PKM2 Determines Myofiber Hypertrophy In Vitro and Increases in Response to Resistance Exercise in Human Skeletal Muscle

Round 1

Reviewer 1 Report

In this manuscript Verbrugge et al. studied the role of PKM isoforms in hypertrophying muscle and found that resistance exercise increases PKM2 expression, which is higher in fast fibers under normal conditions, and that PKM2 positively regulates myotube growth.

Manuscript is clear, concise and well-written. However, there are some limitations:

• The authors show Pkm1 and Pkm2 relative expression levels in human skeletal muscle during resistance training, but T0 protein levels of both isoforms are not shown. It would be interesting to see their ratio before training, as it would give us a more complete picture.
• Protein expression levels do not tell us anything about Pkm1 and Pkm2 activity. Did the authors maybe perform some preliminary experiments on this?
• The authors should show how did siRNA treatment affect Pkm1 and Pkm2 protein levels, not only mRNA. We know now that mRNA levels do not necessarily reflect protein levels.
• It is known that Pkm2 activity is greatly regulated by allosteric effectors as well as intracellular signaling, but the study lacks any mechanistic experiment.

Minor points:

• I’d delete “The muscle size-regulating enzyme” from the title, it’s obsolete.
• Line 34 – “expression is higher” instead of “is higher expressed”.
• Line 51 – define “mTOR”, since it is mentioned for the first time.
• Ines 53-4 – Type I and II fibers should be defined here.
• Line 72 – by LDH is meant LDHA, correct?
• Lines 74-5 – “Collectively this suggests…” – this phrase is repetitive and should be deleted.
• Line 86 – delete “has”.
• Line 99 – “hypothesis” instead of “aim”.
• Line 116 – “following” instead of “after”.
• Line 147 – define “MOPS buffer”.
• Line 148 – define “PVDF”.
• Line 198 – add “rat muscles” at the end of the phrase.
• Line 202 – “following” or “using” instead of “after”.
• Line 204 – closed bracket is missing.
• Line 238 – names of the genes should be always written in italics.
• Line 252 – each well contained exactly what?
• Line 266 – delete the last “expressed”.
• Line 284 – delete “and”.
• General comment for Figure legends – they should describe what is represented in the figure, not describe results. They should be corrected.
• Lines 301-3 – 39% and 95% reduction in myotube size does not seem to correspond to what is shown in the Figure (3D).
• Line 395 – delete “of”.

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Reviewer 2 Report

Verbrugge et al., sought to determine whether PKM 1 and/or 2 would play a role in mediating the hypertrophy observed with resistance training in humans. To this end, they used both human muscle biopsies and rodent animal/cell culture work. The results of the study are interesting and the document is well written. I do have some concerns that should be addressed and suggestions that can improve the manuscript.

Major

1. For the cell culture work - what is the passage number? For the siRNA treatments does the n represent technical replicates over varying passage numbers? 
2. For the rat soleus and EDL data in Figure 1, how come protein amounts of PKM2 and PKM1 were not probed for? I also only see Pkm2 mRNA, what about Pkm1?
3. For the human resistance training study: were there changes in fibre type distribution?
4. The authors should also consider that based on their findings in cell models, the reduction in PKM1 after resistance training may also contribute to muscle hypertrophy albeit through unknown mechanisms.
5. How come IGF-1 treatment did not increase PKM2 expression? This would help support its role in mediating muscle hypertrophy.
6. What happens to MHC isoform expression in cells that were singly or doubly silenced for PKM1 and/or PKM2?
7.  

Minor

1. Please include a figure timeline showing the biopsy collection - it will be easier for the reader.
2. In the abstract, the authors conclude that PKM2 is an important mediator in contributing to enhanced muscle growth in developing and adult human skeletal muscle. This line should be reworded as your mechanistic exploration were performed in rodent (rat and mouse) models. 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report