Skeletal Muscle Androgen-Regulated Gene Expression Following High- and Low-Load Resistance Exercise

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript examines the role of volume equated high load (HL) and low load (LL) resistance exercise (RE) on serum and intramuscular androgen related variables. Overall, the research question is novel and important. I commend the authors. On this study due to the significant time and resources required to pull this off. In addition, the question elucidates an often-contested area of debate in our filed on 1) the role of RE load in adaptations, and the role of endocrine responses (androgens) and their contribution to RE adaptations. I believe this manuscript would provide much needed area of investigation to address the molecular mechanisms on these topics.

Given this, I must emphasize there are numerous instances in the manuscript that require additional detail and clarification, particularly in the Methods and Results sections.  There are also other places that require citations for statements, and on one instance misinterpretation of research findings where the reference was used. Significant revision is needed. However, I believe this paper would add value to the field if many of the points below are clarified.

Biggest question:  For the blood and muscle TST, AR, and DHT data, were samples 1) assayed again with remaining tissue from the Cardaci 2020 study, or 2) are the values here re-reporting the data that was published in Cardaci 2020 and using the values from that paper?

If all these values are from new experiments on remaining tissue, then you need to provide the inter- and intra-assay coefficient of variation for these variables, and present them graphically in the manuscript (currently none of the intramuscular TST,DHT, AR protein are presented). As is, it is clear myogenic gene expression data are a new analysis. However, for the serum and intramuscular TST, AR, DHT, and AR-DNA binding data it is not clear where the data were obtained (additional analysis of remaining tissue vs just using the values from Cardaci et al. (2020)). Clarification, and additional details in these points in the manuscript would greatly address many of the comments provided below.

ABSTRACT

Line 36: “This reinforces prior LBM-strength relationship evidence, …” This is evidence of…?  LBM & Strength, or Endocrine concentrations & strength? Or both?  Could edit to be more specific here.

INTRODUCTION

Line 45: “Each emphasizes physical attributes — hypertrophy, maximal strength, and/or explosive power — along a performance spectrum.” add "respectively" here to clarify. BB=hyp, PL=strength, OWL=power.

Lines 52-52: Add References here. A review and an original research article.

Line 84: Typo “following HL versus LL RE but focused more the potential individual”. Add "on".  Focused more (on) the...

METHODS

Line 191: “vastus lateralis muscle on each participant’s dominant leg using the TRU-CORE 1..”. What gauge biopsy needles were used for this device?

Line 204: what speed and duration were the samples centrifuged at?

Line 205: Were the blood samples taken at 3hr and 24hr (next morning) also fasted? If so, for how long?

Lines 208-215: 2.8 Serum and Intramuscular Hormone Analysis. There are numerous things requiring clarification here.

Line 209: “As previously described [13], Serum androgenic hormone concentrations (both free and total TST) were measured using enzyme-linked immunosorbent assay (ELISA) kits (MyBiosource, San Diego, CA, USA).”

But this is incorrect, the Cardaci et al. 2020 says “these hormones were analyzed with different kits "(Eagle Biosciences Incorporation, Nashua, NH, USA". So were your serum TST (free & total) measured with kits from MyBiosource?, or were they from Eagle Biosciences?

Lines 212-213: “Similarly, intramuscular AR, TST, and DHT levels were assessed by the same ELISA kits.”

Which kits? In the methods from Cardaci 2020:

AR kit = MyBioSource Incorporation, San Diego, CA, USA);

TST kit = Eagle Biosciences Incorporation, Nashua, NH, USA;

DHT kit = Eagle Biosciences Incorporation, Nashua, NH, USA.

So from this current manuscript’s methods you say serum TST was measured with kit from MyBiosource, and then say intramuscular TST was measured with the same kit. But intramuscular TST (in Cardaci) says the kit was from Eagle Biosciences. So which kits and companies were used to measure the serum and muscle androgenic data? This is especially confusing because as stated earlier it is not clear if this is 1) re-analysis of tissue, which would require new kits, or 2) re-reporting values from Cardaci 2020, which would suggest you used the companies and kits from that paper. But if this were the case the serum TST kits should be the same but here they are different companies.

Even more importantly, you did not report any inter- and intra-assay CVs for your ELISAS. These are required to include in this manuscript if they were new experiments done on remaining tissue.

Having reviewed the ELISA protocols, you should also include methods on muscle sample preparation for the assays. You do not include that information currently. The sample collection procedures suggested that assay performance is best on fresh samples, but the samples were frozen first (which would be expected in our field). Did you add protease inhibitors to the sample preparation for AR protein ELISAs?

Moreover, there are no AR-DNA binding activity methods provided in the methods section.  I assume you measured these with kits again, but they were not mentioned in the methods although you report AR-DNA binding data in the results.  Please include the AR-DNA binding activity methods as well.

Line 265: Were the data analyzed for normality? Was there an a priori power analysis performed? There was no mention in the Cardaci 2020 paper about a power analysis. Testing for normality would be essential for RMANOVA considering the sample size. Also normality testing would influence whether Pearson Correlations or Spearman Correlations should have been used for certain correlational analyses.

Line 268: What about intramuscular AR protein, TST, and DHT? Were these variables also run with a 2X3 RMANOVA? (I assume yes, but this wasn’t stated in the stats section).

Line 271: Why was a Tukey post hoc used here instead of Bonferroni like in Cardaci et al. 2020?

You could also use Holm’s sequential Bonferroni procedure for post-hoc comparisons. Not a requirement for these revisions, it but may offer an intermediary adjustment between Tukey and Bonferroni given the number of comparisons performed with this type of design.  I’ve provided the citation and abstract to paper explaining the procedure below.

Eichstaedt KE, Kovatch K, Maroof DA. (2013) A less conservative method to adjust for familywise error rate in neuropsychological research: the Holm's sequential Bonferroni procedure. NeuroRehabilitation. 32(3):693-6. doi: 10.3233/NRE-130893.

Abstract

Background: The traditional Bonferroni method is a commonly used post hoc hypothesis test to adjust for familywise error rate inflation; however, a less well-known derivative of this test, the Holm's sequential procedure, provides an alternative method for familywise error rate correction. This less conservative approach is particularly relevant to studies investigating neuropsychological functioning because of the extent to which neuropsychological datasets tend to include interrelated outcome measures, reducing the relevance of the universal null hypothesis assumption, on which the traditional Bonferroni method relies.

Methods: Mathematical illustrations demonstrating how to compute the two adjustments are provided. The two methods are compared using a simple hypothetical dataset.

Results: By using the divisors (n - j + 1) in lieu of n, Holm's sequential procedure is guaranteed to never reject fewer hypotheses than the traditional Bonferroni adjustment.

Conclusions: The Holm's sequential procedure corrects for Type I error as effectively as the traditional Bonferroni method while retaining more statistical power. Although the Holm's sequential procedure is more complicated to compute than the traditional Bonferroni method, the Holm's sequential procedure may be a more appropriate method for adjusting familywise error rate inflation in many types of neuropsychological research.

Line 274: “lat pulldown and unilateral leg press 10RM, serum hormone markers (free TST, TST and DHT”. Based on your methods looks like only these are serum. DHT is intramuscular. Need to differentiate that here.

Line 279: The Results section is missing the results from AR protein, intramuscular TST and DHT. Need to present those variables here in the results as well (preferably as a figure).

Line 290-291: Not necessary to report these results exact p-values here since this was already stated in the results text above.

y-axis label should include /B-actin next to gene since all the values were normalized to a HKG.

Line 290: Asterisks (*) denote statistically significant findings, whereby..” Should edit to "statistically different from PRE at p<0.05)".

Line 294: Why were there no correlations done between myogenic genes and TST,AR,DHT as well?

Line 299: Did not include AR-DNA binding activity in your methods. Need to include the methods for that assay in the methods, and report the results in AR-DNA binding activity in the results section.

Line 301: Intramuscular DHT and AR protein results should be reported in the results section (as a figure).

Line 301: How were these correlations on the serum and intramuscular values performed? Did you pool all timepoints (PRE, 3HR,24HR) and loadings (HL & LL) together or were these correlations performed only at specific post exercise timepoints? It’s not clear what values were used for correlational analysis.

Line 311:

In the correlation matrix AR-DNA Binding Activity and AR protein had a r=0.526, but were not significant? Also, serum total TST and muscle TST had a r=-0.615, but were not significant?  

This is interesting since these are some of the highest Pearson correlation r's in the correlational data set (r=0.526 & r=-0.615), besides BP, LP, LBM, TST (r=0.909, r=0.705, r=0.605). 

Moreover, the (r=0.526 & r=-0.615) were not significant, while other correlations you reported had much lower r’s but were significant (r=0.28, r=0.33, 0.39, r=0.41, r=0.42).

It may be worthwhile to double check and verify that [AR-DNA Binding Activity vs. AR protein; r=0.526] and [serum total TST vs. muscle TST; r=-0.615] were indeed not significantly related. I only recommend this due to the r’s being relatively higher compared to the other significant androgen relationships reported.

Were the serum TST values that correlated with LBM the resting TST at PRE, or values at 3HR, or 24HRs? Were they AUC, or were they % increase from PRE values? Not clear here.

While the correlation matrix and heatmap show all of the correlations performed (55 total), I suggest to present the 8 significant correlations as scatterplots with individual panels (A-H) so the readership can see the linearity of the relationships visually.

Alternatively, you could present significant relationships with common variables on the same scatterplot such as one variable on the x-axis, one other variable the primary y-axis, and the third variable on the secondary y-axis with trendlines showing the line of best fit between the two pairs of relationships.  

For example:

Muscle DHT (x-axis), AR protein (left y-axis), muscle TST (right y-axis); then two trendlines (1. muscle DHT vs AR protein), (2. Muscle DHT vs Muscle TST).

LBM (x-axis), LP 1RM (left y-axis), BP 1RM (right y-axis); then two trendlines (1. LBM vs LP1RM), (2. LBM vs BP1RM).

Serum Free TST (x-axis), AR mRNA (left y-axis), AR-DNA Binding (right y-axis); then two trendlines (1. Serum Free TST vs AR mRNA), (2. Serum Free TST vs AR-DNA Binding).

DISCUSSION

Line 326: “…a concept supported by Willoughby and Taylor [8], who demonstrated temporal downregulation of AR following sequential bouts of RE.”

This is not correct and this sentence needs to be revised.  Actually Willoughby & Taylor (2004) show the opposite. I’ve provided the excerpt from the discussion in the original paper Willoughby & Taylor (2004) here:

"Our present results illustrate that heavy resistance exercise in humans resulted in increases in AR protein expression of 40% and 100% 48 h after the first and second exercise bouts respectively. However, a peak increase in AR protein expression of 202% (P <0.05) occurred 48 h after the third exercise bout compared with 6% for the control." 

The response was upregulation, not downregulation as you wrote it. They also showed a similar pattern with AR mRNA increasing sequentially after repeated bouts, not a down-regulation as this sentence suggests that you wrote.

Lines 381-384: These are great points here.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The study entitled “Skeletal Muscle Androgen-Regulated Gene Expression Following High- and Low-Load Resistance Exercise” aimed to investigate changes in gene expression at 3 and 24 hours following two acute resistance training sessions using different protocols (high- and low-load).

The manuscript is well-written and easy to follow. My main comment concerns the statistical analysis—specifically, the correlation between gene expression and other parameters analyzed. The authors did not specify which time point was used for the correlation assessment (i.e., 3 or 24 hours post-exercise) and reported AR mRNA, but not the others. Could the authors clarify these two aspects?

Additionally, have the authors considered including both gene expression changes at “3h vs pre” and “24h vs pre” as two separate variables in the correlation analysis?

Please see below some minor comments/suggestions:

• Lines 103–104: “Light load” and “high-load” were previously defined, so please use LL and HL, respectively.
• Lines 110–116 (“During LL, … LL volume-load”): Consider moving and combining this section with “2.4 Resistance Exercise Protocol.”
• Line 322, Reference 18: It is worth noting that this study analyzed only the 48-hour post-exercise time point.
• I am unsure whether this issue is specific to the reviewer version, but Figure 2 appears to be blurry and of low resolution.

Author Response

see attachment

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The following comments refer to specific lines in the manuscript. There are minimal concerns.

Line 182 How were "hydration levels" measured? What constituted abnormal results?

Line 194 Stab incision first? How was depth determined in the first biopsy, and then how was it controlled subsequently?

Line 202 Vacutainer is a registered trademark, so if that company's tubes were used, the manufacturer (Becton Dickinson) and source should be explained. If not that company, then cite the correct company and source.

Line 202 "guage" should be "gauge."

Line 202 With what anticoagulant in the tube? If no anticoagulant, is 10 minutes sufficient to allow clot formation and clot retraction before separation of serum from the clot?

Line 203 "wherein" should be "after which."

Line 204 Serum or plasma?

Line 212 "levels" should be "concentrations."

Line 215 What are the coefficients of variation for these hormone assays? And what are the validation data for each assay?

References 13, 30, 33, 37 Be consistent in the capitalization of the first word and no others (other than proper names) in titles of articles cited in the references.

Author Response

see attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

I appreciate the thorough and timely revisions made to the manuscript. The authors have addressed all comments and I have no further comments or revisions necessary for acceptance.