Both Acute and Consecutive Days of Formoterol Stimulation Influence Myogenic, Mitochondrial, and myomiR Gene Expression in Human Skeletal Muscle Cells

Round 1

Reviewer 1 Report

Gordon and colleagues present good justification to assess the effect of formoterol and illustrate a detailed explanation on the process of myogenesis and how formoterol treatment may act as an “exercise mimetic”. Thank you for your work on this important topic. However, some work is needed to bring more clarity to the research conducted. Please see a list of points below:

1.    Authors fail to state the experimental n replicate, yet each graph is presented with error bars – this should be addressed.

2.    Have the primers used within these experiments passed MIQE guidelines? If so, this should be stated, along with their efficiency to ensure that they are targeted and not off-target

3.    More information should be given on the cell used within these experiments. If they are purchased through a source, the reference for this source should be given (more than is currently mentioned). Or are they primary? If so, the subject characteristics and method used for isolation should be given.

4.    Authors do not state the passage number of these cells and whether they maintained the cells within an appropriate passage range, as human skeletal muscle cells begin to lose their robustness with passaging – this should be addressed

5. Further to point 4 - given you are comparing mRNA expression on day 6 to day 8 with different conditions, experiments to show how you measured mRNA expression of markers of myotube maturation/myoblast inhibition should be included to prove that Pax7 (for example) was higher at day 1 differentiation to day 6.

6.    Images of myotube differentiation through day 0 to day 8 should be provided. The literature is quite vague on the time it takes to differentiate human skeletal muscle cells, with very typical inter-individual variation on time to differentiate.

7.    The author points to a manuscript currently in review for justification on results presented in this manuscript. Effort should be made to avoid this, particularly when it has yet to be published.

8.    A paragraph on the statistics used in this work should be included within the methods section for clarity.

9.    There is a paragraph titled "gene expression of proteins" - can you elaborate on what this means? I would advise using the term "markers" as opposed to protein, as you have not measured any protein in these experiments.

10. Did you complete a concentration curve for formoterol? - this should be included in the supplementary/emphasized within the "exercise mimetic" section of the methods.

11. To add to point 10, was there an effect of 30nM formoterol on human skeletal muscle cell viability? Was this assessed?- how?

12. The authors reflect on the effect of formoterol on transcriptional markers relating to mitochondrial activity (line 24). More work is needed in order to come to this conclusion. Firstly, only one marker (PGC-1alpha) is assessed to come to this rationale and as authors are aware, PGC-1alpha is a regulator of biogenesis and biogenesis does not always indicate an increase in function/activity. The effect of biogenesis on mitochondrial function is indirect. Given there is no measure of function in this work, the use of this term is misplaced. “Formoterol stimulation promotes PGC-1alpha transcriptional expression” is more appropriate. Secondly, more assessments on mitochondrial biogenesis are needed to be comfortable that this is having such an effect. Further, the relationship between B2AR and PGC-1alpha seems vague and is not explained, given the sub heading and its position within the manuscript, readers could be confused to think B2AR is a mitochondrial marker which in fact it is not – this should be separated.

13.  You briefly discuss chronic/acute exercise and resistance exercise within the manuscript introduction and this is supported by the measure of the likes of mTOR, FOXO & MuRF1. Yet the increase in PGC-1alpha could potentially more encompass an effect on aerobic capacity. Given this is where you noted an increase in transcriptional expression, further analysis of these markers of aerobic capacity (i.e. AMPK signaling, more measures of biogenesis etc.) would be warranted.

Author Response

Point 1: I've included an (n) representing sample size in the methodology. 

Point 2: Per Bustin et al., 2009; the primers used in this study meet MIQE guidelines. I do not have efficiency values available and do not have the capability currently to assess it. 

Point 3 and 4: Cells were purchased through Sigma Aldrich (stated in Methodology). The cells came to us de-identified (Sigma isolate them, not us) and were primary human skeletal muscle myoblasts (passage 4-6 -- which I've included the passage range now in the methodology). 

Point 5: We did not measure expression at Day 1 or earlier (before Day 6) within the myogenic sequence. This was primarily due to the assumption that expression of these markers (e.g., Pax7, MyoD) are expressed early in myogenesis during the proliferative/early-differentiation phases as has been shown/reported in studies previously. We are unable to go back and add these experiments to this study as I no longer have the capability to do so. I will mention that we had measured some of these myogenic markers at various points of myogenesis previously in our lab (observations only) and found typical patterns of sequential expression for these myogenic markers. 

Point 6: Images representing various stages of differentiation have been provided in the methods section. 

Point 7: I agree with your statement here. The information (sentence) is additive to the manuscript, and the information referenced is currently in review (though it's not been accepted yet). So I changed the (currently in review) to (unpublished findings). I've seen this type of context used previously in other manuscripts. 

Point 8: I've included the statistical analysis as a subsection at the end of the methodology. 

Point 9: I agree. I've changed this subtitle "Gene Expression of Proteins" to Gene Expression of Markers Related to Myogenesis, Muscle Growth, and Mitochondrial Activity. 

Point 10: We did not create a concentration curve for formoterol; thus, we cannot provide this information. To clarify, we essentially incubated wells/cells with 30 nM of formoterol for 3 hours on days in which formoterol was used. 

Point 11: We did not directly assess the viability of formoterol on cells in this study. Images were captured at relevant points throughout this study's design to assess cell health, adherence, and growth. 

Point 12: These are fair and understandable points. I've re-phrased some of this material to separate any connection/distinguish between PGC-1a and B2AR in this study.

Point 13: This is a good point. I added a statement addressing this in the discussion. I no longer have the capability/capacity to further assess expression of additional genes (this data is from my dissertation). But I made a point that this should be addressed in future investigations. 

 

Reviewer 2 Report

Gordon et al present a qPCR analysis of Formoterol-induced gene expression changes. The study is interesting but require some revision.

 

 

Here my minor concerns

 

Please include relevant p-values above the graphs (replacing symbols), makes study easier to appreciate.

 

It is unclear why stats on some comparison are missing, eg D8CON vs D8FORM (same for D7).

Comparing D6CON with D7FORM or D8FORM is unfair, as gene expression in CON may fluctuate as cells are kept in culture (ie MYOD, MYOG, PAX7, FOXO3)

 

Explain DxCON vs DxFORM at the start of results

A diagram depicting experimental design at the start of figure 1 would help in understanding data.

Thus, I suggest to place Figure 5 as diagram in Figure 1, to better present the study

 

Authors claim that MAFbx expression is unchanged, but graph shows strong upregulation in D6FORM compared to D6CON, is this not significant?

Author Response

Point 1: As the p values are presented in the figure legends for each individual figure, I don't believe adding the p values to the actual figures adds any additional value to this manuscript. The figures were generated using high-quality software and, to me as the author, I believe any significant results in this study are presented clearly without needing any further modifications. 

Point 2: For these cell culture studies, and our experience with inducing myogenesis in human skeletal muscle cells, myogenesis takes approximately 6 days of time for the cells to fully/mostly mature. So D6 Con represents mature myotubes that have gone through myogenic activity without any type of stimulation other than growth/differentiation media. Knowing this, and since this study's design was exploratory (we didn't know what may happen to genes/muscle beyond this 6th day), we referenced D6CON as our control. So all conditions were expressed compared to D6CON as this is our primary reference/control point. 

Point 3: I can understand the confusion as you get to the results section of this manuscript. However, I believe that is largely due to the format of this journals sections (methods explained later on). Figure 5 is purely methodology and I believe it best fits within the methods portion of this manuscript. To add to this further, I believe the methods section does a good job (I refined this several times to try and make it more succinct) of explaining what we did to readers. Though reading Figure 5 will help readers understand the study design better, I believe it must stay place in the methods section as it is purely methodology. 

Point 4: That is correct. MAFbx expression was elevated but deviation for this condition was too high. 

Round 2

Reviewer 1 Report

Thank you for making these edits re. the manuscript - "Both acute and consecutive days of formoterol stimulation influence myogenic, mitochondrial, and myomiR gene expression in human skeletal muscle cells"

I am satisfied with the changes and believe they strengthen your manuscript.

I appreciate that some of the points made may not be able to be rectified, particularly that in relation to formoterol concentration curves & cell viability assays.

I have left one or two points below which might also be important to rectify prior to publication:

1. Figure 5 - include a scale bar

2. Although you mention the n in the materials and methods, from a reader’s perspective, seeing the n within the figure helps to put clarity on the data, and interpret the SD with more accuracy.

 

3. The product number for the cells should be included. It is not enough to just have the company where you purchased these cells from. I believe it is either C-12530 or C-14060. The product name should also be mentioned “SkMC”(found within the SDS). Having used these cells myself before, it is important to have all this information present as there are differences with different companies – this could be due to the donor etc.

4. Please check the font for the Pax7 reverse primer and insure it is consistent with everything else (I might be wrong here).

Many thanks for your manuscript and all of your work on this topic.

Author Response

1. I am unable to add a scale bar, unfortunately. These images were collected previously without including the scale bar and I no longer have the capabilities to add it retroactively.
2. I included (n = 5) in legend/description for Figures 1-4. 
3. I included the cell type as requested in the methods section. I thought this was a good and thoughtful addition for future replication. 
4. I adjusted the Pax7 primer sequence.