Muscle Fiber Hypertrophy and Myonuclei Addition - A Systematic Review and Meta-Analysis PDF

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Muscle Fiber Hypertrophy and Myonuclei Addition:
A Systematic Review and Meta-analysis
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Miguel S. Conceição1, Felipe C. Vechin1, Manoel Lixandrão1, Felipe Damas1, Cleiton A.
Libardi2; Valmor Tricoli1, Hamilton Roschel1, Donny Camera3, and Carlos Ugrinowitsch1
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School of Physical Education and Sport, University of São Paulo, São Paulo, Brazil;
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Laboratory of Neuromuscular Adaptations to Resistance Training, Department of
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Physical Education, Federal University of São Carlos, São Carlos, Brazil; 3Mary
MacKillop Institute for Health Research, Centre for Exercise and Nutrition, Australian
Catholic University, Melbourne, Australia
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Accepted for Publication: 22 February 2018

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Copyright © 2018 American College of Sports Medicine

Medicine & Science in Sports & Exercise, Publish Ahead of Print
DOI: 10.1249/MSS.0000000000001593
Muscle Fiber Hypertrophy and Myonuclei Addition:
A Systematic Review and Meta-analysis
Miguel S. Conceição1, Felipe C. Vechin1, Manoel Lixandrão1, Felipe Damas1,
Cleiton A. Libardi2; Valmor Tricoli1, Hamilton Roschel1,
Donny Camera3, and Carlos Ugrinowitsch1
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1
School of Physical Education and Sport, University of São Paulo, São Paulo, Brazil;
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Laboratory of Neuromuscular Adaptations to Resistance Training, Department of Physical
Education, Federal University of São Carlos, São Carlos, Brazil; 3Mary MacKillop Institute for
Health Research, Centre for Exercise and Nutrition, Australian Catholic University, Melbourne,
Australia
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Author for correspondence
Hamilton Roschel, Ph.D
School of Physical Education and Sport, University of São Paulo

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Av. Prof. Mello Moraes, 65, São Paulo, SP, Brazil

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Cep: 05508-030
Email: hars@usp.br
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The authors would like to express gratitude for the São Paulo Research Foundation (FAPESP),
grant #2016/09759-8 and grant #2015/19756-3, and CNPq, grants #448387/2014-0 and
#406609/2015-2. The authors declare that the results of the study are presented clearly, honestly,
and without fabrication, falsification, or inappropriate data manipulation. The authors declare no
conflicts of interest. The results of the present study do not constitute endorsement by ACSM.
Copyright © 2018 by the American College of Sports Medicine. Unauthorized reproduction of this article is prohibited.
ABSTRACT
Introduction: The myonuclear domain theory postulates that myonuclei are added to muscle
fibres when increases in fibre cross-sectional area (i.e. hypertrophy) are ≥26%. However, recent
studies have reported increased myonuclear content with lower levels (e.g. 12%) of muscle fibre
hypertrophy. Purpose: To determine whether a muscle fibre hypertrophy “threshold” is required
to drive the addition of new myonuclei to existing muscle fibres. Methods: Studies of resistance
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training (RT), endurance training (ET), with or without nutrient (i.e., protein) supplementation
and steroid administration, with measures of muscle fibre hypertrophy and myonuclei number, as
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primary or secondary outcomes, were considered. Twenty-seven studies incorporating 62
treatment groups and 903 subjects fulfilled the inclusion criteria and were included in the
analyses. Results: Muscle fibre hypertrophy ≤ 10% induces increases in myonuclear content
although a significantly higher number of myonuclei are observed when muscle hypertrophy is
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~22%. Additional analyses showed that age, sex and muscle fibre type do not influence muscle
fibre hypertrophy nor myonuclei addition. Conclusion: Even though a more consistent myonuclei
addition occurs when muscle fibre hypertrophy is >22%, our results challenge the concept of a
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muscle hypertrophy threshold as significant myonuclei addition occurs with lower muscle
hypertrophy (i.e. <10%).

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Key words: myonuclear domain, satellite cell, muscle hypertrophy, myonuclear.

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1 Introduction
Satellite cells (SC) are the muscle stem cell population mainly committed to the skeletal
muscle lineage (1-3). When exposed to mechanical loading, SC become activated, enter the cell
cycle, proliferate and may eventually fuse and donate their nuclei to existing muscle fibres (3).
The donation of myonuclei to existing muscle fibres is required in situations where increased
transcription capacity is required, such as repair to damaged muscle cells, or to promote protein
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accretion over a given physiological cellular myonuclear domain limit. The later concept is based
on the theory that each myonucleus controls mRNA transcription and protein
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synthesis/breakdown over a finite volume of cytoplasm (i.e. myonuclear domain) (4, 5). Thus,
increases in muscle cell volume [i.e. increase in muscle fibre cross-sectional area (CSA)]
expands the myonuclear domain over the physiological range, therefore requiring the addition of
new myonuclei to muscle fibres (6-9).

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Petrella et al. (9) and others (9-12) have described the existence of a theoretical
hypertrophy “threshold” after which new myonuclei should be added to muscle fibres to increase
transcriptional capacity. When muscle fibres transcriptional capacity is not increased, further
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expansions of muscle fibres CSA may be limited. This threshold seems to be ≥26% of fibres‟
CSA, but not less than 15% (9, 12). Nonetheless, evidence for such a threshold is equivocal (8,
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13, 14). For instance, Bellamy et al. (8) reported significant increases in type I (~13%) and II
(~20%) fibre CSA, with concomitant increments in myonuclei number per muscle fibre,
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following a 16 wk resistance training (RT) program. In addition, 12 wk of endurance training
(ET) increased type I (~12%) and IIA (~16%) fibres CSA with significant addition of myonuclei
in type I fibres only (15). Other studies have also failed to detect myonuclei accretion after RT,
even though muscle fibres achieved the supposed hypertrophy threshold (i.e. 26%) (13, 16, 17).
Taken together, particular original studies do not suggest the existence of a minimum muscle
fibre hypertrophy threshold (i.e. upper limit of the myonuclear domain) for myonuclear addition,
challenging the myonuclear domain theory. Clustering studies based on the magnitude of muscle
fibre hypertrophy may therefore help determining the required muscle fibre hypertrophy level
inducing myonuclei addition.
The variability reported in original studies with regard to a myonuclear threshold may be
attributed to several factors such as low statistical power, pre-intervention myonuclei number
and domain size, age, sex, the type and duration of training interventions, and fibre type
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differences. Meta-analyses summarize data from different studies and allow testing for the
effects of moderator variables (i.e. age, sex, and muscle fibre type) thereby providing a better
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estimate of the factors affecting the magnitude of the muscle fibre hypertrophy and addition of
new myonuclei to muscle fibres. Accordingly, the purpose of the current meta-analysis was to
determine a muscle fibre hypertrophy threshold driving the addition of new myonuclei to muscle
fibres. To account for the high heterogeneity between-studies, we explored the effects of
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potential moderators (i.e. pre-intervention myonuclei number and domain size, age, sex,
intervention model, and fibre type) on muscle fibre hypertrophy and myonuclear content.
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2 Methods
2.1 Search strategy

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Literature searches were performed on PubMed, Web of Science and Google Scholar
databases and conducted on studies published between January 1980 to March 2016.
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Combinations of the following keywords were used as search terms related to muscle
hypertrophy: “muscle hypertrophy”, “satellite cell”; “myonuclei”; “myonuclear domain” and
“muscle stem cell” and related to training: “resistance training”; “resistance exercise”; “strength
training”; “power training”; “endurance training” and “endurance exercise”. Endurance training
studies were included in the present meta-analysis for two reasons. Myonuclei may be added to
muscle cells to control both hypertrophic and non-hypertrophic processes. It is also reasonable to
assume that hypertrophic and non-hypertrophic processes are enhanced after both endurance
training and resistance training, although in different magnitudes, as cell‟s biological processes
should be augmented as individuals impose higher energy demands and mechanical stress to
muscle cells (i.e. training load progression). Therefore, including endurance training studies data
in our meta-analysis increases its external validity, as it may show that increasing myonuclei
number may occur even when myonuclear domain is not reaching physiological ceiling and
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muscle hypertrophy is small. Additional support to this matter is provided in Appendix,
Supplemental Digital Content 1, Effect sizes, http://links.lww.com/MSS/B220, which displays
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data from several studies in which muscle hypertrophy and myonucelar addition were small and
similar between resistance training and endurance training studies (small cluster studies –
described in the statistical analysis section).
Furthermore, reference lists of articles were reviewed in which relevant articles to the
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topic were added to the analyses and new articles published between April 2016 and April 2017
were included when inclusion criteria were reached.

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2.2 Inclusion Criteria
Inclusion criteria were limited to studies involving healthy human subjects, independent
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of age, sex or intervention (i.e. at least 4 weeks of resistance training, endurance training or
steroid administration intervention) that evaluated chronic changes in muscle fibre CSA and
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myonuclear content.
Methodological quality was calculated using the Physiotherapy Evidence Database
(PEDro) scale, which is based on the Delphi list (18). PEDro score was estimated by two
independent researchers and only studies that presented a score ≥ 4.0 were included for analyses.
Disagreements were resolved in a consensus meeting.
2.3 Study Selection and Data Extraction
Initially, 1,632 studies were retrieved. After removing duplicates and studies unrelated to
muscle fibre hypertrophy and myonuclei addition, 274 studies were evaluated for design quality
following the procedures previously described. To reduce the selection bias potential, titles and
abstracts of all studies were independently evaluated by two investigators (M.S.C and F.C.V),
and a mutual decision was made as to whether or not they met the inclusion criteria (described
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above). Sixty-three studies were selected and each study was independently evaluated by the
same investigators (M.S.C and F.C.V). Twenty studies met the inclusion criteria were therefore,
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added to the analyses. After reading all articles and reviewing the reference list, an additional
two articles were included. Five more articles were published after finishing searching and
finally 27 studies met the inclusion criteria and outcomes of each study were recorded and used
for future analyses (Figure 1).

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Two independent researchers (M.S.C. and F.C.V.) extracted data for the following variables:
number of subjects per group, age, sex, intervention model, muscle fibre CSA, myonuclear
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content and domain size per muscle fibre. To test for possible coder drift, we randomly selected
30% of the studies for recoding following procedures outlined by Cooper‟s et al., (19). Mean
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agreement between coders was 95%. Some of the studies did not present mean and standard
deviation (SD) values of the dependent variables. Despite email contact with the corresponding
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author of these studies requesting data for muscle fibre CSA and myonuclei number, no
information was received in the majority of cases. Thus, when possible, we used a recently
validated (20, 21) online platform, Webplotdigitizer, (Version 3.9, available at
http://arohatgi.info/WebPlotDigitizer/index.html) to obtain mean and SD values from these
studies‟ figures. Table 1 summarises studies included in the analyses. While some studies pooled
muscle fibre CSA and myonuclei number data among fibre types, others studies did not pool
muscle fibre CSA data among muscle fibres types (e.g. fibre type I, fibre type II) but pooled
myonuclei number data across muscle fibre types. Yet, others studies pooled muscle fibre CSA
but did not pool myonuclei number. Whenever muscle fibre CSA and myonuclei data did not
present the same level of analysis (i.e. per fibre type or pooled), data was pooled for analyses.
2.4 Statistical analyses
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Meta-analytic analyses were conducted using Comprehensive Meta-analysis software
version 2.2 (Biostat Inc., Englewwod, NJ, USA). Given that few studies included in the present
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meta-analysis had a control group, data were analysed using a pre-to post-intervention model,
considering only the intervention groups. In this sense, effect sizes (ES) for muscle fibre
hypertrophy and number of myonuclei were calculated for each study, using pre- and post-
intervention means, pre-intervention SD, sample size and pre- to post-correlation. As none of the
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studies included in the meta-analyses reported pre- to post-correlation and that the model used
for data analyses requires this moderation, the correlation was estimated as follow:
r = (S2pre + S2post – S2D) / 2 * (Spre* Spost).

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SD is the standard deviation of the difference score (pre- to post-intervention), defined by:
SD = square root [(S2pre / n) + (S2post / n)] and S is the standard deviation.

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All ES were corrected for small sample size bias with the following formula:
[1 – (3 / (4 * (n1 + n2 – 2) – 1)].
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The between-study heterogeneity was verified with the I2 statistics. Pre- to post-intervention ES
were calculated for each intervention on each study. Following, hypertrophy ES values from all
the studies were separated using a k-means cluster analysis. Three clusters explained >90% of
the data variance and produced significantly different centroids between the three clusters (small-
moderate- and large-hypertrophy) while maximizing the cubic clustering criteria (i.e. -2.6) being
selected for subsequent analyses. The heterogeneity threshold were set as I2 ≤ 25% (low), I2 ≥25%
to ≤ 50% (moderate) and I2 ≥ 75% (high) (22). As the heterogeneity of the muscle fibre
hypertrophy and myonuclei number per muscle fibre data was high (I2 ≥ 96.58 and I2 ≥ 92.53,
respectively), random effects models were implemented, as recommended (23).
To determine possible sources of the observed heterogeneity, additional analyses were
undertaken to test the effects of the following moderators on muscle fibre CSA: age, sex,
intervention model and muscle fibre type. For this purpose, all of the studies were allocated to
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their respective cluster and then, mean effect sizes of myonuclei number were compared between
clusters of muscle fibre CSA. This analysis determined if distinct magnitudes of muscle fibre
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hypertrophy (i.e. centroids of the three clusters) affected the addition of myonuclei to the muscle
fibres. Additional analyses were undertaken to test for the effect of the moderators cited above on
myonuclei number. Whenever ES confidence intervals of the levels of a given moderator factor
did not overlap, levels were considered as influential. As pre-intervention myonuclear content is
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a continuous variable, a fixed effect meta-regression was implemented to test for the effects of
this variable in the addition of new myonuclei to muscle fibres.
A sensitivity analysis was performed to identify the presence of highly influential studies
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which could potentially bias the analyses. Thus, an analysis removing one study at a time was
performed, and then examining its effect on between-group comparisons (i.e. overall effect).
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Studies were considered as influential if removal resulted in a change of the ES going from
significant (P ≤ 0.05) to non-significant (P > 0.05) or if their removal caused a large change in
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the magnitude of the coefficient. This procedure has been adopted elsewhere (24). The
significance level adopted was P < 0.05. All data are present as mean ± standard error (SE).
3 Results
Twenty-seven studies investigated the changes in muscle fibre CSA (i.e., hypertrophy) as
well as the number of myonuclei after a period of intervention (e.g. RT, ET, with or without
nutritional supplementation, or testosterone administration), and therefore were included in the
present meta-analysis. This resulted in 62 effect sizes and a total sample size of 903 subjects. The
mean rating of study quality assessed by the PEDro scale was 5.
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3.1 Muscle Fibre Cross Sectional Area
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The mean ES for muscle fibre hypertrophy was 0.45 ± 0.03 (95%CI: 0.37 - 0.52) (Figure
2). However, the funnel plot indicated the occurrence of publication bias as several studies were
outside the confidence limits of the plot (τ=0.13, P=0.12; Egger‟s regression intercepts of 3.17,
P=0.01). Thus, a fill and trim procedure was implemented to minimise the magnitude of the
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publication bias, producing more conservative ES estimates (ES before fill and trim: 0.45, 95%CI
0.37 - 0.52; ES after fill and trim: 0.48, 95%CI 0.40 - 0.56). As the effect of the fill and trim
procedure on the ES estimates was trivial, ES estimates before the fill and trim procedure were
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considered as appropriate.
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Thereafter, a cluster analysis was used to allocate studies according to the magnitude of
muscle fibre CSA hypertrophy. The percentage change in muscle fibre hypertrophy in each
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cluster was classified as follows: small-cluster: 10% of hypertrophy, ES: 0.17 ± 0.02, 95%CI: 0.13
- 0.22; moderate-cluster: 22% of hypertrophy, ES: 0.59 ± 0.02, 95%CI: 0.55 - 0.63; and large-
cluster: 27% of hypertrophy, ES: 1.03 ± 0.04, 95%CI: 0.95 - 10) (Figure 3). Clustering muscle
fibre hypertrophy data across studies greatly reduced heterogeneity in the model (small-cluster -
Q: 172.3, df: 26, P: 0.000, I2: 84.9%; moderate-cluster - Q: 56.76, df: 27, P: 0.001, I2: 52.43%;
large-cluster - Q: 11.89, df: 6, P: 0.065, I2: 49.52%).
3.2 Effects of age, sex, intervention model and fibre type on CSA hypertrophy
Muscle fibre CSA increased significantly and by a similar magnitude in younger (i.e.,
between from 17 to 60 yr) and older individuals (i.e., older than 60 yr) (ES: 0.41 ± 0.04, 95%CI:
0.33 - 0.49, I2: 94.65% and ES: 0.50 ± 0.07, 95%CI: 0.35 - 0.64, I2: 98.26%, respectively). Muscle
fibre CSA also increased significantly and by a similar extent for both men and women (ESMen:
0.42 ± 0.04, 95%CI: 0.34 - 0.51, I2: 96.09%, ESWomen: 0.64 ± 0.12, 95%CI: 0.40 - 0.88, I2: 92.66%).
0.45 - 0.63, I2: 97.40%) and
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Regarding intervention model, RT (ES: 0.54 ± 0.04, 95%CI:
testosterone treatment (ES: 0.35 ± 0.06, 95%CI: 0.24 - 0.47, I2: 40.50%) produced greater muscle
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fibre hypertrophy than ET (ES: 0.14 ± 0.03, 95%CI: 0.06 - 0.19, I2: 65.37%). Furthermore, RT
presented a trend toward greater muscle fibre hypertrophy than testosterone treatment as there is
a small overall between their respective confidence intervals. Even though RT produced the
highest muscle fibre hypertrophy, it also yielded the highest heterogeneity value (I2=97.40%),
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while testosterone treatment and ET showed lower values (I2=40.50% and I2=65.37,
respectively). Interestingly, type I (ES: 0.42 ± 0.08, 95%CI: 0.26 - 0.58, I2: 97.17%), and type II
muscle fibres (ES: 0.54 ± 0.06, 95%CI: 0.43 - 0.65, I2: 96.41%) presented similar magnitudes of
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increase in muscle fibre size. Pooled muscle fibre analysis resulted in significant increases in
CSA (ES: 0.36 ± 0.04, 95%CI: 0.27 - 0.45, I2: 92.92%).

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3.3 Myonuclei Number

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The mean ES across all the studies was 0.47 ± 0.05 (95%CI: 0.37 - 0.57), demonstrating
that the number of myonuclei significantly increased after interventions (Figure 4). Sensitivity
analysis (i.e., removing one study at a time and re-analysing the data) revealed that number of
myonuclei were not highly affected by any single study (data not shown). Even though no single
study affected the analysis, funnel plot revealed that three studies greatly inflated publication
bias (three studies outside the right confidence limit of the funnel plot, τ=0.21, P=0.02 and
Egger‟s regression intercepts of 3.97 P=0.005). Removing these studies improved the visual
profile of the funnel plot, but Egger‟s regression intercept remained significant (τ=0.09, P=0.29
and Egger‟s regression intercepts of 2.37, P=0.005). Similar to muscle fibre hypertrophy, a fill
and trim procedure was implemented to minimize publication bias, however ES estimates did not
change based on the random model. Thus, as these studies have unusually high effect sizes they
were not considered for the final analysis.
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Myonuclei addition occurred in all three clusters (small-cluster: 10% of hypertrophy, ES:
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0.25 ± 0.03, 95%CI: 0.18 - 0.32; moderate-cluster: 22% of hypertrophy, ES: 0.46 ± 0.04, 95%CI:
0.36 - 0.56; large-cluster: 27% of hypertrophy, ES: 0.47 ± 0.10, 95%CI: 0.27 - 0.67). Yet, the
moderate cluster showed a significantly greater ES compared to the small-cluster (Figure 5). The
heterogeneity data for small-, moderate-, and large- clusters were; Q: 291.42, df: 25, P: 0.000, I2:
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91.42%; Q: 220.51, df: 25, P: 0.000, I2: 88.66%; Q: 104.80, df: 6, P: 0.000, I2: 94.28%,
respectively. Even though the magnitude of muscle fibre hypertrophy seems to coordinate the
addition of new myonuclei to muscle fibres, the high heterogeneity observed on within each
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cluster suggests other sources of variance.

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3.4 Effects of age, sex, intervention model and fibre type on myonuclei number.
Age did not affect the increase in myonuclei number (ES: 0.37 ± 0.04, 95%CI: 0.29 - 0.45,
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I2: 93.33% and ES: 0.35 ± 0.04, 95%CI: 0.27 - 0.42, I2: 89.18%, for young and older adults,
respectively). In regard to sex, the number of myonuclei increased similarly after intervention for
both men (ES: 0.41 ± 0.03, 95%CI: 0.33 - 0.49, I2: 92.70%) and women (ES: 0.27 ± 0.08, 95%CI:
0.10 - 0.45, I2: 69.00%). Regarding the intervention model, RT (ES: 0.41 ± 0.03, 95%CI: 0.34 -
0.49, I2: 93.04%), ET (0.16 ± 0.05, 95%CI: 0.05 - 0.27, I2: 93.27%) and testosterone treatment
(ES: 0.34 ± 0.04, 95%CI: 0.25 - 0.43, I2: 0.00%) significantly increased myonuclei number. Also,
RT showed higher values compared to ET. Fibre type did not affect the addition of myonuclei as
it increased similarly for both type I (ES: 0.40 ± 0.05, 95%CI: 0.30 - 0.50, I2: 88.52%), type II
(ES: 0.43 ± 0.06, 95%CI: 0.32 - 0.55, I2: 94.75%) and pooled muscle fibre types (ES: 0.27 ± 0.05,
95%CI: 0.20 - 0.35, I2: 88.37%).
3.5 Effects of pre-intervention myonuclear domain on myonuclei addition.
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The meta-regression produced a non-significant model (Q=1.57, P=0.21) and slope (-
0.0001, P=0.21; i.e., effects not different from zero), but a significant intercept (0.64, P<0.0001)
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was observed.
4 Discussion
It has been proposed that a theoretical muscle fibre hypertrophy threshold of ~26% is
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required before the addition of new myonuclei to muscle fibres (9-12). The major findings from
our meta-analyses show that myonuclei addition does occur when muscle fibre hypertrophy is
≤10% although consistent myonuclear addition is observed when muscle fibre hypertrophy is
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>22%. We also demonstrated that a) age, sex and muscle fibre type do not affect myonuclei
addition; b) RT is more effective than ET to induce muscle fibre hypertrophy and myonuclei
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addition after training; and c) steroid administration produced the lowest heterogeneity value
(I2=40.50%) for myonuclei addition. Our findings therefore extend and add new information to
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the present knowledge regarding the muscle fibre hypertrophy necessary to myonuclei addition.
Results of several studies have indicated that myonuclei addition is necessary for muscle
fibre hypertrophy to occur (6, 7, 9, 25, 26). In contrast, evidence also suggests that existing
myonuclei are able to increase rates of protein synthesis to elicit moderate level of muscle fibre
hypertrophy (9-14). Cluster analysis allowed us to determine how distinct levels of muscle fibre
hypertrophy (i.e. small, moderate and large) may affect myonuclei addition. Overall, our data
suggest that muscle fibre hypertrophy ≤10% may induce myonuclei addition, which is lower than
the threshold value (≥ 26%) or even the minimum value (i.e. 15%) reported elsewhere (7, 9).
The presence of myonuclei addition with such a small amount of muscle fibre hypertrophy
indicates that reaching a ceiling of the myonuclear domain is not required. Accordingly, the meta
regression showed that myonuclei addition is not influenced by initial myonuclear domain. The
fact that some studies did not find increases in myonuclear content with low-to-moderate
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magnitude of hypertrophy could be due to the lack of sensitivity in the immunohistochemical
procedures to detect small changes in myonuclear content. All studies are based on cross-
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sections, obtained from muscle biopsies, to evaluate muscle fibre size, myonuclear content
and/or domain size. However, it is important to consider that changes in muscle architecture are
not occurring in only two dimensions. The theory is that every myonucleus controls a certain
cytoplasmic volume (three dimensions) of the muscle fibre, which is not accounted in the two
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dimensions of the histochemical analyses. Furthermore, it is not clear if myonuclei are
homogenously distributed along the muscle fibre length. Thus, it is possible that cross-section
slices used in the immunohistochemical analyses may not contain any myonucleus just by
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chance. Taking these limitations into consideration, our two-dimensional analyses suggested that
the moderate and large- clusters produced similar ES for myonuclei addition, supporting that
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22% of muscle fibre hypertrophy does require robust addition of new myonuclei to muscle
fibres. It is important to note that the high heterogeneity values for myonuclei addition among
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the hypertrophy clusters (large-cluster I2: 94.28; moderate-cluster: I2: 88.66; small-cluster: I2
91.42) indicate that other moderate variables may be affecting myonuclei addition, such as the
quality of each study, measured here by PEDro scale. When we separated only studies qualified
as high quality (PEDro scale higher than 6), seven studies and 28 treatments (data not shown)
were identified and the overall heterogeneity test to myonuclei number decreased from high (I2 ≥
97.62) to moderate (I2 ≥ 65.62), suggesting that „low‟ quality study design (e.g., no control
group) can definitely influence the myonuclei number results. Accordingly, we moderated the
myonuclei number by hypertrophy cluster (small hypertrophy: 10%; moderate hypertrophy: 22%
and large hypertrophy: 27%) only in „high‟ quality studies and thus, the heterogeneity test
generally decreases from high to moderate (small: from I2 ≥ 91.42 to I2 ≥ 61.58; moderate: from
I2 ≥ 88.66 to I2 ≥ 68.49 and large: from I2 ≥ 94.28 to I2 ≥ 69.88). Taken together, we suggest that
≤10% of muscle fibre hypertrophy can induce the addition of new myonuclei to the muscle fibre,
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however significantly higher myonuclei addition is observed with larger muscle fibre
hypertrophy (i.e. 22%).
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Small cohort studies have reported that age and sex may affect myonuclear content,
although evidence supporting such a notion is equivocal (9, 10). SC and myonuclear content,
specifically in type II muscle fibres, have been shown to decrease with age (27). In addition,
some studies have shown that RT can increase the myonuclear content in younger, but not older,
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individuals (9, 13). Our results contrast these findings as ES confidence intervals overlapped
when age was considered as a moderator factor, suggesting that the elderly maintain the capacity
to increase the number of myonuclei per muscle fibre when data on the topic are pooled. Karlsen
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et al. (2015) (28) did not specifically compared the ability of young and older individuals to add
myonuclei to muscle fibres, but showed that regardless of the training status (i.e. sedentary or
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endurance trained) a large cohort of young and older individuals has similar number of
myonuclei per muscle fibre. Furthermore, they showed that myonuclei number has a very high
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positive relationship with muscle fibre size (r=0.99). Taken together, ours and Karlsen‟s et al.
(2015) findings suggest that myonuclear content is not affected by age but by fibre size, which
does not corroborate with the findings of some independent studies (9, 13, 17, 29). Low
statistical power may be an issue when assessing the effect of sex in myonuclei addition. For
example, Petrella et al. (9) reported that the number of myonuclei increased after RT only in
young men (not in women), while muscle fibre CSA increased independently of sex. In contrast,
our findings point to no differences either in muscle fibre hypertrophy or myonuclei addition
when sex is considered as a moderator factor. The high heterogeneity values obtained at each
level of age and sex provide additional support to the lack of appropriate statistical power in
small trials. It is important to highlight that, due to the inclusion criteria, only studies
investigating muscle fibre CSA and myonuclei number were included in our analyses, suggesting
that other studies evaluating muscle fibre CSA or myonuclei number in isolation could exist.
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Therefore, the effects of age and sex on muscle fibre CSA and myonuclei addition needs to be
further investigated with larger trials and the inclusion of appropriate control groups in order to
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improve the precision of within- and between-subjects variability.
Another interesting finding of the present study were the effects of training model and
testosterone treatment on muscle fibre CSA and myonuclei addition. Our analyses demonstrated
that RT, ET and testosterone treatment increase muscle fibre CSA and number of myonuclei.
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However, as would be expected, RT has a greater effect than ET on muscle CSA and myonuclei
number. Accordingly, it is a reasonable assumption that myonuclei addition observed with
endurance training may not be directed towards muscle hypertrophy. Considering the importance
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of muscle protein synthesis to muscle hypertrophy (30-33), such findings are entirely plausible.
While rates of myofibrillar protein synthesis are highly associated with muscle fibre hypertrophy
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(31, 33), most of the protein synthesis is directed toward mitochondrial biogenesis when ET is
undertaken (33). Additional myonuclei-driven biological processes can be enhanced after ET to

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meet the higher energy demands to muscle cells imposed by the progression of the training load.
For instance, McKenzie et al., (2016) investigated the effects of carbohydrate (CHO) and protein
supplementation after 10 days of intensified ET in well trained cyclists. Interestingly, it was
found a significant increment in myonuclei number without any significant changes in muscle
fibre CSA, after CHO supplementation. These results suggest that increments in myonuclei
number may be necessary to support biological processes other than muscle hypertrophy.
Furthermore, our results show that testosterone treatment increases muscle fibre CSA and
myonuclei number, confirming previous study findings (34-36). Testosterone treatment
presented virtually no heterogeneity in the data, while RT and ET induced high heterogeneity
(I2>93%), suggesting that testosterone treatment produces a very homogenous effect on muscle
fibre CSA and myonuclei number increases. On the other hand, moderator factors not tested in
the present study (i.e volume, intensity and frequency of training) may have driven the high
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heterogeneity following both RT and ET. Taken collectively, we confirm that testosterone
treatment can increase muscle fibre CSA and myonuclei addition and that RT is more effective
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to induce muscle fibre hypertrophy and increase number of myonuclei than ET.
Previous reports have suggested that myonuclei addition may present a fibre type-specific
response (37). Fry et al., (2014) showed that 12 weeks of ET significantly increased type I and II
muscle fibre CSA, while myonuclei number increased only in type I fibres, suggesting that type I
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fibres are more responsive to ET in regards to myonuclei addition. Bellamy et al. (2014) showed
that muscle fibre CSA increased after RT independently of muscle fibre type and that myonuclei
addition similarly occurred only in type I fibres. The results of these studies demonstrate that
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muscle fibre hypertrophy can occur independently of fibre type and that myonuclei addition
occurs more frequently in type I muscle fibres, independently of training model. However, and in
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contrast to these findings, our analyses indicate that fibre hypertrophy and myonuclei addition
are not fibre type-dependent. The reason for such a discrepancy is hard to reconcile, but the
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ability of meta-analyses to summarise the available data and to minimize possible effects of
small trials should be taken into account to support a non-specific fibre type response for muscle
fibre hypertrophy and myonuclei addition.
Conclusion
Even though a more consistent myonuclei addition occurs when muscle fibre hypertrophy
is >22%, our results challenge the concept of a muscle hypertrophy threshold as evidenced by
our analyses reporting significant myonuclei addition with lower muscle hypertrophy increases
(i.e. ~10%). Age, sex and fibre type do not affect myonuclei addition, while RT seems to induce
higher muscle fibre hypertrophy and increases in myonuclear content compared with ET.
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Fundamental to future studies, we recommend the inclusion of a reproducibility measure (i.e.,
coefficient of variation) to myonuclei number and inclusion of a control group to allow for a
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direct comparison of myonuclei number to any intervention. Furthermore, studies should always
include a statistical power analysis to estimate the number of subjects needed to decrease the
probability of occurrence of type I or II errors. From a statistic power analyses of our data having
β of at least 90% for the interaction effect, type I (α) error rate of 5%, moderate correlation
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among repeated measures (r = 0.8), nonsphericity correction of 1, and a large partial eta-squared
of 0.3 (38) a total sample size of 32 subjects (16 per group) was estimated. However, this
estimative is also imprecise, as we do not have the random error associated with the myonuclei
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assessments due to the lack of control groups in most of the trials used herein.
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Acknowledgments
The authors would like to express gratitude for the São Paulo Research Foundation
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(FAPESP), grant #2016/09759-8 and grant #2015/19756-3, and CNPq, grants #448387/2014-0
and #406609/2015-2. The authors declare that the results of the study are presented clearly,
honestly, and without fabrication, falsification, or inappropriate data manipulation. The authors
declare no conflicts of interest. The results of the present study do not constitute endorsement by
ACSM.
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Figure Captions
Figure 1. Schematic of search process.
Figure 2. Overall ES for muscle fibre hypertrophy. Letters A to J represent different treatments
within the same study.
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Figure 3. Muscle fibre hypertrophy data clustered according to the magnitude of the ES. Letters
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A to J represents different treatments within the same study.
Figure 4. Overall ES of number of myonuclei. Letters A to J represent different treatments
within the same study.

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Figure 5. Overall and individual effect sizes (ES) for the number of myonuclei per cluster of
muscle fibre hypertrophy. Letters A to J represents the different treatments in the same study.
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Supplemental Digital Content

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SDC 1_Appendix-Effect Sizes.docx

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Table 1. The respective characteristics of each treatment from studies included.
Age Intervention
Study Participants (yrs) Sex Type
Bellamy et al. (2014) 23 24±3 M RT
Charifi et al. (2003) 11 73±3 M ET
Dirks et al. (2017) A 17 76±2 6M 11W RT
Dirks et al. (2017) B 17 71±1 6M 11W RT
Frese et al. (2015) 8 17±0 M ET
Fry et al. (2014) 23 47±3 6M 17W ET
Herman-Montemayor et al. (2015) A 9 20±2 W RT
Herman-Montemayor et al. (2015) B 7 22±4 W RT
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Herman-Montemayor et al. (2015) C 10 19±1 W RT
Hikida et al. (1998) A 7 22±5 M RT
Hikida et al. (1998) B 8 65±6 M RT
Joanisse et al. (2013) 15 27±8 W ET
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Joanisse et al. (2015) A 19 21±4 16M 3W ET
Joanisse et al. (2015) B 10 21±2 M ET
Kadi and Thornell (2000) 10 38±6 W RT
Kadi et al. (2004) 15 24±1 M RT
Leenders et al (2013) A 24 70±1 W RT
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Leenders et al (2013) B 29 71±1 M RT
McKenzie et al (2016) A 8 25±7 2M 6W ET
McKenzie et al (2016) B 8 25±7 2M 6W ET
Mackey et al. (2007) 29 75±4 13M 16W RT
Mackey et al. (2011) A 16 74±3 W RT
Mackey et al. (2011) B 12 25±3 M RT
Nielsen et al. (2012) 18 22±2 M RT-BFR
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Olsen et al. (2006) A 9 24±2 M RT

Olsen et al. (2006) B 8 23±2 M RT
Olsen et al. (2006) C 8 23±2 M RT
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Petrella et al. (2006) A 13 26±1 M RT

Petrella et al. (2006) B 13 27±1 W RT
Petrella et al. (2006) C 13 64±1 M RT
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Petrella et al. (2006) D 13 62±1 W RT

Reidy et al. (2017) A 22 24±0.6 M RT
Reidy et al. (2017) B 17 25±1 M RT
Roberts et al. (2015) A 12 21±1 M RT
Roberts et al. (2015) B 12 25±2 M RT
Snijders et al. (2016) 22 23±1 M RT
Sinha-Hikim et al. (2002) A 9 27±1 M TT
Sinha-Hikim et al. (2002) B 7 30±1 M TT
Sinha-Hikim et al. (2002) C 8 29±1 M TT
Sinha-Hikim et al. (2002) D 9 29±1 M TT
Sinha-Hikim et al. (2002) E 6 26±1 M TT
Sinha-Hikim et al. (2006) A 7 68±2 M TT
Sinha-Hikim et al. (2006) B 4 67±3 M TT
Stec et al. (2016) 42 60-75 - RT
Verdijk et al. (2009) 13 72±2 M RT
Verdijk et al. (2014) 51 71±6 - RT
Verdijk et al. (2016) 16 72±1 M RT
M: Men; W: Women; RT: Resistance training; RT-BFR: Resistance training with blood flow
restriction; ET: Endurance Training, TT: Testosterone Treatment. A, B, C, D and E represent
different interventions used in the same study.
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Appendix: Effect Sizes
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Table 1 below clearly shows that Effect Sizes are similar between the resistance training studies in the small hypertrophy cluster (20 data lines- no highlight and blue highlight) and the
endurance training studies within the same cluster (7 data lines – yellow highlight) (trivial difference in ES – 0.044), suggesting that the latter group of studies does not affect our results and
that myonuclei addition may also occur to optimize biological processes (other than muscle hypertrophy) after resistance training. When the same analysis is performed for myonuclei addition,
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Frese‟s et al. (2015) [1] study should be carefully considered as it produced the highest ES among all of the studies included in the present meta-analysis. Thus, for the purpose of presenting our
point, we removed Frese‟s data (red highlight on the right-hand side of the table) and the difference in average ES between resistance training and endurance training studies in the small
hypertrophy cluster is also trivial (ES=0.11). Finally, we would like to point out that within the 20 data lines of resistance training studies in the small hypertrophy cluster, 8 out of the 20 (blue
highlight) displayed muscle hypertrophy and myonuclei addition (green highlight) at similar, or even lower, levels to the endurance training studies (yellow highlight).
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Table 1. Individual and average Effect Sizes and Standard Errors for the small hypertrophy cluster (cluster #1) separated by resistance training (no highlight and blue highlight) and endurance
training (yellow highlight) studies.
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MUSCLE HYPERTROPHY MYONUCLEI ADDITION ENDURANCE TRAINING - MUSCLE HYPERTROPHY ENDURANCE TRAINING - MYONUCLEI ADDITION
Studies Hedges's g Lower limit Upper limit % Hypertrophy Hedges's g Lower limit Upper limit Hedges's g Lower limit Upper limit Hedges's g Lower limit Upper limit
Charifi et al. (2003) 0.128 0.051 0.206 21 0.09 0.01 0.16 Charifi et al. (2003) 0.128 0.051 0.206 Charifi et al. (2003) 0.09 0.01 0.16
Dirks et al. (2017) A 0.010 -0.054 0.074 1.0 0.56 0.40 0.71 Frese et al. (2015) 0.036 -0.051 0.124 Frese et al. (2015) 15.24 12.06 18.42 REMOVED
Frese et al. (2015) 0.036 -0.051 0.124 0.00 15.24 12.06 18.42 Joanisse et al. (2013) 0.179 -0.002 0.360 Joanisse et al. (2013) 0.03 -0.04 0.10
Fry et al. (2014) A 0.151 0.095 0.207 12.0 0.27 0.21 0.33 Joanisse et al. (2015) A 0.018 -0.048 0.083 Joanisse et al. (2015) A 0.20 0.13 0.26
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Fry et al. (2014) B 0.256 0.142 0.369 16.0 0.00 -0.05 0.06 Joanisse et al. (2015) B 0.213 0.064 0.361 Joanisse et al. (2015) B 0.38 0.31 0.45
Herman-Montemayor et al. (2015) B 0.275 0.193 0.357 11.0 0.09 -0.14 0.32 McKenzie et al (2016) A 0.023 -0.252 0.299 McKenzie et al (2016) A 0.24 -0.03 0.51
Herman-Montemayor et al. (2015) C 0.337 0.251 0.424 38.0 0.21 0.04 0.38 McKenzie et al (2016) B 0.339 0.040 0.639 McKenzie et al (2016) B 0.09 -0.21 0.39
Hikida et al. (1998) A 0.362 0.267 0.457 23.0 0.35 -0.01 0.70 Average 0.134 -0.028 0.296 Average 0.171 0.030 0.313
Hikida et al. (1998) B 0.279 0.189 0.368 43.0 0.33 0.18 0.49
Joanisse et al. (2013) 0.179 -0.002 0.360 -5 0.03 -0.04 0.10
Joanisse et al. (2015) A 0.018 -0.048 0.083 1.00 0.20 0.13 0.26 RESISTANCE TRAINING - MUSCLE HYPERTROPHY RESISTANCE TRAINING - MYONUCLEI ADDITION
Joanisse et al. (2015) B 0.213 0.064 0.361 6.00 0.38 0.31 0.45 Hedges's g Lower limit Upper limit Hedges's g Lower limit Upper limit
McKenzie et al (2016) A 0.023 -0.252 0.299 1.00 0.24 -0.03 0.51 Dirks et al. (2017) A 0.010 -0.054 0.074 Dirks et al. (2017) A 0.56 0.40 0.71
McKenzie et al (2016) B 0.339 0.040 0.639 12.00 0.09 -0.21 0.39 Mackey et al. (2007) 0.080 0.030 0.130 Mackey et al. (2007) 0.22 0.14 0.29
Mackey et al. (2007) 0.080 0.030 0.130 4.0 0.22 0.14 0.29 Mackey et al. (2011) A 0.014 -0.060 0.089 Mackey et al. (2011) A 0.00 -0.21 0.21
Mackey et al. (2011) A 0.014 -0.060 0.089 -1.0 0.00 -0.21 0.21 Mackey et al. (2011) B 0.052 -0.145 0.249 Mackey et al. (2011) B 0.00 -0.07 0.07
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Mackey et al. (2011) B 0.052 -0.145 0.249 -2.0 0.00 -0.07 0.07 Sinha-Hikim et al. (2002) C 0.133 -0.171 0.436 Sinha-Hikim et al. (2002) C 0.13 -0.17 0.44
Roberts et al. (2015) B 0.331 0.254 0.407 12.0 0.24 0.06 0.43 Sinha-Hikim et al. (2002) F 0.011 -0.266 0.288 Sinha-Hikim et al. (2002) F 0.23 -0.05 0.51
Snijders et al. (2016) A 0.235 0.177 0.293 18.0 0.96 0.81 1.12 Verdijk et al. (2009) A 0.286 0.185 0.386 Verdijk et al. (2009) A 0.25 0.18 0.32
Sinha-Hikim et al. (2002) A 0.249 -0.033 0.531 10.0 0.41 0.13 0.68 Verdijk et al. (2016) A 0.118 0.052 0.184 Verdijk et al. (2016) A 0.38 0.21 0.55
Sinha-Hikim et al. (2002) C 0.133 -0.171 0.436 5.0 0.13 -0.17 0.44 Average 0.088 -0.054 0.230 Average 0.220 0.054 0.387
Sinha-Hikim et al. (2002) F 0.011 -0.266 0.288 0.0 0.23 -0.05 0.51
Sinha-Hikim et al. (2002) H 0.196 -0.110 0.501 8.0 0.15 -0.16 0.45
Sinha-Hikim et al. (2006) A 0.342 0.001 0.683 17.0 0.58 0.24 0.91
Verdijk et al. (2009) A 0.286 0.185 0.386 4.0 0.25 0.18 0.32
Verdijk et al. (2014) 0.210 0.171 0.248 21.0 0.28 0.25 0.32
Verdijk et al. (2016) A 0.118 0.052 0.184 3.0 0.38 0.21 0.55
Average 0.196 0.058 0.334 12.150 0.282 0.099 0.464
C
C
Reference
A
1. Frese S, Ruebner M, Suhr F, Konou TM, Tappe KA, Toigo M, et al. Long-Term Endurance Exercise in Humans Stimulates Cell Fusion of Myoblasts along with Fusogenic
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Accepted for Publication: 22 February 2018

Copyright © 2018 American College of Sports Medicine

Muscle Fiber Hypertrophy and Myonuclei Addition:

A Systematic Review and Meta-analysis

Miguel S. Conceição1, Felipe C. Vechin1, Manoel Lixandrão1, Felipe Damas1,

Cleiton A. Libardi2; Valmor Tricoli1, Hamilton Roschel1,

Donny Camera3, and Carlos Ugrinowitsch1

Hamilton Roschel, Ph.D

School of Physical Education and Sport, University of São Paulo

Av. Prof. Mello Moraes, 65, São Paulo, SP, Brazil

hypertrophy. Purpose: To determine whether a muscle fibre hypertrophy “threshold” is required

hypertrophy (i.e. <10%).

Key words: myonuclear domain, satellite cell, muscle hypertrophy, myonuclear.

new myonuclei to muscle fibres (6-9).

following a 16 wk resistance training (RT) program. In addition, 12 wk of endurance training

inducing myonuclei addition.

2.1 Search strategy

hypertrophy: “muscle hypertrophy”, “satellite cell”; “myonuclei”; “myonuclear domain” and

Supplemental Digital Content 1, Effect sizes, http://links.lww.com/MSS/B220, which displays

described in the statistical analysis section).

were included when inclusion criteria were reached.

2.2 Inclusion Criteria

Methodological quality was calculated using the Physiotherapy Evidence Database

Disagreements were resolved in a consensus meeting.

for future analyses (Figure 1).

validated (20, 21) online platform, Webplotdigitizer, (Version 3.9, available at

http://arohatgi.info/WebPlotDigitizer/index.html) to obtain mean and SD values from these

2.4 Statistical analyses

r = (S2pre + S2post – S2D) / 2 * (Spre* Spost).

SD = square root [(S2pre / n) + (S2post / n)] and S is the standard deviation.

respectively), random effects models were implemented, as recommended (23).

To determine possible sources of the observed heterogeneity, additional analyses were

this variable in the addition of new myonuclei to muscle fibres.

nutritional supplementation, or testosterone administration), and therefore were included in the

mean rating of study quality assessed by the PEDro scale was 5.

large-cluster - Q: 11.89, df: 6, P: 0.065, I2: 49.52%).

0.45 - 0.63, I2: 97.40%) and

CSA (ES: 0.36 ± 0.04, 95%CI: 0.27 - 0.45, I2: 92.92%).

3.3 Myonuclei Number

were not considered for the final analysis.

cluster suggests other sources of variance.

95%CI: 0.20 - 0.35, I2: 88.37%).

3.5 Effects of pre-intervention myonuclear domain on myonuclei addition.

hypertrophy (i.e. 22%).

number. Accordingly, it is a reasonable assumption that myonuclei addition observed with

undertaken (33). Additional myonuclei-driven biological processes can be enhanced after ET to

supplementation after 10 days of intensified ET in well trained cyclists. Interestingly, it was

myonuclei number, confirming previous study findings (34-36). Testosterone treatment

fibre hypertrophy and myonuclei addition.

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