements in the Introduction and Discussion to avoid overinterpretation and to present the PGAM2–JAK2/STAT3 axis as a potential mechanism that requires further validation in future studies.

Comments 2: The abstract lacks specific quantitative results (e.g., percentage changes in CSA or protein levels), making it purely descriptive.

Response 2: Thank you for this valuable comment. We agree that the original Abstract was mainly descriptive and did not provide sufficient quantitative information. In the revised manuscript, we have added representative quantitative results to the Results section of the Abstract. Specifically, we now report that CLP caused an approximately 38% reduction in diaphragm weight, an approximately 37% decrease in muscle fiber cross-sectional area, and an approximately 105% increase in PGAM2 protein expression compared with the sham group. These additions make the Abstract more informative and better reflect the main quantitative findings of the study.

Comments 3: For several critical experiments, the sample size is limited to n=3 or n=4. In biological systems with inherent variability like the CLP model, such small numbers may lack sufficient statistical power.

Response 3: Thank you for this important comment. We agree that the CLP model has inherent biological variability and that relatively small sample sizes may limit statistical power, particularly for molecular analyses. In the revised manuscript, we have clarified the sample size used for each experimental analysis in both the Methods section and figure legends. For the main in vivo phenotypic analyses, including body weight, diaphragm weight, and muscle fiber cross-sectional area, 6–7 animals per group were included as independent biological replicates. For some molecular experiments, including Western blotting and RT-qPCR, the sample size was limited to n = 3–4 per group because of the limited availability of remaining diaphragm tissue and sample quality after tissue processing. To address this concern, we have revised the relevant statements to avoid overinterpretation of these molecular findings. We now present these results as supportive evidence rather than definitive standalone proof. In addition, we have added this issue as a limitation in the Discussion, emphasizing that future studies with larger sample sizes and independent validation are needed to further confirm the role of PGAM2 in CLP-induced diaphragmatic atrophy.

Comments 4: The authors list primer sequences but omit the sequences for the siRNAs used to knock down PGAM2. This hinders the reproducibility of the study.

Response 4: Thank you for this helpful comment. We have added the siRNA sequences used for PGAM2 knockdown in the revised Methods section to improve the reproducibility of the study.

Comments 5: The methodology focuses entirely on histological (CSA) and molecular markers. Including physiological assessments, such as ex vivo diaphragmatic force production, would significantly strengthen the clinical relevance of the findings.

Response 5: Thank you for this valuable suggestion. We agree that physiological assessment of diaphragmatic function, such as ex vivo diaphragmatic force production, would further strengthen the functional and clinical relevance of our findings. The present study primarily focused on histological evidence of diaphragmatic atrophy and the associated molecular mechanism involving PGAM2 and JAK2/STAT3 signaling. Due to limitations in tissue availability and experimental setup, ex vivo diaphragm contractile force measurements were not performed in the current study. We have added this point as a limitation in the Discussion and emphasized that future studies should incorporate direct functional assessments of diaphragmatic contractility to further validate the physiological significance of PGAM2-mediated diaphragmatic injury in sepsis.

Comments 6: In the Results text, the authors state that PGAM2 overexpression exacerbated atrophy and E3 ligase expression. However, the legend for Figure 4 (Lines 246-248) explicitly states that PGAM2 overexpression suppressed the elevation of MAFbx and MuRF1. This is a fundamental error that confuses the entire narrative of the paper.

Response 6: Thank you for pointing this out. We acknowledge that the word "suppressed" in the original figure legend was incorrect and inconsistent with our experimental results. We have corrected this wording in the revised manuscript. The sentence now states that PGAM2 overexpression further increased the TNF-α-induced elevation of MAFbx and MuRF1 protein levels.

Comments 7: Similarly, the legend for Figure 5 (Lines 278-279) claims that PGAM2 knockdown exacerbated CLP-induced phosphorylation of JAK2/STAT3, whereas the actual data and Results text claim the opposite (reduction). Such inconsistencies must be addressed to ensure data integrity.

Response 7: Thank you for pointing out this important inconsistency. We acknowledge that the wording in the original Figure 5 legend was incorrect. The revised legend now states that PGAM2 knockdown reduced the CLP-induced phosphorylation of JAK2 and STAT3.

Comments 8: Figure 1G cites n=3, which appears to be a typo. Additionally, Figure 1 legend mentions "SD" while the methods section specifies "SEM".

Response 8: Thank you for pointing out these inconsistencies. We acknowledge that the sample size stated for Figure 1G was a typographical error, and we have corrected it to the actual n value in the revised figure legend. In addition, we have revised the Figure 1 legend to ensure consistency with the Methods section. The data are now consistently described as mean ± SEM throughout the manuscript.

Comments 9: The authors speculate that PGAM2 might directly bind to JAK2 or STAT3, but provide no experimental proof (e.g., Co-IP or molecular docking). Without this, the link remains correlative rather than causative.

Response 9: Thank you for this important comment. We agree that the relationship between PGAM2 and JAK2/STAT3 signaling should not be interpreted as a direct causal interaction without experimental support. To address this concern, we have added exploratory Co-IP data in the supplementary file, which provide preliminary evidence suggesting an association between PGAM2 and phosphorylated STAT3. We have also added this point as a limitation in the Discussion, noting that future studies using repeated Co-IP experiments, molecular docking, and functional validation are needed to confirm whether PGAM2 directly regulates JAK2/STAT3 signaling.

Comments 10: As PGAM2 is a glycolytic enzyme, the Discussion should more thoroughly address how sepsis-induced metabolic shifts (like the Warburg-like effect in muscle) relate to PGAM2 upregulation. It is counter-intuitive that a "growth-promoting" glycolytic enzyme would be upregulated in an atrophic state.

Response 10: Thank you for this insightful comment. We agree that the relationship between PGAM2 upregulation and sepsis-induced muscle atrophy requires further discussion, especially given the classical role of PGAM2 in glycolysis. In the revised manuscript, we have expanded the Discussion to address this issue. We now explain that sepsis can induce profound metabolic remodeling in skeletal muscle, including mitochondrial dysfunction and a compensatory or maladaptive shift toward glycolysis, which resembles a Warburg-like metabolic pattern. In this context, PGAM2 upregulation may not necessarily indicate a growth-promoting state, but rather reflect stress-induced glycolytic remodeling and inflammatory adaptation in septic muscle.

Author Response File: Author Response.docx

Reviewer 4 Report

Comments and Suggestions for Authors

 

The study is dedicated to a vital clinical problem of muscle atrophy induced by sepsis. The authors investigate a novel signaling pathway that may contribute to the sepsis-induced atrophy of diaphragm. However, there are some points that should be improved in methods description.

How many mice were in each experimental group? In some figures there are four data points per group, in some – 3, and in some – even 6. Why for different types of analyses there are different sample numbers?

Please, describe the experimental procedures for sham operated group in detail.

Please indicate how many muscle fibers from each sample were analyzed to determine fibers cross-sectional area

For PGAM2 immunostaining the picture of negative control (without primary antibodies) should be represented with the pictures of experimental group samples.

The scale bars in all the immunohistochemical pictures are unreadable. Please correct.

Please specify the content of loading buffer in Western blot description

Why not analyze TNFalpha mRNA expression, as it is a direct upstream for the PGAM?

It is written that C2C12 samples were tripled, so it should be three data points of myotubes diameter (mean +- SEM) in Figure 2 F (it may be F but the letter is lost after E). But there are far more sample points in this figure. What are they? In figure 2 A there are 6 points in control group and 5 in every other group. Shouldn’t there be three points in each group?

Minor:

Space between letter and square or round bracket. Example: not “something(Figure 1)”,

but “something (Figure 1).”

Lines 255-256: ‘’Representative immunoblots and densitometric quantification showing that PGAM2 overexpression significantly suppressed the TNF-α-induced elevation of MAFbx and MuRF1 protein levels’’ – misprint? The study pvoves that PGAM2 enhances

Author Response

Comments 1: How many mice were in each experimental group? In some figures there are four data points per group, in some – 3, and in some – even 6. Why for different types of analyses there are different sample numbers?

Response 1: Thank you for this important comment. We have clarified the number of mice in each group and the exact n value for each analysis in the revised Methods section and figure legends. The different sample numbers among figures were mainly due to tissue loss or sample-quality issues during experimental processing, such as consumption of diaphragm tissue in previous assays, failed frozen section preparation, poor section quality, or insufficient remaining tissue for molecular analyses. Each data point represents one independent mouse.

Comments 2: Please, describe the experimental procedures for sham operated group in detail.

Response 2: Thank you for this helpful comment. We have added a detailed description of the sham operation procedure in the revised Methods section, including anesthesia, laparotomy, cecum exposure without ligation or puncture, cecum replacement, abdominal closure, and postoperative care.

Comments 3: Please indicate how many muscle fibers from each sample were analyzed to determine fibers cross-sectional area.

Response 3: Thank you for this helpful comment. We have clarified in the revised Methods section that, for each mouse, at least four diaphragm sections were analyzed, three fields were captured from the middle costal region of each section, and approximately 200 muscle fibers were measured per section to determine muscle fiber cross-sectional area.

Comments 4: For PGAM2 immunostaining the picture of negative control (without primary antibodies) should be represented with the pictures of experimental group samples.

Response 4: Thank you for this helpful suggestion. We have added representative negative control images for PGAM2 immunostaining, in which the primary antibody was omitted, to the supplementary materials.

Comments 5: The scale bars in all the immunohistochemical pictures are unreadable. Please correct.

Response 5: Thank you for pointing this out. We have revised all immunohistochemical/immunofluorescence images to improve the readability of the scale bars. The scale bars have been enlarged and adjusted for better contrast in the revised figures.

Comments 6: Please specify the content of loading buffer in Western blot description.

Response 6: Thank you for this helpful comment. We have specified the composition of the 5× SDS-PAGE loading buffer in the revised Western blot Methods section.

Comments 7: Why not analyze TNFalpha mRNA expression, as it is a direct upstream for the PGAM?

Response 7: Thank you for this valuable comment. We agree that TNF-α mRNA expression in diaphragm tissue would provide additional evidence for the upstream inflammatory regulation of PGAM2. Previous studies have shown that TNF-α is increased in CLP-induced sepsis models, and we used TNF-α stimulation in vitro to mimic this inflammatory condition. However, we acknowledge that not measuring TNF-α mRNA in the diaphragm tissue was an oversight in the current study. We have clarified this point in the revised Discussion and added it as a limitation, noting that future studies should directly assess TNF-α and other inflammatory mediators in septic diaphragm muscle to better define the upstream regulation of PGAM2.

Comments 8: It is written that C2C12 samples were tripled, so it should be three data points of myotubes diameter (mean + SEM) in Figure 2 F (it may be F but the letter is lost after E). But there are far more sample points in this figure. What are they? In figure 2 A there are 6 points in control group and 5 in every other group. Shouldn't there be three points in each group?

Response 8: Thank you for pointing out this important issue. We agree that the original presentation of the C2C12 myotube diameter data was unclear and could be misleading, as some individual myotube measurements were displayed rather than the independent experimental replicates used for statistical analysis. Following your suggestion, we have reanalyzed the data and replaced the statistical graphs in the revised Figure 2. In the revised version, each data point represents one independent experiment/biological replicate, and the data are presented as mean ± SEM. We have also corrected the missing panel label and revised the figure legend to clearly state how the data points were defined.

Comments 9 (Minor): Space between letter and square or round bracket. Example: not "something(Figure 1)", but "something (Figure 1)."

Response 9 (Minor): Thank you for pointing out these issues. We have carefully checked the manuscript and corrected the missing spaces between text and brackets throughout the revised version, such as changing "something(Figure 1)" to "something (Figure 1)."

Comments 10 (Minor): Lines 255-256: "Representative immunoblots and densitometric quantification showing that PGAM2 overexpression significantly suppressed the TNF-α-induced elevation of MAFbx and MuRF1 protein levels" – misprint? The study proves that PGAM2 enhances.

Response 10 (Minor): Thank you for pointing out this issue. We acknowledge that the word "suppressed" in the Figure legend was a misprint and inconsistent with our results. It has been corrected to indicate that PGAM2 overexpression further enhanced the TNF-α-induced elevation of MAFbx and MuRF1 protein levels.

Reviewer 5 Report

Comments and Suggestions for Authors

PGAM2 is an enzyme that participates in glycolysis, and quite surprisingly, the authors describe that in muscle atrophy induced by sepsis-induced inflammation (cecal ligation/puncture) the expression levels of PGAM2 go up, quite contrary to the common knowledge that severe inflammation shuts down most of the protein synthesis machinery.

To test the role of PGAM2, the authors used C2C12 cells, a suitable in vitro model of skeletal fibers to dissect putative pathways. By using TNF-α, a well-known inflammatory cytokine, the authors showed that PGAM2 expression levels increased with respect to non-treated cells; the same was observed with IL-1 and IL-6.

Using loss-of-function (knock-down) and gain-of-function (overexpression) experiments, the authors suggested that PGAM2 could modulate cell atrophy. Moreover, they showed evidence that the phenomenon could be due to the activation of the STAT3 signaling pathway, a pathway known to participate in muscle atrophy.

Despite the functional novelty suggested for PGAM2, it is unclear why the authors did not test their hypothesis using the in vivo model. Furthermore, the authors did not perform gain- or loss-of-function experiments to validate the overall pathway proposed. Results seem to be specific to a particular group of muscle fibers, apparently not affecting small fiber sizes at all.

Overall, the work is interesting but, in my opinion, additional experiments are required to support their hypothesis.

Specific comments: 

Authors report major changes in muscle mass as early as 3 days. Given the magnitude of this phenotype, have the authors examined different muscle groups (e.g., soleus vs. tibialis anterior) to determine whether atrophy is uniform across muscle types?

The magnitude of the reported effect is unclear. Is the body weight loss only 0.3%? This seems inconsistent with the substantial muscle loss described. Please explain.

A temporal analysis of enzyme expression changes is encouraged. Since surgical time-course data are already available, this would help establish when PGAM2 up-regulation occurs relative to the onset of atrophy.

The order of panels B and C should be revised: time-course data should be presented first, followed by endpoint measurements.

Did the authors observe uniform PGAM2 over-expression across all fiber types, or were there size-dependent differences? The size distribution graph suggests that small fibers are less affected. This should be clarified.

Units in all figures should be clearly separated and standardized.

Regarding fiber type quantification (Figure F), please provide the absolute number of fibers analyzed per condition, either in the figure legend or methods section.

Please explain how intensity measurements were obtained, including the calibration method used and whether analysis was performed on whole images or restricted to specific regions or fiber types.

There are typographical errors and concatenated words throughout the manuscript. Authors should carefully proofread the text; examples can be found in lines 168–170, 177, 179, 243, and 275.

Line 218: The phrase "Dissolving muscle protein is a key protein that leads to muscle atrophy" is unclear. Please rephrase.

Line 219: The text states that Figure 3A and 3B show both MAFbx and MuRF1, but only MuRF1 appears to be shown. Please clarify.

All images appear to be low resolution. Authors should improve image quality and include high-magnification insets of representative fibers. Calibration bars are barely visible and should be made clearly apparent throughout. Zoomed-in regions would also be helpful, as panels E and H appear to represent distinct anatomical regions that are difficult to interpret in the current format.

Representative images of proteins that are downregulated or unchanged in the model should be included.

RT-qPCR: please indicate the reference gene(s) used for normalization.

Figure 2: Statistical significance should be clearly indicated. Calibration bars are not visible. If expression is normalized to tubulin, data should be presented as fold-change or percentage, with units clearly stated. Panel 2E should show a more representative image, as the current one displays an apparently severe atrophic phenotype. Panel 2B: IL-1 and IL-6 labels should be placed above the plots rather than on the x-axis to avoid confusion. Additionally, are the TNF-α concentrations used in vitro comparable to those reported in vivo?

Figure 3: Panel 3C appears redundant with 3D, as the control condition is already shown and may be omitted. Panel 3E: experimental conditions should be clearly labeled above each panel. Panel 3G: contrast and gamma correction should be improved, as cell morphology and DAPI staining are difficult to visualize.

Figure 4: "In vitro gain-of-function" should be explicitly stated in the figure legend.

Figure 5: What is the effect of TNF-α in the presence of antisense PGAM2? Is the signaling pathway altered under these conditions?

Figure 6: The data presented are comprehensive; however, the lack of gain- or loss-of-function experiments substantially limits the validation of the proposed hypothesis.

It is unclear why gain- and loss-of-function experiments were not performed in vivo to validate the in vitro findings. Including such experiments would substantially strengthen the manuscript's conclusions.

Comments on the Quality of English Language

Author should proofread some of the sentences for clarity 

Author Response

Comments 1: Authors report major changes in muscle mass as early as 3 days. Given the magnitude of this phenotype, have the authors examined different muscle groups (e.g., soleus vs. tibialis anterior) to determine whether atrophy is uniform across muscle types?

Response 1: We thank the reviewer for this important comment. We agree that sepsis-induced muscle wasting may not occur uniformly across different muscle groups, particularly because muscles differ in fiber-type composition, metabolic profile, mechanical loading, and susceptibility to inflammatory and catabolic signaling. In the present study, we primarily focused on the diaphragm muscle because of its direct relevance to sepsis-associated respiratory muscle dysfunction. Therefore, we did not systematically examine other limb muscles, such as the soleus or tibialis anterior, in the current experimental design. Accordingly, we have revised the manuscript to avoid overgeneralizing our findings to all skeletal muscles. We now specify that the observed phenotype reflects changes in the diaphragm under our CLP model and have added this point as a limitation in the Discussion. Future studies comparing muscles with distinct fiber-type composition, such as oxidative slow-twitch muscles and glycolytic fast-twitch muscles, will be necessary to determine whether PGAM2-related metabolic remodeling and muscle atrophy occur in a muscle-type-specific manner during sepsis.

Comments 2: The magnitude of the reported effect is unclear. Is the body weight loss only 0.3%? This seems inconsistent with the substantial muscle loss described. Please explain.

Response 2: We thank the reviewer for pointing this out. The apparent body weight loss of 0.3% was due to an error in figure preparation. The values were calculated as proportions but were not multiplied by 100 when presented as percentages. Therefore, a value of approximately 0.3 actually represents approximately 30% body weight loss, not 0.3%. We have corrected the figure and the corresponding labels. This correction does not affect the raw data, statistical analysis, or the conclusions of the study.

Comments 3: A temporal analysis of enzyme expression changes is encouraged. Since surgical time-course data are already available, this would help establish when PGAM2 up-regulation occurs relative to the onset of atrophy.

Response 3: We thank the reviewer for this helpful suggestion. We agree that a temporal analysis of PGAM2 expression would provide valuable information regarding whether PGAM2 up-regulation precedes or follows the onset of muscle atrophy. However, in the present study, molecular analyses of PGAM2 expression were performed mainly at the endpoint, and we did not collect sufficient diaphragm samples at multiple postoperative time points for a reliable temporal analysis. We have therefore revised the Discussion to acknowledge this limitation. In the revised manuscript, we clarify that our current data demonstrate an association between PGAM2 up-regulation and diaphragm muscle atrophy in CLP-induced sepsis, but do not define their precise temporal sequence. Future studies with serial tissue collection will be required to determine the timing of PGAM2 induction relative to the development of muscle wasting.

Comments 4: The order of panels B and C should be revised: time-course data should be presented first, followed by endpoint measurements.

Response 4: We thank the reviewer for this suggestion. We have revised the order of panels B and C so that the time-course data are presented before the endpoint measurements.

Comments 5: Did the authors observe uniform PGAM2 over-expression across all fiber types, or were there size-dependent differences? The size distribution graph suggests that small fibers are less affected. This should be clarified.

Response 5: We thank the reviewer for this comment. We did not perform fiber-type-specific staining or size-stratified PGAM2 analysis in the current study. Therefore, we cannot determine whether PGAM2 over-expression is uniform across all fiber types or differs according to fiber size. We have clarified this limitation in the revised manuscript. Our data support an overall increase in PGAM2 expression after CLP, while its fiber-type- or size-dependent pattern requires further study.

Comments 6: Units in all figures should be clearly separated and standardized.

Response 6: We thank the reviewer for this suggestion. We have carefully checked all figure labels and revised the units to ensure that they are clearly separated from the variable names and consistently standardized throughout the figures.

Comments 7: Regarding fiber type quantification (Figure F), please provide the absolute number of fibers analyzed per condition, either in the figure legend or methods section.

Response 7: We thank the reviewer for this suggestion. We have added the absolute number of fibers analyzed per condition in the Methods section.

Comments 8: Please explain how intensity measurements were obtained, including the calibration method used and whether analysis was performed on whole images or restricted to specific regions or fiber types.

Response 8: We thank the reviewer for this helpful comment. We have revised the Methods section to clarify how intensity measurements were obtained. Briefly, images were acquired from the middle costal region of the diaphragm under identical exposure settings within each experiment. PGAM2 fluorescence intensity was quantified using ImageJ by whole-image analysis, rather than by selected fiber types. The same thresholding and background subtraction procedures were applied to all images within the same experiment.

Comments 9: There are typographical errors and concatenated words throughout the manuscript. Authors should carefully proofread the text; examples can be found in lines 168–170, 177, 179, 243, and 275.

Response 9: We thank the reviewer for pointing this out. We have carefully proofread the manuscript and corrected the typographical errors, spacing issues, and concatenated words throughout the text, including the examples noted in the indicated lines.

Comments 10: Line 218: The phrase "Dissolving muscle protein is a key protein that leads to muscle atrophy" is unclear. Please rephrase.

Response 10: We thank the reviewer for pointing this out. We have rephrased this sentence to improve clarity. The revised sentence now reads: "Muscle protein degradation is a key process contributing to muscle atrophy, and the ubiquitin–proteasome pathway plays an important role in this process."

Comments 11: Line 219: The text states that Figure 3A and 3B show both MAFbx and MuRF1, but only MuRF1 appears to be shown. Please clarify.

Response 11: We thank the reviewer for pointing this out. We have corrected the text to accurately match the figure content and clarified the corresponding figure references.

Comments 12: All images appear to be low resolution. Authors should improve image quality and include high-magnification insets of representative fibers. Calibration bars are barely visible and should be made clearly apparent throughout. Zoomed-in regions would also be helpful, as panels E and H appear to represent distinct anatomical regions that are difficult to interpret in the current format.

Response 12: We thank the reviewer for this helpful suggestion. We have re-imported the original high-resolution fluorescence images and improved the overall image quality in the revised figures. The calibration bars have also been enlarged and made more clearly visible throughout the figure. In addition, we have clarified in the Methods section that all diaphragm images were obtained from the middle costal region under the same magnification to ensure anatomical consistency. Although we did not add separate high-magnification insets, the revised higher-resolution images allow clearer visualization of representative muscle fibers and staining patterns.

Comments 13: Representative images of proteins that are downregulated or unchanged in the model should be included.

Response 13: We thank the reviewer for this suggestion. In the revised manuscript, we have improved the presentation of the representative images for the key proteins showing significant changes in the CLP model. However, representative images for downregulated or unchanged proteins were not included as separate panels in order to keep the figure concise and focused on the main findings. The quantitative data for these proteins are presented in the corresponding figure panels to support the interpretation.

Comments 14: RT-qPCR: please indicate the reference gene(s) used for normalization.

Response 14: We have added the reference gene(s) used for normalization in the RT-qPCR section of the Methods.

Comments 15: Figure 2: Statistical significance should be clearly indicated. Calibration bars are not visible. If expression is normalized to tubulin, data should be presented as fold-change or percentage, with units clearly stated. Panel 2E should show a more representative image, as the current one displays an apparently severe atrophic phenotype. Panel 2B: IL-1 and IL-6 labels should be placed above the plots rather than on the x-axis to avoid confusion. Additionally, are the TNF-α concentrations used in vitro comparable to those reported in vivo?

Response 15: We thank the reviewer for the constructive comments. We have revised Figure 2 as follows: statistical significance is now clearly indicated; calibration bars have been made visible; the normalized expression data (to tubulin) are now presented as fold-change with units clearly stated; a more representative image has been provided for panel 2E; the labels for IL-1 and IL-6 have been moved above the plots; the TNF-α concentrations used in vitro were determined through preliminary cell-based experiments, and they are consistent with those reported in the literature (DOI: 10.3390/ijms23073878). We have now added the TNF-α concentrations in the figure legend of Figure 2 for clarity.

Comments 16: Figure 3: Panel 3C appears redundant with 3D, as the control condition is already shown and may be omitted. Panel 3E: experimental conditions should be clearly labeled above each panel. Panel 3G: contrast and gamma correction should be improved, as cell morphology and DAPI staining are difficult to visualize.

Response 16: We appreciate the reviewer's suggestion. However, we respectfully prefer to retain Panel 3C because it more intuitively demonstrates the successful knockdown of PGAM2, which is a key experimental validation. The experimental conditions for Panel 3E are already described in the figure legend and in the main text. We have uniformly adjusted the brightness and contrast for all panels in Figure 3, including Panel 3G, to improve the visibility of cell morphology and DAPI staining. We have now added the TNF-α concentrations in the figure legend of Figure 3 for clarity.

Comments 17: Figure 4: "In vitro gain-of-function" should be explicitly stated in the figure legend.

Response 17: We have explicitly stated "in vitro gain-of-function" in the corresponding figure legend as requested.

Comments 18: Figure 5: What is the effect of TNF-α in the presence of antisense PGAM2? Is the signaling pathway altered under these conditions?

Response 18:  Thank you for this important question. As shown in revised Figure 5, PGAM2 knockdown using antisense siRNA significantly reduced CLP- and TNF-α-induced phosphorylation of JAK2 and STAT3, indicating that the JAK2/STAT3 signaling pathway is altered in the presence of antisense PGAM2. These findings suggest that TNF-α-induced signaling activation is at least partially dependent on PGAM2 expression. We have clarified these results in the revised manuscript and added corresponding limitations in the Discussion.

Comments 19: Figure 6: The data presented are comprehensive; however, the lack of gain- or loss-of-function experiments substantially limits the validation of the proposed hypothesis. It is unclear why gain- and loss-of-function experiments were not performed in vivo to validate the in vitro findings. Including such experiments would substantially strengthen the manuscript's conclusions.

Response 19: We thank the reviewer for these important comments. Based on the current experimental design, we are unable to definitively answer whether TNF-α alters the signaling pathway in the presence of antisense PGAM2 or to validate the hypothesis through in vivo gain- or loss-of-function experiments; therefore, we have added this limitation to the Discussion and clarified that further mechanistic in vivo studies are needed.

 

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Thank you for the thorough and constructive responses to my review; you have effectively addressed each of my concerns, and the manuscript is significantly improved as a result. I particularly appreciate the inclusion of specific quantitative data in the abstract and the correction of the critical inconsistencies in the figure legends regarding the direction of PGAM2’s effects, which were essential for the integrity of your findings. Your transparency regarding sample size limitations and the inclusion of siRNA sequences greatly enhances the reproducibility and rigor of the study. Furthermore, the expanded discussion on metabolic remodeling and the "Warburg-like" shift in septic muscle provides a much-needed theoretical framework that reconciles the upregulation of a glycolytic enzyme with an atrophic state. While I acknowledge that the PGAM2–JAK2/STAT3 axis remains partly exploratory and would benefit from direct functional force measurements in the future, you have appropriately addressed these points as limitations and supported your claims with the newly provided preliminary Co-IP data. The manuscript is now much clearer and well-supported, and I have no further concerns.

Reviewer 5 Report

Comments and Suggestions for Authors

Overall, the authors have improved the article by incorporating several of the suggested comments, although the more substantial ones were not addressed, which is understandable given the amount of work they would require. The manuscript provides an interesting perspective on accelerated muscle fiber atrophy due to inflammation. Although the mechanistic basis of this biological phenomenon remains underdeveloped, the topic is highly relevant. Thus, I believe this descriptive work is worthy of publication in its current form.

Comments on the Quality of English Language

Authors have improved the text, and their outcomes.