Electronic Cigarette Exposure Induces Adverse Cellular Alterations in Skeletal Muscle in Male Mice Subjected to a High-Fat Diet

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Electronic-Cigarette Exposure Induces Adverse Cellular Alterations in Skeletal Muscle in Male Mice Subjected to a High-Fat Diet

Thank you for the opportunity to review this interesting manuscript, which investigates an important and timely topic. The study design to probe the interaction between E-Cigarette exposure and diet is a strength. Below are comments aimed at further strengthening the manuscript.

Abstract:

• The authors should consider revising the self-reference to 'Our laboratory' to a more general statement about the existing literature. In this sentence (Our laboratory demonstrated the adverse effects of E-Cig on the liver and heart, but their effects on skeletal muscle have not been well studied)
• I recommend integrating the high-fat diet context into the aim sentence.
• Please clarify the scientific rationale for including the Normal Chow Diet (NCD) group in the methodology.
• Please specify the exact skeletal muscle (or muscles) that were harvested and analyzed in this study.
• Please specify the exact analytical techniques used to evaluate skeletal muscle alterations.
• Please define the abbreviations "LC3-I" and "LC3-II" in full upon their first use in the text.
• Please revise the sentences to eliminate the repetitive use of "additionally’’ and also “In addition” in the introduction.

Introduction:

• Page 2, line 61, please remove an editing error differences
• Page 2, lines 64 and 65: please modifythe  sentence into (Skeletal muscle is essential for maintaining a healthy quality of life, enabling physical activity, and reducing the risk of metabolic and chronic diseases).
• Page 2, lines 87 to 90: (In the present study, using a model of HFD-induced obesity in mice, we tested the hypothesis that nicotine delivered via E-Cig and HFD will cause skeletal muscle abnormalities, while E-Cig plus normal chow diet (NCD) will be without effect on several muscle parameters) The hypothesis that "E-Cig plus NCD will be without effect" requires clarification, as it is central to the study's conclusion. Please clarify the scientific value of this finding. If the result is indeed null, does this imply that E-Cig use is only harmful in the context of pre-existing metabolic stress (e.g., obesity), and is this a novel and significant insight for public health?
• The Discussion would be significantly strengthened by the addition of a dedicated paragraph that synthesizes the findings for the specific parameters investigated.

 

Methode

• Please provide a clear scientific rationale for the exclusive use of male mice in this study, as metabolic and musculoskeletal pathophysiology, areas where significant sex differences are well-documented.
• Specify the material of the cage used in the experiment.
• The use of a 0% nicotine, flavored E-Cigarette group as a control requires further justification and discussion. The study design correctly uses this group to isolate the effects of nicotine. However, a growing body of literature indicates that the chemical constituents of E-Cigarette flavorings (e.g., aldehydes, volatiles) can themselves induce cellular toxicity and oxidative stress, independent of nicotine.
• The manuscript should include data on body weight progression throughout the study. To confirm the successful induction of obesity and metabolic dysfunction in the HFD groups, it is essential to present the body weights of the mice.
• Please specify the exact skeletal muscles that were harvested and analyzed in the Methods section.
• The manuscript would be greatly enhanced by the inclusion of a graphical timeline or table summarizing the experimental design.
• Please improve the legibility of text in Figure 5, especially by ensuring a high-contrast color scheme (small white text inside black boxes).
• Please add a conclusion section.
• Please add a list of abbreviations.

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This paper describes a study in which male mice were exposed to e-cigarettes and a high fat diet to determine the effects on skeletal muscle.  The authors conducted an evaluation of the soleus muscle for p-AMPK, total AMPK, p-38 MAPK, SOD2, skeletal muscle triglycerides, and mitochondrial damage.  Strengths of the paper include a testable hypothesis and some data to support the notion that HFD exacerbates the effects of e-cigarette exposure.

The following comments are provided to strengthen the paper:

Abstract

Lines 23-25: the abstract is not clear as written.  For example, the study could be described as "C57BL/6 mice fed either a normal 24 chow diet (NCD) or high fat diet (HFD) were exposed to 1) saline aerosol control, 2) E-Cig 0%, or 3) E-Cig 2.4% nicotine, respectively) for 12 weeks."

Line 24: what does "Blue PLUS E-Cig, 0% or 2.4% nicotine, respectively" indicate?

Line 25: consider grammar for this sentence (i.e., the skeletal muscle itself was not directly exposed to the E-cig 2.4%)

Line 31: in addition to the abstract, it is not clear why the authors only evaluated mitochondrial structure and did not conduct any mitochondrial functional assays.

Introduction:

Line 40: replace comma with period after "nicotine".

Line 46: what is meant by "6.1% and 2%" (i.e., these numbers increased from what?)

Line 47: "now affecting a wider range of ages" is unclear

Line 50: this is not clear (did you mean that the mice were on the Western diet, not the hepatic steatosis)

Lines 58 - 59: this sentence seems out of place.

Line 61: change to "less engaged" and state compared to who

Line 61: differences is crossed out, which must be a typo

Line 68: "unhealthy" is a subjective term - consider removing

Line 83: do the authors compare the metabolism associated with nicotine administered i.p. versus inhalation in the discussion of the paper?

Methods

Line 101: did the mice gain weight with the HFD?

Line 103: why do the authors say "In some experiments"?

Lines 107 - 109: the experimental paradigm does not seem equivalent to a human scenario.  Could the authors please justify this exposure design?

Line 118: why was the soleus only utilized; suggest that the authors also evaluate a mixed muscle type and a fast twitch as well; in addition, hopefully other tissues were harvested for further study

Line 120: while Western blots are still in use, these types of analyses do not get to the mechanisms - were any other analysis conducted (e.g., PCR?)

Line 159: what was the TEM conducted for (implied for mitochondrial structure analysis, but this should be stated in the text)?

Results: 

Line 183: why did the authors decide to place the triglyceride data in the supplement?

Figures: all of the figures should be resubmitted as high resolution (i.e., they appear fuzzy to the reader), especially Figure 4.

Line 205: it appears that the authors are including previously published data in the paper.

Line 234: was this data collected under the same exposure scenario?

Discussion:

Line 290: could the authors elaborate on how they think the HFD is exacerbating the effects of the E-cig exposure?

Line 296: did the authors measure the serum-free fatty acids in this study?

Line 300: it is not clear why the authors mentioned a lipolysis inhibitor in this section.

Lines 313 - 315: it is not clear if these variables were measured in this study.

Line 325: please clarify "old proteins, cellular trash" - this does not sound very scientific.

Line 334: mitochondrias is a typo

Line 337: why is the text right justified?

Line 339: change "less resistance to" to "more"

Lines 341 - 342: not sure that this point adds anything to their hypothesis

Lines 346 - 354: Could the authors briefly describe how their system differs from what other researchers are using.  It is very frustrating when authors tell the reader to refer to other papers.

Line 355: change "it's" to " which is an"

Lines 357 - 358: this sentence is unclear

Lines 361- 363: this part of the sentence is unclear

Line 366: what evidence do the authors have to speculate that the propylene glycol and glycerol components affect muscle?

Line 375: change "in a future" to "in the future"

Lines 384 - 386: it is not clear why this text was included - suggest removing.

Lines 393 - 395: unsure why this text is necessary

Line 396: remove "deglamorization of E-Cig use" - this is not very scientific.

Line 400: this figure seems out of place.  In addition, generally at the end of a paper, there are overall conclusions, which were not clearly provided in this paper.

 

 

 

Comments on the Quality of English Language

The paper should be revised for grammatical and structural issues as well as the scientific premise (i.e., it appears that the authors are including previously published data in the paper and unclear if some of the data was collected using the same exposure scenario).  The authors may want to work with an scientific writer to improve the content of the paper.

Author Response

Reviewer 2

Comments and Suggestions for Authors

This paper describes a study in which male mice were exposed to e-cigarettes and a high fat diet to determine the effects on skeletal muscle.  The authors conducted an evaluation of the soleus muscle for p-AMPK, total AMPK, p-38 MAPK, SOD2, skeletal muscle triglycerides, and mitochondrial damage.  Strengths of the paper include a testable hypothesis and some data to support the notion that HFD exacerbates the effects of e-cigarette exposure. The following comments are provided to strengthen the paper:

 

Response: We appreciate the reviewer’s comments, which improved our manuscript. Our changes are made with tracking to facilitate re-review of the manuscript.

 

Abstract

Comment: Lines 23-25: the abstract is not clear as written.  For example, the study could be described as "C57BL/6 mice fed either a normal 24 chow diet (NCD) or high fat diet (HFD) were exposed to 1) saline aerosol control, 2) E-Cig 0%, or 3) E-Cig 2.4% nicotine, respectively) for 12 weeks."

Response: The sentence has been corrected as “C57BL/6 mice, fed either a normal chow diet (NCD) or HFD, were exposed to either saline aerosol control or aerosol generated from Blu PLUS^TM containing 0% or 2.4% nicotine for 12 weeks” (lines 23-25).

 

Comment: Line 24: what does "Blue PLUS E-Cig, 0% or 2.4% nicotine, respectively" indicate? Blu PLUS

Response: We apologize for the ambiguity and the misspelling. It has been corrected as “C57BL/6 mice, fed either a normal chow diet (NCD) or HFD, were exposed to either saline aerosol control or aerosol generated from Blu PLUS^TM e-cigarettes containing 0% or 2.4% nicotine for 12 weeks.”

 

Comment: Line 25: consider grammar for this sentence (i.e., the skeletal muscle itself was not directly exposed to the E-cig 2.4%).

Response: We have corrected the sentence as “The skeletal muscle from HFD-fed mice exposed to E-Cig 2.4% had ....” (line 29-30).

 

Comment: Line 31: in addition to the abstract, it is not clear why the authors only evaluated mitochondrial structure and did not conduct any mitochondrial functional assays.

Response: We appreciate the reviewer's comment. We are seeking additional funding to perform these important functional experiments, which require fresh tissue.

 

Introduction:

Comment: Line 40: replace comma with period after "nicotine".

Response: We thank you for the correction, and we have changed it (line 45).

 

Comment: Line 46: what is meant by "6.1% and 2%" (i.e., these numbers increased from what?)

Response: We corrected and rewrote the sentence as “In fact, E-Cig consumption in young adults (18-24 years) increased from 2.7% in 2017 to 10.3% in 2023 and in adults (25-44 years) from 1.5% in 2017 to 6.1% in 2023” (lines 50-52).

 

Comment: Line 47: "now affecting a wider range of ages" is unclear.

Response: We revised the sentence as “These trends pose a national health problem, affecting a wider age-range of E-Cig users, from 18 to 44 year-olds.” (lines 52-53).

 

Comment: Line 50: this is not clear (did you mean that the mice were on the Western diet, not the hepatic steatosis).

Response: We corrected the sentence as “In animal models on a high-fat diet (HFD), E-Cig have been shown to produce detrimental consequences on metabolic functions, including hepatic steatosis and …“ (lines 54-56).

 

Comment: Lines 58 - 59: this sentence seems out of place.

Response: We corrected the sentence (line 60-61).

 

Comment: Line 61: change to "less engaged" and state compared to who

Response: We thank you for the suggestion, and we have made the change as “adolescents, who use E-Cig, are less engaged in moderate to vigorous physical activities compared with never-users” (lines 62-64).

 

Comment: Line 61: differences is crossed out, which must be a typo

Response: We thank you for the correction, and we have deleted it.

 

Comment: Line 68: "unhealthy" is a subjective term - consider removing

Response: We thank you for the correction, and we have deleted it.

 

Comment: Line 83: do the authors compare the metabolism associated with nicotine administered i.p. versus inhalation in the discussion of the paper?

Response: We did not perform a head-to-head metabolic comparison between routes. However, in the Discussion section, we referenced prior i.p. nicotine + HFD effects (liver/heart/muscle) to contextualize our present findings. We also noted that our inhalation system yields human-like nicotine pharmacokinetics. We added a clarifying sentence to state this explicitly (line 297-300).

 

Methods

Comment: Line 101: did the mice gain weight with the HFD?

Response: This is a very pertinent and thoughtful comment. Yes, we recorded weekly body weight throughout the study. The HFD groups exhibited significant weight gain, we have published this information in Ref. #2 and manuscript Ref. #37 (Hasan et al, PLoS One, 2020).

 

Comment: Line 103: why do the authors say "In some experiments"?

Response: We have removed “in some experiments” to avoid confusion.

 

Comment: Lines 107 - 109: the experimental paradigm does not seem equivalent to a human scenario. Could the authors please justify this exposure design?

Response: Thank you for raising this. Our exposure design had been studied and validated to approximate human vaping patterns and doses³ (manuscript Ref. #27). This design mimics human E-Cig behavior (vaping episodically during the 12-hour exposure) with increased plasma nicotine and cotinine concentrations.

 

Comment: Line 118: why was the soleus only utilized; suggest that the authors also evaluate a mixed muscle type and a fast twitch as well; in addition, hopefully other tissues were harvested for further study.

Response: We appreciate the reviewer’s suggestion. Given that the published data from our group demonstrated that nicotine injections damaged the mitochondria, we selected the soleus as the first target due to its high concentration of mitochondria (oxidative muscle), making it particularly sensitive to mitochondrial insults. The primary objective of this manuscript was to assess whether E-Cig exposure, as a novel nicotine delivery model produces similar damage to nicotine injections. The mixed muscles (tibialis anterior and gastrocnemius) will be analyzed in the further studies to broaden the relevance of our initial findings.

 

Comment: Line 120: while Western blots are still in use, these types of analyses do not get to the mechanisms - were any other analysis conducted (e.g., PCR?)

Response: We agree that mechanistic depth benefits from multi-modal approaches. In addition to Western blot analysis of activity pathways (p-AMPK, p-ATGL, p-p38; LC3-I/II; p62; and antioxidant enzyme levels), we performed TEM and intramuscular biochemical quantification of triglycerides. We did not perform qPCR analysis in this study because our primary endpoints focused on post-translational regulation. The soleus is a small muscle, limiting parallel extraction for all modalities without compromising key assays. However, we will be analyzing the tibialis anterior and gastrocnemius using RT-qPCR and include in a follow-up manuscript.

 

Comment: Line 159: what was the TEM conducted for (implied for mitochondrial structure analysis, but this should be stated in the text)?

Response: Thank you for this suggestion. We used TEM specifically to evaluate mitochondrial ultrastructure. We have added explicit statements in Methods (line 172) and in Results (lines 276-277).

 

Results

Comment: Line 183: why did the authors decide to place the triglyceride data in the supplement?

Response: Thank you for the suggestion. We treated intramuscular triglycerides as a supporting/secondary endpoint—used to contextualize the signaling (p-AMPK/p-ATGL)—so we placed it in the Supplement to preserve flow in the main Results. The triglyceride data are fully reported (means ± SEM and statistics) and mirror the main conclusions (higher triglycerides with HFD). If the reviewer prefers, we will be happy to move this panel into the main figures (e.g., as an additional panel in Figure 1).

 

Comment: Figures: all of the figures should be resubmitted as high resolution (i.e., they appear fuzzy to the reader), especially Figure 4.

Response: We apologize and have improved the quality of the figures in the resubmission.

 

Comment: Line 205: it appears that the authors are including previously published data in the paper.

Response: Thank you for raising this. All figures and quantitative results in this manuscript are original and were generated from the animals described herein. Prior publications are cited only to justify the exposure paradigm and to contextualize our findings.

 

Comment: Line 234: was this data collected under the same exposure scenario?

Response: We thank the reviewer for this necessary clarification. Yes, all data presented in this manuscript were collected under the same exposure protocol. Animals exposed to saline, E-Cig 0% nicotine, and E-Cig 2.4% nicotine were under identical conditions, including chamber settings and exposure duration. This consistency ensured that the differences were attributed to the treatment groups.

 

Discussion

Comment: Line 290: could the authors elaborate on how they think the HFD is exacerbating the effects of the E-cig exposure?

Response: We appreciate the request for clarification. We have added in Discussion that HFD acts as the metabolic “second hit” that unmasks E-Cig 2.4% effects, i.e. E-Cig 2.4% + HFD compared with E-Cig 2.4% + NCD affected coherent stress signature p-AMPK and p-ATGL suppression, oxidative stress with p-p38 activation, reduced autophagy (LC3-II reduction; p62 increase), and mitochondrial damage, indicating that diet-induced obesity amplifies nicotine/aerosol damage. We have now consolidated these points into a brief paragraph to make the mechanism explicitly (lines 320-324).

 

Comment: Line 296: did the authors measure the serum-free fatty acids in this study?

Response: This is a very pertinent and thoughtful comment. Yes, we assessed serum-free fatty acids (FFA) in this experimental set. The E-Cig 2.4% nicotine plus HFD group exhibited a significant increase in the FFA; we have published this information². Our publications have shown an increase in the FFA after E-Cig 2.4% nicotine exposure^4,5.

 

Comment: Line 300: it is not clear why the authors mentioned a lipolysis inhibitor in this section.

Response: We thank you for noting this. The reference to acipimox (a lipolysis inhibitor) is contextual, citing prior work showing that pharmacologic suppression of lipolysis attenuated nicotine-associated injury, thereby supporting our mechanistic interpretation that nicotine increased circulating free fatty acids, worsened outcomes under HFD. It was not used in the present experiments. We have revised the sentence to make this explicit and relocated it to the mechanistic synthesis in the Discussion (lines 318-320).

 

Comment: Lines 313 - 315: it is not clear if these variables were measured in this study.

Response: We appreciate the meticulous review. We measured the protein levels of HO-1, SOD1, and SOD2 as oxidative stress markers (Fig. 2A, B, C, and D).

 

Comment: Line 325: please clarify "old proteins, cellular trash" - this does not sound very scientific.

Response: We thank you for the correction, and we have deleted this citation (lines 347-350).

 

Comment: Line 334: mitochondrias is a typo

Response: We thank you for the correction, and we have edited it (line 355).

 

Comment: Line 337: why is the text right justified?

Response: We apologize for the formatting issue. The right justification is an artifact from the template conversion during journal formatting. We will correct the paragraph to the journal’s standard in the revised manuscript.

 

Comment: Line 339: change "less resistance to" to "more".

Response: We have improved the sentence to clarify it. “E-Cig users demonstrated reduced endurance capacities and earlier onset of fatigue during performance testing and higher levels of lactate compared to non-smokers” (lines 361-362).

 

Comment: Lines 341 - 342: not sure that this point adds anything to their hypothesis

Response: We have removed the sentence.

 

Comment: Lines 346 - 354: Could the authors briefly describe how their system differs from what other researchers are using. It is very frustrating when authors tell the reader to refer to other papers.

Response: Thank you for this suggestion. We now include a brief description of our exposure system and how it differs from commonly used set-ups (lines 367-373).

 

Comment: Line 355: change "it's" to " which is an"

Response: We thank you for the correction, and we have changed it (line 374).

 

Comment: Lines 357 - 358: this sentence is unclear

Response: We have clarified the sentence to “Oxidative fibers support superior endurance performance, whereas a shift toward glycolytic (type IIb) fibers diminishes exercise capacity” (lines 376-377).

 

Comment: Lines 361- 363: this part of the sentence is unclear

Response: We have clarified the sentence to “effects of E-Cig on the proportion of the muscle fiber types and exercise endurance are still not clear” (lines 379-380).

 

Comment: Line 366: what evidence do the authors have to speculate that the propylene glycol and glycerol components affect muscle?

Response: Our speculation is based on the effects observed with the E-Cig 0% nicotine group, the published chemistry showing that heating propylene glycol and vegetable glycerin generates toxic aldehydes (e.g., acrolein, formaldehyde) during vaping⁶, and published data demonstrating direct adverse effects of these aldehydes on skeletal muscle cells⁷.

We have rewritten our sentence and added the references #53 and 54 in the manuscript (line 383-387).

 

Comment: Line 375: change "in a future" to "in the future"

Response: We thank you for the correction, and we have changed it (line 393).

 

Comment: Lines 384 - 386: it is not clear why this text was included - suggest removing.

Response: We have removed the sentence.

 

Comment: Lines 393 - 395: unsure why this text is necessary

Response: We posit that this sentence is necessary to highlight the public health issue and the translational relevance of our findings (now lines 401-402).

 

Comment: Line 396: remove "deglamorization of E-Cig use" - this is not very scientific. “reducing the perceived appeal of E-Cig use”.

Response: We thank you for the correction, and we have changed it to “for reducing the perceived appeal of E-Cig use” (line 403).

 

Comment: Line 400: this figure seems out of place. In addition, generally at the end of a paper, there are overall conclusions, which were not clearly provided in this paper.

Response: Thank you. We have added the conclusion section (lines 408-418).

 

Comments on the Quality of English Language

Comment: The paper should be revised for grammatical and structural issues as well as the scientific premise (i.e., it appears that the authors are including previously published data in the paper and unclear if some of the data was collected using the same exposure scenario). The authors may want to work with an scientific writer to improve the content of the paper.

Response: We carefully reviewed the revised paper for grammatical and structural issues and posit that it is much improved.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The study evaluates the effect of e-cigarettes on the musculoskeletal system in a mouse model.

The authors must justify the number of animals used in the study and show the resource calculation according to the established formula in the manuscript.
Furthermore, the authors must also show the ARRIVE form, which describes all animal handling procedures.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

In this study, the authors investigated the changes induced by the nicotine delivered by e-cigarettes to skeletal muscle structure. For the study, C57BL/6 mice fed a high fat die or a chow diet were exposed to aerosol containing saline, E-Cig 0%, or E-Cog 2.4 % 12 hours/day for 12 days. The results provided in the manuscript indicate that E-Cig 2.4% reduced phospho-AMP and phospho-adipose tryglyceride lipase only in the presence of a HFD. These impairments were associated with an increased oxidative stress and increased expression of phospho-p38 MAPK, which resulted in autophagy deregulation, decreased LC3-II/LC3-I ratio, p62 accumulation, and increased mitochondrial damage. The conclusion of the authors is that exposure to nicotine through e-cigarette damages skeletal muscles in addition to other organs as liver and heart.

Comments: The study is properly planned and executed. The manuscript is properly written. The data are properly reported and the conclusions of the authors is consistent with the data reported here. 

Title: it conveys the scope of the study clearly

Abstract: it summarized properly the background, the material, the results and the conclusions of the study. 

Materials and Methods: provide sufficient amount of details to reproduce the study.

Statistical analysis: appropriate.

Figures and Results: The figures are clear and the Results can be followed and appreciated easily. 

Discussion: appropriate

Minor comment: Do the authors have any data suggesting that CD36 and FATP protein expression and/or activity is increased in the skeletal muscle to result in the micro-myosteatosis observed by the authors? 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf