The EMBO Journal Peer Review Process File - EMBO-2013-86145
Manuscript EMBO-2013-86145
Creb coactivators direct anabolic responses and enhance
performance of skeletal muscle
Nelson E. Bruno, Kimberly A. Kelly, Richard Hawkins, Mariam Bramah-Lawani, Antonio L.
Amelio, Jerome C. Nwachukwu, Kendall W. Nettles and Michael D. Conkright
Corresponding author: Michael Conkright, The Scripps Research Institute, Scripps Florida
Review timeline:
Submission date:
Editorial Decision:
Revision received:
Editorial Decision:
Accepted:
01 July 2013
25 July 2013
01 February 2014
11 February 2014
20 February 2014
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
Editor: Thomas Schwarz-Romond
1st Editorial Decision
25 July 2013
Thank you very much for submitting your study on the role of CREB transcriptional co-activator to
direct anabolic responses in skeletal muscle for consideration to The EMBO Journal editorial office.
Three referees have provided comments on your dataset and find the major message relevant and of
potential interest for a general audience. However, particularly the comments from ref#1 express
significant concerns on the asymmetric results from hormonal stimulation in cell culture versus
transgenic expression in animals.
It would thus be of critical importance for the final decision on publication, to overcome this
problem by providing the requested kinetic analysis of Crtc2 activation upon exercise in vivo
(please refer to ref#1's comments for details).
I realize that these are demanding and time-consuming experiments. I would thus understand if you
were to seriously consider more rapid presentation of your paper in a less-selective, physiologyoriented title.
If you were to attempt experimental revisions for The EMBO Journal, please make sure to address
ref#1's comments in full, while integrating your new results much better, into the existing,
conceptual framework as outlined by refs#2 and #3.
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Please be reminded that The EMBO Journal considers only one round of revisions and the ultimate
decision on publication will dependent on the outline and strength of the revised manuscript.
I am very much looking forward to hear from you on potential timeline/feasibility of the requested
experimental expansions (preferably via E-mail) and remain with best regards.
REFEREE REPORTS:
Referee #1:
Summary
In the present study, Bruno et al. describe the function of the transcriptional co-activators Crtc2/3 in
hormone-dependent growth/metabolic responses of skeletal muscle. In particular, the authors
demonstrate that sympathetic signaling via beta-adrenergic receptors activates the cAMP-PKACREB signaling pathway in muscle cells, including the activation of CREB-associated Crtc coregulators in response to exercise. Crtc co-activators are found to serve as a point of convergence
between the cAMP- and calcium-dependent pathways in myocytes, triggering a downstream PGC1alpha-IGF1-Akt signaling axis. In this respect, transgenic animals over-expressing Crtc2
specifically in skeletal muscle display muscle hypertrophy and enhanced storage of energy
substrates. Overall, the authors propose a biphasic control of muscle function through sympathetic
signaling, comprising a transient catabolic phase followed by anabolic changes for recovery,
mediated through the Crtc transcriptional co-activator complex.
General comments
Given the overall importance of proper skeletal muscle function for systemic energy homeostasis,
molecular pathways controlling both catabolic and anabolic processes clearly represent an important
area of research with immediate implications for a number of muscle-related pathologies. In this
regard, the current manuscript by Bruno et al. clearly presents an important and interesting concept
in hormone-dependent molecular adaptations of skeletal muscle with immediate consequences for
muscle function and associated metabolic disorders. Overall, the study is well structured, employs
state-of-the-art technology, and the conclusions are generally supported by the experimental data.
However, two major points require additional attention by the authors: 1) The proposed biphasic
model is largely based on the combinatorial interpretation of in vitro and in vivo data, i.e. hormonal
stimulation experiments in isolated myocytes vs. transgenic animals, respectively. As a
consequence, the observed phenotypes are recorded on quite different time scales. Whereas effects
of hormone stimulation on CREB activation occur within minutes to hours, transgenic
overexpression of Crtc2 in mice represents a long-term chronic condition. Thus, the authors should
provide a more detailed and extended kinetic analysis of Crtc2 activation (e.g. nuclear localization)
in response to exercise in vivo. Does Crtc2 activation in vivo match the proposed biphasic model, do
Crtc2 target genes as explored in this study (PGC1, DGAT) respond accordingly? 2) The current
study does not employ the available transgenic model in exercise studies to further support the
author's hypothesis. How do Crtc2 transgenic mice respond to exercise, do they even display a
catabolic phase, is their endurance capacity enhanced? Experimental data to address these issues
would clearly strengthen the case for publication. Additional minor comments are listed below.
Specific comments
Figure 2C: Please include data on Crtc2/3 phosporylation/localization.
Figure 2D: Please include the resting control groups for each gene individually.
Figure 2E: Are the shown differences statistically significant. Please provide information.
Figure 3B: Can you block the Iso+KCL effect by siRNA against Crtc2/3? Please provide additional
experimental data.
Figure 3C: It is not evident from the figure or legend which Crtc bands are shown exactly (phosphor
vs. total, nuclear, cytoplasmic?). Please clarify. Also, the figure call out in the text is wrong, please
correct.
Figure 4: Please provide Western Blot data on the degree of overexpression of Crtc2 in transgenic
animals.
Figure 4G: Please indicate at which time point after Crtc3 transfection hypertrophy was observed.
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Figure 5: Please confirm major findings (HDAC phosphorylation, translocation) in skeletal muscle
from transgenic Crtc2 animals.
Figure 6C: Please perform quantitative PCR analysis to allow for the comparison between proximal
and distal promoter enrichment.
Figure 6F: Can you block the effects on Pdpk1 and Akt phosphorylation by siRNA against PGC1alpha as proposed?
Figure 6G: How do you explain the enhance phosphorylation of Akt by Crtc2/3 overexpression?
Please discuss.
Figure 8E: The legend for this figure is missing.
All figures: Please provide statistics for all figures.
All text: Please check for spelling as there are quite a number of spelling errors in the text.
Referee #2:
This study deals with an interesting and important, but very confusing topic. It is well documented
that endurance exercise induces a PGC-1 -mediated increase in muscle mitochondria, but does not
cause muscle hypertrophy. PGC-1 4, discovered by Ruas et al., is a very different protein from
PGC-1 1. It is induced by strength training-weight-lifting and catecholamines and causes muscle
hypertrophy. A currently unexplained, but well documented, finding is that strenuous endurance
exercise results in large increases in epinephrine and norepinephrine but does not result in muscle
hypertrophy, while typical body building weight training, such as a 8 repetitions of a bench press
with a heavy weight results in a much smaller increase in catecholamines than, for example, running
a 10,000 meter race, and yet causes muscle hypertrophy.
Comments.
1. The exercise that you had your mice do, if performed repeatedly, would result in an increase in
muscle mitochondria but would certainly not result in muscle hypertrophy. As shown in Figure 2,
the treadmill running resulted in an increase in PGC-1 1, not PGC-1 4 expression. This component
should be taken out of your paper, as it has no relevance to PGC-1 4 induced muscle hypertrophy.
2. The concept developed in your paper regarding the role of catecholamines in regulating
glycogenolysis in muscle during exercise would probably seem reasonable to people not familiar
with this area. Actually, however, glycogenolysis in muscle during exercise is primarily regulated
by the increases in cytosolic Ca2+ and the availability of inorganic phosphate. Catecholamines play
a rather minor role, and beta-blockade actually accelerates glycogen depletion. The most rapid
glycogen breakdown occurs at the onset of exercise, before there is much increase in
catecholamines. You need to revise this component of your manuscript. See, for example: AC
Juhlin-Dannfelt, et al. Effects of b-adrenergic receptor blockade on glycogenolysis during exercise. J
Appl Physiol 53: 549-554, 1982.
Referee #3:
The authors describe the effects of Crtc over expression on skeletal muscle phenotype. The finding
that the muscles hypertrophy is quite exciting. They have also made an effort to demonstrate that
exercise could activate this pathway. The specificity finding (co-activator role of calcium) is very
nice. However, even though they have made a concerted effort to understand the exercise and
muscle mass literature, there are some significant oversights that need to be corrected.
General comments
1. The authors use high intensity running to stimulate epinephrine release. Running is well known to
cause an increase in epinephrine. However, this type of exercise is also well known to result in a
loss of muscle fiber size and prevent muscle hypertrophy (see for example Kraemer et al. J Appl
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Physiol. 1995 Mar;78(3):976-89.) How does this affect the proposed model?
2. The hormonal explanation of muscle hypertrophy has recently been challenged in a series of
articles that would completely disagree with the authors discussion about the importance of the
hormonal state (i.e. epinephrine) on muscle hypertrophy.
a: West DW, Phillips Eur J Appl Physiol. 2012 Jul;112(7):2693-702.
b: West et al. J Appl Physiol. 2010 Jan;108(1):60-7.
c: West et al. J Physiol. 2009 Nov 1;587(Pt21):5239-47
These papers need to be discussed.
3. The authors argue that the SNS through Crtc induce hypertrophy by increasing the amount of
Igf1. However, Igf1 is not required for load-induced muscle hypertrophy (see Spangenburg et al J
Physiol. 2008 Jan 1;586(1):283-91.). As the authors state, IGF-1 can most definitely induce
hypertrophy, but only when expressed at a high level in muscle for a long time. This is not what
occurs physiologically with exercise.
4. The authors state that Crtc drive hypertrophy by increasing synthesis and decreasing degradation.
However, resistance exercise is known to increase both synthesis AND degradation (see Phillips et
al Am J Physiol. 1997
Jul;273(1 Pt 1):E99-107).
How much of the hypertrophic effect can be blocked by rapamycin? The authors should treat the DT
mice with Dox and rapamycin and see the effect on hypertrophy.
5. Christian Handschin (and others) have data in the PGC-1a knockout mice, where PGC-1a4 is not
expressed, showing that muscle hypertrophy occurs normally with loading. This data suggests that,
as far as exercise-induced hypertrophy is concerned, the PGC1a4story might be a red herring.
6. PGC-1 is known to directly control glycogen levels (Wende et al J Biol Chem. 2007 282(50):
36642-51). Is it possible that the glycogen effects of Crtc are indirectly mediated through PGC-1?
7. The authors state that "While full-length Pgc-1 (Pgc-1 1) is regulated by p38 MAP kinase
phosporylation of ATF2 in skeletal muscle" this is a very limited view of the control of PGC-1 1/2.
Further, the authors state that "Only the distal promoter was responsive to isoproteronol
stimulation". However, Chinsomboon in PNAS 2009 showed that bAR stimulation could regulate
the PGC-1a2 promoter as well. Since this is the isoform of PGC-1 that is induced by endurance
exercise, and likely controls glycogen, this isoform should be measured.
8. The calcium from exercise does not regulate calcineurin activity (see Garcia-Roves et al.
Diabetes. 2005 54(3):624-8.) during exercise, calcium signaling is more likely to be the result of
CaMKII (see Smith et al. Am J Physiol Endocrinol Metab. 2008 Sep;295(3):E698). CAMK can also
regulate PGC-1 and this pathway is known to be involved in the adaptation to endurance exercise
NOT strength.
9. Instead of focussing on the exercise angle, the authors should focus on the G protein coupled
receptors and hypertrophy angle. Between the work of Minetti on Galphai in Science Signaling (Sci
Signal. 2011 Nov 29;4(201):ra80), von Maltzahna on Wnt couple to G proteins in Nature (Nat Cell
Biol. 2011 Dec 18;14(2):186-91), the Ghrelin receptor preventing atrophy, there is a lot of work that
indicates that G proteins are somehow promoting the maintenance of muscle mass. The current
manuscript has a potential mechanism for these observations. None of these things seem to be
regulated by classical exercise paradigms. Low levels of exercise may be needed for them to work,
but likely not. The authors should challenge their mice with Ghrelin, Wnt, etc. and see whether in
the presence of Crtc none of these factors have additional benefits. This likely would provide a more
interesting pharmacological finding and not have the problems that exist with the exercise issues.
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1st Revision - authors' response
01 February 2014
We sincerely thank the Reviewers for their insightful comments and suggestions and the Editor for
the opportunity to submit a revised manuscript. We have made substantial changes in response to
the very helpful critiques. In particular we now provide an extended kinetic analysis of activation of
Crtc/Creb and several of its target genes in vivo following exercise, and now demonstrate a
significant improvement in high-intensity exercise performance in the skeletal muscle-specific Crtc2
transgenic mice, which is accompanied by significant reductions in serum lactate. To our knowledge
this is the very first description of a physiological role of the Crtc/Creb transcriptional complex in
skeletal muscle.
Conceptually, we have completely rewritten the Discussion in response to the suggestions of the
Referees, where we now specifically address the unique muscle adaptations that are directed by the
SNS-to-Crtc/Creb circuit in response to high intensity exercise. Further, we discuss how the
Crtc/Creb transcriptional complex simultaneously promotes muscle hypertrophy and increased
mitochondrial biogenesis, and how this function of the SNS in response to high intensity exercise is
distinct from the adaptations manifest following extensive aerobic or resistance training.
Referee 1:
Major comment 1: The proposed biphasic model is largely based on the combinatorial
interpretation of in vitro and in vivo data, i.e. hormonal stimulation experiments in isolated
myocytes vs. transgenic animals, respectively. As a consequence, the observed phenotypes are
recorded on quite different time scales. Whereas effects of hormone stimulation on CREB activation
occur within minutes to hours, transgenic overexpression of Crtc2 in mice represents a long-term
chronic condition. Thus, the authors should provide a more detailed and extended kinetic analysis of
Crtc2 activation (e.g. nuclear localization) in response to exercise in vivo. Does Crtc2 activation in
vivo match the proposed biphasic model, do Crtc2 target genes as explored in this study (PGC1,
DGAT) respond accordingly?
Response:
As suggested, we completed and now provide an extended kinetic analysis of
Crtc2 dephosphorylation (activation) and sub-cellular localization in response to high-intensity
exercise. Complementing these data, the relative transcript levels of several Crtc2/Creb-responsive
genes were measured in the same cohort of exercised mice. These results are provided in Expanded
View Figure E3A-C and are described in the text as follows:
“An extended kinetic analysis of these transcripts showed that maximal induction occurred between
1-2 hr post-exercise and that transcript levels returned to resting levels four to eight hours later
(Expanded View Figure E3A). Moreover, dephosphorylation and nuclear translocation of Crtc2
followed a similar temporal pattern (Expanded View Figure E3B-C).
Major comment 2: The current study does not employ the available transgenic model in exercise
studies to further support the author's hypothesis. How do Crtc2 transgenic mice respond to
exercise, do they even display a catabolic phase, is their endurance capacity enhanced?
Experimental data to address these issues would clearly strengthen the case for publication.
Response: The Reviewer suggests very important analyses, particularly since enforced Pgc1α
expression in skeletal muscle increases glycogen stores but renders the mice unable to utilize these
stores during exercise (Wende et al, 2007). We have addressed the Reviewer’s concern/suggestion
by providing two additional experiments.
First, we tested if the glycogen stores could be utilized in the Crtc2-expressing mice. The glycogen
content in skeletal muscle was quantitated in DTg mice at rest and after a bout of high intensity
exercise. The glycogen content decreased 48% immediately after exercise and by six hr the total
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glycogen was restored to levels greater than pre-exercise amounts. Figure 8 has been amended to
include these new results, which demonstrate that Crtc2-expressing mice do undergo a catabolic
phase in response to exercise.
Second, we evaluated the exercise capacity of the Crtc2-expressing mice, and these data are in
Figure 9 of the revised manuscript. Specifically, we utilized an exercise protocol that would elicit a
large SNS response and that could be repeated multiple times before and after induction of the Crtc2
transgene. Maximum exercise capacity was determined utilizing a treadmill-based exercise stress
test that consisted of an escalating belt speed that increased by 2 m/min every 2 minutes at a 15
degree incline after an initial warm-up at 20 m/min for 5 minutes. Mice were exercised to
exhaustion during three trials prior to induction of the Crtc2 transgene followed by an additional
three trials two weeks following administration of Dox. Both groups displayed some signs of
training effect, but the Crtc2 transgenic mice exhibited a significant increase in performance that did
not occur with the wild type cohort. These data are presented in Figure 9B. Moreover, lower serum
lactate levels occurred after 20 minutes of exercise when the Crtc2 transgene was expressed,
suggesting that the same amount of work induced less metabolic stress in these mice. These data are
provided in Figure 9C.
Minor comments:
Figure 2C: Please include data on Crtc2/3 phosphorylation/localization.
Response: As requested, additional data has been provided that shows that the Crtc coactivators are
dephosphorylated and activated in response to exercise. These new data are provided in Figure 2C,
and Expanded View Figure E3B-C.
Figure 2D: Please include the resting control groups for each gene individually.
Response: The requested analyses of resting control groups are now provided Figure 2D.
Figure 2E: Are the shown differences statistically significant. Please provide information.
Response: Statistical analyses show that the differences are significant. These data are now provided
in Figure 2E.
Figure 3B: Can you block the Iso+KCL effect by siRNA against Crtc2/3? Please provide additional
experimental data.
Response: We have attempted these studies but dual knockdown of Crtc2 and Crtc3 appears toxic to
the myotubes and to our knowledge no group has succeeded in such analyses in muscle cells. We
are unsure if the deleterious effects are the result of silencing of both Crtc2 and Crtc3 or if they are
due to the high load of adenovirus required to deplete both RNA transcripts.
As an alternative approach, we assessed the effects of forced expression of dominant negative ACreb, and demonstrated that the co-stimulation of Iso and KCl is indeed dependent upon Creb.
These data are now provided in Expanded View Figure E4. Finally, we previously published in Cell
(Screaton et al, 2004) that the Crtc coactivators function as coincidence detectors of cAMP and
calcium signaling in pancreatic beta cells.
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Figure 3C: It is not evident from the figure or legend which Crtc bands are shown exactly (phosphor
vs. total, nuclear, cytoplasmic?). Please clarify. Also, the figure call out in the text is wrong, please
correct.
Response: We thank the reviewer for pointing out these ambiguities, which have now been
corrected. We have provided arrows to define the phospho-Crtc band and dephosphorylated band in
Figure 3C.
Figure 4: Please provide Western Blot data on the degree of overexpression of Crtc2 in transgenic
animals.
Response: We have added the requested data, which are provided in Expanded View Figure E5.
Figure 4G: Please indicate at which time point after Crtc3 transfection hypertrophy was observed.
Response: The muscle tissue was harvested ten days after electroporation of the DNA into the TA
muscle. The legend for Figure 4G has been revised to now state, “(G) Wet weight of TA muscles
after ten days of Crtc3 expression. The muscle was electroporated with either empty vector or a Crtc3
expression plasmid. Each line represent the difference in mass relative to the contralateral muscle of
each animal, n = 10 per group.”
Figure 5: Please confirm major findings (HDAC phosphorylation, translocation) in skeletal muscle
from transgenic Crtc2 animals.
Response: As requested by the Reviewer, we have added a western blot that shows the
phosphorylation status of the HDAC proteins in wild type and DTg mice after two weeks of Dox
administration, which is now provided in Figure 5F. We have also analyzed subcellular localization
of HDACs in double transgenic mice after 3 and 7 days of Dox administration, and these data are
now provided in Expanded View Figure E6C.
Figure 6C: Please perform quantitative PCR analysis to allow for the comparison between proximal
and distal promoter enrichment.
Response: As requested, we have conducted quantitative PCR analysis comparing Pgc-1α isoform
transcripts from the proximal and distal promoters. For these experiments we used the primers
described by Miura et al, 2008 and in a second series of experiments we used the primers described
by Ruas et al, 2012. The data from these two independent experiments show that Crtc-dependent
transcription is primarily dependent on regulation of the distal promoter. Moreover, our data are
strikingly similar to the effects of clenbuterol described in Figure 3 in the study by Miura et al,
2008. Our data are now provided in Expanded View Figure E7B-C.
Figure 6F: Can you block the effects on Pdpk1 and Akt phosphorylation by siRNA against PGC1alpha as proposed?
Response: We have been unsuccessful in obtaining the necessary reagents to replicate the previously
published finding that Pgc-1α4 induces AKT phosphorylation in skeletal muscle. We have modified
the text within the manuscript to reflect the fact we cannot show that increased expression of Pgc-1α
causes the increased phosphorylation of Akt1 or the increased expression of Pdpk1.
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Figure 6G: How do you explain the enhance phosphorylation of Akt by Crtc2/3 overexpression?
Please discuss.
Response: We have revised the text to include the following statement, “Several upstream activators
of Akt1 phosphorylation, including Igf1 and Pdpk1 are up regulated by Crtc/Creb signaling but
whether the hypertrophy observed is the direct result of these genetic changes remains unclear
(Rommel et al, 2001; Musarò et al, 2001; Ruas et al, 2012).”
Figure 8E: The legend for this figure is missing.
Response: This oversight has been corrected.
All figures: Please provide statistics for all figures.
Response: Statistics are now provided for all of the figures.
Referee 2:
This study deals with an interesting and important, but very confusing topic. It is well documented
that endurance exercise induces a PGC-1α-mediated increase in muscle mitochondria, but does not
cause muscle hypertrophy. PGC-1α4, discovered by Ruas et al., is a very different protein from
PGC-1α1. It is induced by strength training-weight-lifting and catecholamines and causes muscle
hypertrophy. A currently unexplained, but well documented, finding is that strenuous endurance
exercise results in large increases in epinephrine and norepinephrine but does not result in muscle
hypertrophy, while typical body building weight training, such as a 8 repetitions of a bench press
with a heavy weight results in a much smaller increase in catecholamines than, for example,
running a 10,000 meter race, and yet causes muscle hypertrophy.
Comments.
1. The exercise that you had your mice do, if performed repeatedly, would result in an increase in
muscle mitochondria but would certainly not result in muscle hypertrophy. As shown in Figure 2,
the treadmill running resulted in an increase in PGC-1α1, not PGC-1α4 expression. This component
should be taken out of your paper, as it has no relevance to PGC-1α4 induced muscle hypertrophy.
Response: We have rewritten the Discussion to address these comments. We do not propose that
SNS activation of the Crtc/Creb transcriptional complex is a mechanism of long-term endurance or
resistance training, but rather the role of the SNS in regulating adaptations in skeletal muscle evoked
during the transition from the untrained state to the early stages of training. The initiation of a new
mode of exercise, whether resistance or aerobic exercise, will cause the release of catecholamines if
a high level of intensity is reached, which is perceived by the muscle as a metabolic stress. We
propose that activation of the Creb/Crtc transcriptional complex is a mechanism to alter gene
expression that will in turn reduce stress during subsequent occurrences.
In support of this notion, there are several studies showing that the initiation of aerobic exercise
increases myofiber hypertrophy concurrent with mitochondrial biogenesis. For example, mice
provided free wheel running access have a significant increase in mitochondrial enzyme expression
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after two weeks and tend to show hypertrophy of hind limb muscle during this interval (Allen et al,
2002; Harrison et al, 2002; Konhilas et al, 2005; Allen et al, 2001). Moreover, 12 weeks of cycle
ergometer training induced myofiber hypertrophy in untrained young men, older men and older
women (Harber et al, 2009; 2012). Collectively, these studies demonstrate that under certain
training conditions, myofiber hypertrophy is induced during the early stages of aerobic exercise.
The Reviewer is correct that the PCR primers originally used in Figure 2 are unable to discriminate
between the different Pgc-1α isoforms. We have conduced an extended kinetic analysis of Pgc-1α4
transcripts and these results are provided in Expanded View Figure E3A and are described in the
revised text of the manuscript as follows: “An extended kinetic analysis of these transcripts showed
that maximal induction occurred between 1-2 hours post-exercise and transcript levels returned to
resting levels four to eight hours later (Expanded View Figure E3A).”
2. The concept developed in your paper regarding the role of catecholamines in regulating
glycogenolysis in muscle during exercise would probably seem reasonable to people not familiar
with this area. Actually, however, glycogenolysis in muscle during exercise is primarily regulated by
the increases in cytosolic Ca2+ and the availability of inorganic phosphate. Catecholamines play a
rather minor role, and beta-blockade actually accelerates glycogen depletion. The most rapid
glycogen breakdown occurs at the onset of exercise, before there is much increase in
catecholamines. You need to revise this component of your manuscript. See, for example: AC JuhlinDannfelt, et al. Effects of b-adrenergic receptor blockade on glycogenolysis during exercise. J Appl
Physiol 53: 549-554, 1982.
Response: There is a large body of literature that shows that epinephrine, through activation of PKA,
causes the phosphorylation and activation of phosphorylase kinase, and breakdown of glycogen.
Moreover, PKA also switches off glycogen synthase via phosphorylation. These mechanisms
induced by catecholamines are indisputable, though glycogen breakdown certainly can occur
independently of PKA activity as well.
We are proposing a biphasic mechanism such that when catecholamines stimulate glycogen
breakdown acutely, the same signaling pathway provides a mechanism for glycogen storage. Modes
of exercise that cause glycogenolysis independent of catecholamines would evoke some alternative
mechanism for replenishment.
Regarding the study cited by the Reviewer, the systemic administration of beta blockers will
attenuate lyposis in adipose tissue and of intramuscular tryglycerides (IMTGs). The decrease in
available fatty acids will force the skeletal muscle to be more reliant on carbohydrates as an energy
source. Studies where pharmacological agents are administered systemically are often difficult to
conclude causation; however, many in vitro studies show that catecholamine-induced glycogen
breakdown and lyposis in skeletal muscle is blocked by beta adrenergic antagonists.
Referee 3:
The authors describe the effects of Crtc overexpression on skeletal muscle phenotype. The finding
that the muscles hypertrophy is quite exciting. They have also made an effort to demonstrate that
exercise could activate this pathway. The specificity finding (co-activator role of calcium) is very
nice. However, even though they have made a concerted effort to understand the exercise and
muscle mass literature, there are some significant oversights that need to be corrected.
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General comments
1. The authors use high intensity running to stimulate epinephrine release. Running is well known to
cause an increase in epinephrine. However, this type of exercise is also well known to result in a
loss of muscle fiber size and prevent muscle hypertrophy (see for example Kraemer et al. J Appl
Physiol. 1995 Mar;78(3):976-89.) How does this affect the proposed model?
Response: The data presented demonstrates that Crtc2 overexpression induces myofiber hypertrophy
and increases mitochondrial biogenesis. Reviewers #2 and #3 point out that these two muscle
adaptations do not occur simultaneously in response to resistance training or long-term endurance.
There are examples of mild myofiber hypertrophy in response to HIT training but we do agree that
chronic aerobic training will result in loss of fiber size. The epinephrine response decreases
dramatically with repetition and the loss of muscle fiber size that occurs is due to protein catabolism.
We do not propose that SNS activation of the CREB/Crtc transcriptional complex is a mechanism of
long-term endurance or resistance training. Rather, our studies establish a role for the SNS-toCrtc/Creb circuit in regulating adaptations in skeletal muscle that are due to the onset of training,
and they are perhaps best viewed a signaling circuit that is evoked during transition from the
untrained state to the early stages of training. The initiation of a new mode of exercise, whether it be
resistance training or aerobic exercise, will cause the release of catecholamines if a high level of
intensity is reached, which is perceived by the muscle as a metabolic stress. We propose that
activation of the Creb/Crtc transcriptional complex alters gene expression to reduce stress during
subsequent occurrences.
In support of this notion, there are several studies showing the initiation of aerobic exercise
increases myofiber hypertrophy concurrent with mitochondrial biogenesis, where: (i) mice provided
free wheel running access have a significant increase in mitochondrial enzyme expression after two
weeks and concomitant hypertrophy of hind limb muscle (Allen et al, 2002; Harrison et al, 2002;
Konhilas et al, 2005; Allen et al, 2001); and (ii) lengthy bouts of cycle ergometer training induced
myofiber hypertrophy in untrained young men, older men and older women (Harber et al, 2009;
2012). Collectively, these studies demonstrate that under certain training conditions, myofiber
hypertrophy can result during the early stages of aerobic exercise. We have made extensive changes
to the Discussion to now clearly present this concept.
2. The hormonal explanation of muscle hypertrophy has recently been challenged in a series of
articles that would completely disagree with the authors’ discussion about the importance of the
hormonal state (i.e. epinephrine) on muscle hypertrophy.
a: West DW, Phillips Eur J Appl Physiol. 2012 Jul;112(7):2693-702. b: West et al. J Appl Physiol.
2010 Jan;108(1):60-7. c: West et al. J Physiol. 2009 Nov 1;587(Pt21):5239-47
These papers need to be discussed.
Response: The reports that the Reviewer notes are three very nice studies demonstrating that
elevations in circulating endogenous hormones do not correlate with muscle hypertrophy. However,
these studies did not examine catacholamines. Moreover, in accord with our studies, these reports
suggest that mechanism must be in place that dictates specificity to only the worked muscle, which
our work suggests is directed by a SNS-to-Crtc/Creb circuit. There are many reports showing that
systemic administration of beta agonists having long half-lives induces muscle hypertrophy in the
absence of exercise, but we feel these effects are pharmacological and do not represent the
physiological response. We have noted these points in our revised Discussion and cite these three
studies.
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3. The authors argue that the SNS through Crtc induce hypertrophy by increasing the amount of
Igf1. However, Igf1 is not required for load-induced muscle hypertrophy (see Spangenburg et al J
Physiol. 2008 Jan 1;586(1):283-91.). As the authors state, IGF-1 can most definitely induce
hypertrophy, but only when expressed at a high level in muscle for a long time. This is not what
occurs physiologically with exercise.
Response: We completely agree with the Reviewer that Igf1 does not appear to be required for loadinduced hypertrophy. We do not demonstrate that Igf1 is required for Crtc2-mediated hypertrophy.
However, it is indisputable that chronic activation of beta-adrenergic receptors or administration of
Igf1 causes hypertrophy. We have amended the text to include the following statement, “Several
upstream activators of Akt1 phosphorylation, including Igf1 and Pdpk1 are up regulated by Crtc/Creb
signaling but whether the hypertrophy observed is the direct result of these changes remains unclear
(Rommel et al, 2001; Musarò et al, 2001; Ruas et al, 2012).”
4.
The authors state that Crtc drive hypertrophy by increasing synthesis and decreasing
degradation. However, resistance exercise is known to increase both synthesis AND degradation
(see Phillips et al Am J Physiol. 1997 Jul;273(1 Pt 1):E99-107). How much of the hypertrophic
effect can be blocked by rapamycin? The authors should treat the DT mice with Dox and rapamycin
and see the effect on hypertrophy.
Response: Although we agree with the Reviewer that in response to some forms of exercise there
are increases in both protein synthesis and degradation, experiments by Kline et al., have shown that
rapamycin cannot block hypertrophy induced by ß-agonists. Further, our data is in accord with a
previous study by Berdeaux et al, 2007, published in Nature Medicine, who showed that expression
of a dominant-negative Creb molecule (A-Creb) increased the protein degradation pathways.
5.
Christian Handschin (and others) have data in the PGC-1a knockout mice, where PGC1a4 is not expressed, showing that muscle hypertrophy occurs normally with loading. This data
suggests that, as far as exercise-induced hypertrophy is concerned, the PGC1a4 story might be a
red herring.
Response: Our studies are in accord with some of the observations published by Ruas et. al.;
however, we do not go to the extent of validating all of their observations. The data presented by
Christian Handschin and other suggests that other pathways are able to circumvent the deficiency in
PGC-1α, and we would agree that there likely exist multiple signaling pathways that can impinge on
protein balance.
6. PGC-1 is known to directly control glycogen levels (Wende et al J Biol Chem. 2007 282(50):
36642-51). Is it possible that the glycogen effects of Crtc are indirectly mediated through PGC-1?
Response: Yes, we agree that it is very possible that at least part of the glycogen effects that we
observed are controlled by Crtcs are indirectly mediated through Pgc-1α and/or Nr4a1. Further,
overexpression of Crtc2 increases expression of multiple Pgc-1α isoforms in addition to the Pgc1α4. These other isoforms appear to function similar to full-length Pgc1α isoforms initiated from the
proximal promoter. One major distinction between our observations and those published by the
Kelly laboratory is Crtc2-expressing mice do not become metabolically inflexible as do the skeletalspecific Pgc-1α transgenic mice.
7. The authors state that "While full-length Pgc-1α (Pgc-1α1) is regulated by p38 MAP kinase
phosporylation of ATF2 in skeletal muscle" this is a very limited view of the control of PGC-1α1/2.
Further, the authors state that "Only the distal promoter was responsive to isoproteronol
stimulation". However, Chinsomboon in PNAS 2009 showed that bAR stimulation could regulate the
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PGC-1a2 promoter as well. Since this is the isoform of PGC-1 that is induced by endurance
exercise, and likely controls glycogen, this isoform should be measured.
Response: As requested, we have conducted quantitative PCR analysis comparing the Pgc-1α
isoforms from the proximal and distal promoters using the primers described by Miura et al., 2008
and in a second experiments we used the primers described by Ruas et al., 2012. The data from these
two independent experiments show that Crtc-dependent transcription is primarily through regulation
of the distal promoter. Moreover, our data are strikingly similar to the effects of clenbuterol
described by Miura et al., 2008.
8. The calcium from exercise does not regulate calcineurin activity (see Garcia-Roves et al.
Diabetes. 2005 54(3):624-8.) during exercise, calcium signaling is more likely to be the result of
CaMKII (see Smith et al. Am J Physiol Endocrinol Metab. 2008 Sep;295(3):E698). CAMK can also
regulate PGC-1 and this pathway is known to be involved in the adaptation to endurance exercise
NOT strength.
Response: The manuscript cited by the Reviewer shows that Glut4 translocation is dependent of
CaMKII, rather than on calcineurin. We agree that calcium by whatever mechanism can also
increase Pgc-1α expression but our data and the work of Miura et. al., 2008 would suggest that this
calcium-dependent regulation is through the proximal promoter and not dependent on the Crtc/Creb
complex, which rather acts through the distal promoter.
9. Instead of focusing on the exercise angle, the authors should focus on the G protein coupled
receptors and hypertrophy angle. Between the work of Minetti on Galphai in Science Signaling (Sci
Signal. 2011 Nov 29;4(201):ra80), von Maltzahna on Wnt couple to G proteins in Nature (Nat Cell
Biol. 2011 Dec 18;14(2):186-91), the Ghrelin receptor preventing atrophy, there is a lot of work
that indicates that G proteins are somehow promoting the maintenance of muscle mass. The current
manuscript has a potential mechanism for these observations. None of these things seem to be
regulated by classical exercise paradigms. Low levels of exercise may be needed for them to work,
but likely not. The authors should challenge their mice with Ghrelin, Wnt, etc. and see whether in
the presence of Crtc none of these factors have additional benefits. This likely would provide a more
interesting pharmacological finding and not have the problems that exist with the exercise issues.
Response: The reviewer proposes some very interesting questions regarding these other signaling
pathways; however, we feel that while these studies will provide some interesting insight into the
regulation of myofiber hypertrophy they extend well beyond the scope of the current manuscript.
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2nd Editorial Decision
11 February 2014
Thank you very much for the revised study that has now been seen by one of the initial referees. I
am pleased to inform you that this scientist did not raise further concerns and we are thus happy to
move forward with publication of your study.
-The EMBO Journal encourages the publication of source data, particularly for electrophoretic
gels/blots, with the aim to make primary data more accessible and transparent to the reader. This
entails presentation of un-cropped/unprocessed scans for KEY data of published work. We would be
grateful for one PDF-file per figure with this information.
-Further, I am not sure whether the graph in figure fig 4C would deserve the indication of standard
deviation/error as indicated in the figure legend?
-we would appreciate if you were to provide a minimally 2, up to 4 'bullet point' synopsis that
highlights the major novelty/advance provided by your study.
Please allow me already at this stage to congratulate you to this paper and be assured that the
editorial office will soon be in touch regarding formal acceptance of your study.
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