Content uploaded by Antonio La Torre
Author content
All content in this area was uploaded by Antonio La Torre on Aug 30, 2014
Content may be subject to copyright.
doi:10.1152/physiolgenomics.00040.2010
43:965-973, 2011. First published 14 June 2011;Physiol. Genomics
Alberti, Stefano Benedini, Andrea Caumo, Isabella Fermo and Livio Luzi
Ileana Terruzzi, Pamela Senesi, Anna Montesano, Antonio La Torre, Giampietro
DNA methylation and synthesis in elite athletes
Genetic polymorphisms of the enzymes involved in
You might find this additional info useful...
42 articles, 14 of which can be accessed free at:This article cites http://physiolgenomics.physiology.org/content/43/16/965.full.html#ref-list-1
including high resolution figures, can be found at:Updated information and services http://physiolgenomics.physiology.org/content/43/16/965.full.html
can be found at:Physiological Genomicsabout Additional material and information
http://www.the-aps.org/publications/pg
This infomation is current as of December 28, 2011.
the American Physiological Society. ISSN: 1094-8341, ESSN: 1531-2267. Visit our website at http://www.the-aps.org/.
July, and October by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2011 by
techniques linking genes and pathways to physiology, from prokaryotes to eukaryotes. It is published quarterly in January, April,
publishes results of a wide variety of studies from human and from informative model systems withPhysiological Genomics
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
Genetic polymorphisms of the enzymes involved in DNA methylation
and synthesis in elite athletes
Ileana Terruzzi,
1
Pamela Senesi,
2
Anna Montesano,
2
Antonio La Torre,
2
Giampietro Alberti,
2
Stefano Benedini,
2,4
Andrea Caumo,
2
Isabella Fermo,
3
and Livio Luzi
2,4
1
Division of Metabolic and Cardiovascular Science, San Raffaele Scientific Institute;
2
Department of Sport Science, Nutrition
and Health, Faculty of Exercise Sciences, University of Milan;
3
Laboratory of Chromatographic and Separative Techniques,
San Raffaele Scientific Institute; and
4
Metabolism Research Centre, San Donato Hospital and Scientific Institute, Milan, Italy
Submitted 18 February 2010; accepted in final form 9 June 2011
Terruzzi I, Senesi P, Montesano A, La Torre A, Alberti G,
Benedini S, Caumo A, Fermo I, Luzi L. Genetic polymorphisms of
the enzymes involved in DNA methylation and synthesis in elite
athletes. Physiol Genomics 43: 965–973, 2011. First published June
14, 2011; doi:10.1152/physiolgenomics.00040.2010.—Physical exer-
cise induces adaptive changes leading to a muscle phenotype with
enhanced performance. We first investigated whether genetic poly-
morphisms altering enzymes involved in DNA methylation, probably
responsible of DNA methylation deficiency, are present in athletes’
DNA. We determined the polymorphic variants C667T/A1298C of
5,10-methylenetetrahydrofolate reductase (MTHFR), A2756G of me-
thionine synthase (MTR), A66G of methionine synthase reductase
(MTRR), G742A of betaine:homocysteine methyltransferase (BHMT),
and 68-bp ins of cystathionine -synthase (CBS) genes in 77 athletes
and 54 control subjects. The frequency of MTHFR (AC), MTR
(AG), and MTRR (AG) heterozygous genotypes was found statisti-
cally different in the athletes compared with the control group (P⫽
0.0001, P⫽0.018, and P⫽0.0001), suggesting a reduced DNA
methylating capacity. We therefore assessed whether DNA hypom-
ethylation might increase the expression of myogenic proteins ex-
pressed during early (Myf-5 and MyoD), intermediate (Myf-6), and
late-phase (MHC) of myogenesis in a cellular model of hypomethy-
lated or unhypomethylated C2C12 myoblasts. Myogenic proteins are
largely induced in hypomethylated cells [fold change (FC) ⫽Myf-5:
1.21, 1.35; MyoD: 0.9, 1.47; Myf-6: 1.39, 1.66; MHC: 1.35, 3.10 in
GMA, DMA, respectively] compared with the control groups (FC ⫽
Myf-5: 1.0, 1.38; MyoD: 1.0, 1.14; Myf-6: 1.0, 1.44; MHC: 1.0, 2.20
in GM, DM, respectively). Diameters and length of hypomethylated
myotubes were greater then their respective controls. Our findings
suggest that DNA hypomethylation due to lesser efficiency of poly-
morphic MTHFR, MS, and MSR enzymes induces the activation of
factors determining proliferation and differentiation of myoblasts
promoting muscle growth and increase of muscle mass.
myogenesis; muscle hypertrophy; homocysteine cycle; muscle differ-
entiation; hypertrophy
PHYSICAL EXERCISE INDUCES considerable metabolic and morpho-
logical adaptive changes leading to an altered muscle pheno-
type with enhanced performance in trained individuals. The
mechanism by which exercise exerts its action in athletes is
still unclear. While environmental influences such as training
scheme and diet are important, there is increasing evidence for
strong genetic influences in athletic subjects. It is suggested
that athletes possess some genetic advantage predisposing
them to better sport performances than nonathletes. Thus,
genotype may predict sport ability and performance (9, 41).
Gene expression is controlled by an adequate supply of
methyl groups to the DNA. Specific mechanisms (Fig. 1) take
part in controlling DNA methylation (35): the methionine
synthase enzyme (MS) uses MS-bound cobalamin cofactor as
intermediate methyl-carrier and N5-methyltetrahydrofolate as
methyl donor, supplied by methylene-tetrahydrofolate reduc-
tase (MTHFR) enzyme. Methionine synthase reductase (MSR)
plays a critical role in maintaining adequate levels of activated
cobalamin, the cofactor for MS, which acts as an intermediate
methyl carrier between methyltetrahydrofolate and homocys-
teine, which is condensed with serine to form cysteine by the
enzyme cystathionine -synthase (CBS). The betaine-homo-
cysteine methyltransferase (BHMT) enzyme uses betaine as a
methyl donor to catalyze an alternative pathway of homocys-
teine remethylation, which, in humans, is mainly confined to
the liver and the kidney (28). But the whole cycle performs
other important functions: it controls gene expression by en-
suring an adequate supply of methyl groups to the DNA; it also
regulates nucleotides synthesis, and then the cell cycle, using
methylene group attached to tetrahydrofolate to convert the
uracil-type base found in RNA into the thymine-type base
found in DNA.
The functional integrity of anyone of the enzymes regulating
these pathways is important in maintaining adequate efficiency
of this cycle: common variants in genes codifying regulatory
enzymes of methylation cycle, caused by missense mutations,
are characterized by a reduced enzyme activity (7, 39, 17, 15),
probably responsible of both DNA methylation and synthesis
modification.
The present work was therefore designed to investigate if the
polymorphic variants of MTHFR C677T and A1298C, of CBS
844ins68, of methionine synthase (MTR) A2756G, of methi-
onine synthase reductase (MTRR) A66G, of betaine:homocys-
teine methyltransferase (BHMT) G742A, and of CBS 68-bp
ins, which disrupt the activity of the respective enzymes (7, 39,
17, 15), are present in the DNA of athletes. The study of the 76
elite athletes herein indicates this is the case. Since the pres-
ence of polymorphic variants in our cohort of athletes that may
induce a reduction in DNA methylation, we also studied an in
vitro model of DNA hypomethylation testing whether DNA
hypomethylation would increase the expression of muscle-
specific genes (Myf-5, MyoD, Myf-6, and MHC) crucial in the
myogenic differentiation. In vitro experiments demonstrated
that the induction of hypomethylation of DNA causes cell
hypertrophy and hyperplasia. Taken together, our in vivo and
in vitro studies indicate that elite athletes possess DNA poly-
morphisms of DNA methylation cycle enzymes that may
Address for reprint requests and other correspondence: I. Terruzzi, Div. of
Metabolic and Cardiovascular Science, San Raffaele Scientific Inst., Via
Olgettina, 60, 20132 Milano, Italy (e-mail: terruzzi.ileana@hsr.it).
Physiol Genomics 43: 965–973, 2011.
First published June 14, 2011; doi:10.1152/physiolgenomics.00040.2010.
1094-8341/11 Copyright ©2011 the American Physiological Society 965
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
predispose them to quicker DNA hypomethylation and, con-
sequently, to an higher rate of DNA synthesis. We also showed
that DNA hypomethylation in vitro determines increase in
muscle cell size.
MATERIALS AND METHODS
Subjects. In a case control study, we examined the allelic frequen-
cies and genotype distributions of restricted fragment length polymor-
phisms (RFLP) in the MTHFR, MTR, MTRR, BHMN, and CBS
genes among elite athletes and sedentary subjects. Elite athlete is
defined as an highly specialized athlete whose performances corre-
spond to the best world results in his or her respective sports or
discipline.
Of the 131 subjects recruited for the study, 54 (mean ⫾SD: BMI ⫽
22.4 ⫾1.8; yr 32.3 ⫾8.1) were studied as the sedentary control group
and 77 subjects (mean ⫾SD: BMI ⫽21.0 ⫾1.8; yr 23.4 ⫾5.0)
represented the elite athlete group, studied independently from their
training stage. All the subjects recruited for the study gave their
informed written consent after being given an explanation of pur-
poses, nature, and potential risks of the study.
In vitro experimental protocol. Myoblastic C2C12 cells were main-
tained in growth medium containing DMEM (Dulbecco’s modified
eagle medium) supplemented with 20% (vol/vol) FBS (fetal bovine
serum) up to 70% of confluence, and then (time 0) maintained in
growth medium or differentiated in DMEM supplemented with 1%
horse serum and antibiotics, both without (GM, DM) or with (GMA,
DMA) 5=-aza-2=-deoxycytidine (AZA, 5 M). At the end of the basal
period (0), early (4 h), intermediate (24 h, 48 h), and late myogenesis
(96 h), cells were lysed and the expression of specific myogenic genes
(Myf-5, MyoD, Myf-6, and MHC) was detected by Western blotting.
During late differentiation (72 and 96 h) morphology of MHC ex-
pressing cells was studied by immunofluorescence analysis. For each
experimental condition the DAPI (4=,6-diamidino-2-phenylindole)-
stained nuclei per myotube and myotube length and diameter were
determined and expressed as percentage increase ⫾SD.
Total homocysteine, vitamin B12, and vitamin B6 determination.
Peripheral blood samples were obtained from the patients in the
fasting state. Total plasma homocysteine levels were determined using
an HPLC (high performance liquid chromatography) method based on
the derivatization with SBD-F (ammonium-7-fluorobenzo-2-oxa-1,3-
diazole-4-sulfonate) (6). Vitamin B12 was assayed by enzyme immu-
noassay using the Advia Centaur Immunoassay system (Bayer Health
Care). Vitamin B6 (pyridoxal 5=-phosphate) was measured by apply-
ing a radioenzymatic assay (3).
Mutation detection. DNA from whole blood nuclear pellets, drown
after an overnight fast, was digested and quantified. For DNA extrac-
tion, all reagents were purchased from Sigma-Aldrich. PCR-based
RFLP analysis was used for genotyping in this study. MTHFR
(C677T) and MTHFR (A1298C) polymorphisms were analyzed as
described (19, 36). Amplification products underwent digestion, re-
spectively with HinfI (MTHFR C677T) and MboII (MTHFR
A1298C) (Promega, Madison, WI), and were then electrophoresed on
a 3% methaphore agarose gel (Cambrex Bio Science Rockland) with
ethidium bromide.
The MTR (A2756G) and MTRR (A66G) polymorphisms were
identified after amplification as described (42, 40). Restriction en-
zymes HaeIII and NdeI (Promega) were used for RFLP analysis of
amplification products. The primers and the conditions used for
BHMT genotyping were the same described (38). The PCR product
was digested with TaqI (Promega). For the 68-bp insertion variant of
CBS gene, DNA was amplified with the primers described (19) and
amplification products were observed on a 3% agarose gel.
Fig. 1. Simplified scheme of DNA methylation/synthesis cycle. Dihydrofolate (DHF), tetrahydrofolate (THF), methionine (MET), S-adenosylmethionine (SAM),
S-adenosylhomocysteine (SAH), homocysteine (Hcy), 5,10-methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS) methionine synthase
(MS), methionine synthase reductase (MSR), betaine:homocysteine methyltransferase (BHMT), cystathionine -synthase (CBS), B6 vitamin (B6), and B12
vitamins (B12).
966 ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
Electrophoretic techniques and immunoblotting analysis. C2C12
myotubes were homogenized as described (32). Aliquots of 30 g
supernatant proteins from the different samples were resolved by 8%
SDS-PAGE. Electrophoresed proteins were transferred to nitrocellu-
lose membrane (Protran, Whatman Schleicher & Schuell) as described
(34). The membranes were incubated with Myf5 (C-20), MyoD
(C-20), Myf6 (C-19), or MHC (H-300) antibodies and then incubated
with horseradish peroxidase-conjugated anti-species-specific second-
ary antibodies. Immunoreactive bands were visualized by an enhanced
chemiluminescence method (Amersham Pharmacia Biotech, Piscat-
away, NJ).
The membranes were stripped and reprobed with an antibody to
␣-tubulin (TU-02) to confirm equal protein loading per sample. All
antibodies were purchased by Santa Cruz Biotechnology (Santa Cruz,
CA). Quantitative measurement of immunoreactive bands was per-
formed by densitometric analysis using the Scion image software
(Scion, Frederick, MD).
Immunofluorescence analysis. For indirect immunofluorescence,
cells were fixed in 4% paraformaldehyde, permeabilized with 0.2%
Triton X-100, and blocked with PBS containing 1% bovine serum
albumin. Cells were then immunostained with anti-MHC rhoda-
mine conjugated (Santa Cruz Biotechnology) and nuclei revealed
with DAPI staining. Cells were observed using fluorescence mi-
croscopy (Leica DM IRE2), and images of myotubes were captured
using IM50 software (Leica Microsystems, Switzerland) for size
comparison. Data were displayed and analyzed using Adobe Pho-
toshop CS4.
Statistical analysis. Statistical differences between control and athlete
genotype frequencies were calculated by using the Exact Test 1.0.0.1 for
Windows 95/98/NT 4.0/ME/2000/XP (http://www.exact-test.com/), gener-
alization of Fisher’s Exact Test for 2 ⫻2 contingency tables. The
exact Pvalue was estimated by generation of 10,000,000 random
samples. Expected genotype frequencies were obtained from the allele
frequencies calculated for each genotype, under the assumption of
Hardy-Weinberg (H-W) equilibrium by using the Exact Test, and
were then compared with the observed frequencies by a Monte-Carlo
simulation.
When the determined deviation between observed and expected
numbers has a probability that is ⬎5%, there is no statistical deviation
from H-W equilibrium. Statistical significance calculated using
Fisher’s Exact Test was interpreted as two-tailed Pvalues of
⬍0.05. Odds ratios (OR) and 95% confidence intervals (95% CI)
were calculated to estimate the correlation between polymorphic
genotypes and athletic phenotypes.
For immunoblotting and immunofluorescence analysis, statistical
evaluations were performed by Student t-test using the SPSS 10.0 for
Windows statistical package. Differences between groups were con-
sidered statistically significant if Pⱕ0.05.
RESULTS
Investigation of polymorphic variants in DNA methylation
cycle genes. The mechanisms by which the DNA methylation
cycle enzymes control DNA synthesis and methylation are
summarized in Fig. 1. Genetic variants of these enzymes are
responsible for a reduced efficiency in DNA synthesis and
methylation. Table 1 shows that plasma levels of B12 (434.5 ⫾
131.4, 457.2 ⫾146.5 pg/ml; P⫽0.58) and vitamin B6 (32.4 ⫾
18.4, 36.4 ⫾15.9 nmol/l; P⫽0.20), well-known cofactors
(25) crucial for the correct activity of MS and CBS enzymes,
were normal in all subjects, and the values were not statisti-
cally different between the athlete group compared with the
control group.
We compared the allele and genotype frequencies of the
MTHFR (C677T and A1298C), MTR (A2756G), MTRR
(A66G), BHMT (G742A), and CBS (68-bp ins) polymorphic
genes in athletes and control subjects (Fig. 2). Both groups
were divided according to their wild-type, heterozygous, and
homozygous status relative to each of the six studied polymor-
phic sites (Table 2). A significantly increased frequency of
only MTHFR (AC), MTR (AG), and MTRR (AG) heterozy-
gous genotypes in the athletes’ group compared with the
control group was observed (P⫽0.0001, 0.018, and 0.0001).
Comparison of the observed and expected frequencies demon-
strated that there was a significant deviation in control subjects
of (MTHFR) C677T and in athletes of (MTHFR) A1298C
genotype frequencies from the H-W equilibrium (P⫽0.03),
probably explained by the small sample size used for this study
and a random genetic selection for individuals with certain
genotypes in the sampling process giving rise to a lower than
expected number of subjects with the MTHFR homozygous
genotypes.
Table 3 provides evidence for an increased predisposition of
the athletic group for carrying a polymorphic genotype for the
single MTHFR (AC), MTR (AG), or MTRR (AG and GG)
gene compared with the sedentary genotypes at the same loci
(P⬍0.001, 0.02, ⬍0.001, and 0.002, respectively) estimated
at OR of 4.76 (95% CI: 2.2–10.3), 2.94 (95% CI: 1.2– 6.9),
4.71 (95% CI: 2.1–10.3), and 17.14 (95% CI: 2.0 –143.5),
respectively.
Myogenic protein expression is increased by AZA-DNA
hypomethylation. To simulate the effect of hypomethylation
due to the less efficient polymorphic MTHFR, MS, and MSR
enzymes, we caused DNA hypomethylation by AZA (5 M) in
murine C2C12 myoblastic cells and studied the regulation of
muscle-specific genes expression during myoblast differentia-
tion. C2C12 cells have been widely used as a model for the
process of myogenesis. Under standard tissue culture condi-
tions (GM), the cells proliferate as single cell myoblasts. When
confluent cultures are transferred from GM to DM, the myo-
blasts exit cell cycle (Fig. 3A) and begin to elongate (early
differentiation). Subsequently, confluent mononucleated myo-
cytes begin to fuse forming multinucleate myotubes (interme-
diate differentiation), which become wider and longer over the
next few days as additional myoblasts fuse (late differentia-
tion).
Figure 3Bshows that the myogenic factors progressively
expressed during early phase (Myf-5 and MyoD) and interme-
diate phase (Myf-6) and the muscle-specific protein (MHC)
Table 1. Clinical characteristics and plasma features of control and athlete groups
Cases nAge, yr BMI Vitamin B12, pg/ml Vitamin B6, nmol/l
Control 54 32.3 ⫾8.1 22.4 ⫾1.8 434.5 ⫾131.4 32.4 ⫾18.4
Athletes 77 24.8 ⫾6.7 21.9 ⫾2.9
P0.58 0.20
Values are means ⫾SD. BMI, body mass index.
967ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
expressed during the late phase of myogenic differentiation are
largely induced by DNA hypomethylation in GMA and DMA
groups (FC ⫽Myf-5: 1.21, 1.35; MyoD: 0.9, 1.47; Myf-6:
1.39, 1.66; MHC: 1.35, 3.10, respectively) compared with the
GM and DM groups (FC ⫽Myf-5: 1.0, 1.38; MyoD: 1.0, 1.14;
Myf-6: 1.0, 1.44; MHC: 1.0, 2.20, respectively). In addition,
the expression of MHC gene results positively regulated by
hypomethylation with respect to the unhypomethylated differ-
entiated muscle cells. Overall, our results demonstrate that
MHC gene expressed in muscle tissues is transcriptionally
regulated by DNA hypomethylation.
The hypertrophic response of myofibers is induced by AZA-
DNA hypomethylation. To validate the effect of DNA hypom-
ethylation on myoblast differentiation and to investigate whether
that effect could improve the recruitment of myoblasts into myo-
tubes, or myotube hypertrophy, C2C12 cells were cultured in GM
Fig. 2. Identification of the polymorphic genotypes. Digestion of amplification PCR-RFLP products were cleaved and electrophoresed. A: MTHFR (C677T)
genotypes. The originated fragments of 198 and 175 bp are both present in the heterozygote genotypes (CT), only 198-bp fragment was present in wild type (CC),
while 175-bp fragment is the only present in the mutant homozygous genotype (TT). B: MTHFR (A1298C) genotypes. Polyacrylamide gel electrophoresis of
the PCR-RFLP product showing the bands of 84 and 56 bp obtained with wild-type (AA), heterozygous (AC), and homozygous (CC) A1298C mutation. C: the
MTR 2,756 wild-type homozygotes (AA) produce a 189-bp fragment, the heterozygotes (AG) produce both 189- and 159-bp fragments, and the variant
homozygotes (GG) produce 159-bp fragments. D: after electrophoresis genotypes of the MTRR (A66G) polymorphism were visualized as a 66-bp band for the
wild type (AA), a 66- and 44-bp band for the heterozygous (AG), and a 44-bp band for the mutant homozygous genotype. E: BHMT (G742A) genotypes. Cleaved
and electrophoresed PCR-RFLP products originated fragments of 163, 90, and 71 bp. All 3 bands were present in the heterozygote genotypes (GA), only 169-
and 71-bp fragment were present in wild type (GG), while 169- and 90-bp fragments were present in the mutant homozygous genotype (AA). F: CBS (68-bp
ins) genotypes. Wild-type genotypes were identified by 184-bp band (wt). Heterozygote genotypes (et) carrying the 68-bp insertion variant were identified by
digestion products of the 252-bp fragment, in addition to the normal 184-bp fragment. The mutant homozygous genotype was not found neither in control subjects
nor in athletes.
Table 2. Allele and genotype frequencies of the polymorphisms of MTHFR, MTR, MTRR, BHMT, and CBS genes in control
and athlete groups
Genotypes
Allele Frequency, n(%)
Genotype Frequency, n(%)
P
a
Control Athletes
Control Athletes Observed Expected Observed Expected
MTHFR (C677T) 0.737
CC C 61 (58.7) C 93 (62.0) 14 (26.9) 16.0 25 (33.3) 23.0
CT T 43 (41.3) T 57 (38.0) 33 (63.5) 31.1 43 (57.3) 44.9
TT 5 (9.6) 4.9 7 (9.3) 7.1
MTHFR (A1298C) 0.0001
AA A 85 (80.2) A 92 (63.0) 34 (64.2) 23.1 21 (28.8) 31.9
AC C 21 (19.8) C 54 (37.0) 17 (32.1) 28.2 50 (68.5) 38.8
CC 2 (3.8) 1.7 2 (2.7) 2.3
MTR (A2756G) 0.018
AA A 91 (89.2) A 119 (79.3) 41 (80.4) 34.8 45 (60.0) 51.2
AG G 11 (10.8) G 31 (20.7) 9 (17.6) 15.4 29 (38.7) 22.6
GG 1 (2.0) 0.8 1 (1.3) 1.2
MTRR (A66G) 0,0001
AA A 88 (83.0) A 86 (57.3) 36 (67.9) 23.6 21 (28.0) 33.4
AG G 18 (17.0) G 64 (42.7) 16 (30.2) 24.8 44 (58.7) 35.2
GG 1 (1.9) 4.6 10 (13.3) 6.4
BHMT (G742A) 0,66
GG G 71 (74.0) G 106 (70.7) 25 (52.1) 24.6 38 (50.7) 38.4
GA A 25 (26.0) A 44 (29.3) 21 (43.8) 19.9 30 (40.0) 31.1
AA 2 (4.2) 3.5 7 (9.3) 5.5
CBS (68-bp ins) 0.734
b
wt Wt 105 (97.2) Wt 144 (96.0) 51 (94.4) 50.2 69 (92.0) 69.8
hetero Ins 3 (2.8) Ins 6 (4.0) 3 (5.6) 3.8 6 (8.0) 5.2
homo 0 (0.0) 0.0 0 (0.0) 0.0
a
Exact test (generalization of Fisher’s exact test for 2x2 contingency tables).
b
Fisher’s exact test for 2x2 contingency tables.
968 ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
or DM in the presence or absence of AZA for 72 or 96 h,
respectively.
Images of MHC-positive myotubes detected by immunoflu-
orescence (Fig. 4, Aand a) showed that the morphology of
cells that were hypomethylated by AZA in growth (GMA) or
differentiation medium (DMA) for 3 and 4 days was severely
altered relative to their respective control GM and DM.
The process by which cells with star-shaped morphology
typical of the early stages of differentiation were substituted
with elongated spindle-shaped cells, which aligned close to
each other in the later stages, was slower in GM and DM (96
h) than in GMA and DMA (72 h). In C2C12 cells cultured in
DM or GM in the absence of AZA, maximal myoblast fusion
and myotube dimension required longer times than in AZA-
stimulated cells. After 72 h of treatment, the length and
diameter percentage increase (Fig. 4, Band C) of the myotubes
cultured in GMA was 34.9 and 57.2% with respect to GM,
whereas that of myotubes in DMA was 25.0 and 23.7% with
respect to DM. At 96 h of differentiation, the length percentage
increase of the myotubes (Fig. 4b) treated with AZA is even
greater than their respective controls (GMA ⫽18.7% with
respect to GM and DMA ⫽17.7% with respect to DM). The
diameter percentage increase of the cells differentiated without
AZA (DM) seems to stop when they reach the same GM
myotube size (DM ⫽3.7% with respect to GM), while the
diameter percentage increase of the AZA-stimulated cells is
still growing (GMA ⫽75.8%, DMA ⫽79.6% with respect to
GM and DM) as shown in Fig. 4c.
DISCUSSION
Our in vivo studies in a cohort of elite athletes demonstrate
the presence of DNA polymorphisms of enzymes involved in
the homocysteine cycle leading to reduced DNA methylation.
Therefore, we set up for in vitro experiments where DNA
hypomethylation was induced in murine myoblasts demon-
strating an higher rate of activation and differentiation in
muscle fibers, leading to muscle hypertrophy and hyperplasia.
Taken together, in vivo and in vitro data indicate that elite
athletes have a genetic predisposition to DNA hypomethylation
Table 3. Ratio of odds of expressing the polymorphic
genotypes in control group to the odds of expressing the
same polymorphysms in athlete group
Genotypes
OR
(control/athletes) 95% CI P
MTHFR (C677T)
CC
CT 0.73 0.3–1.6 0.44
TT 0.78 0.2–2.9 0.74
b
MTHFR (A1298C)
AA
AC 4.76 2.2–10.3 ⬍0.001
CC 1.62 0.2–12.4 0.64
b
MTR (A2756G)
AA
AG 2.94 1.2–6.9 0.02
b
GG 0.91 0.1–15.0 1.00
b
MTRR (A66G)
AA
AG 4.71 2.1–10.3 ⬍0.001
GG 17.14 2.0–143.5 0.002
b
BHMT (G742A)
GG
GA 0.94 0.4–2.0 0.87
AA 2.30 0.4–12.0 0.47
b
CBS (68-bp ins)
wt
hetero 1.48 0.4–6.2 0.73
b
homo
OR, odds ratio; CI, confidence interval.
b
Fisher’s exact test (2 sided)
computed for 2x2 contingency table.
Fig. 3. A: when the growth factors are used up, the committed myoblasts exit cell cycle, cease dividing and begin to elongate (early differentiation). In a second
stage mononucleated myocytes begin to fuse, forming fused multinucleated myocytes (intermediate differentiation) that become wider and longer, becoming
organized into multinucleate myotubes (late differentiation). B: the basic helix-loop-helix (bHLH) transcription factors function as dominant activators of skeletal
muscle differentiation during early differentiation (MyoD, Myf-5), intermediate differentiation (Myf-6) and stimulate MHC gene expression in myotubes during
the late phase of muscle differentiation. The histograms depict the concentration of Myf-5, MyoD, Myf-6, and MHC proteins in untreated and AZA-treated
myoblasts at 4, 24, 48, and 96 h, respectively . The data of 3 independent experiments are expressed as fold changes. Protein quantification was adjusted for the
corresponding ␣-tubulin level, and the data are expressed as means ⫾SD. The Pvalue was obtained by comparing GMA, DM, and DMA vs. GM (⫽1).
969ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
Fig. 4. Effects of AZA on myoblast differentiation. MHC in myotubes of control (GM, DM) and treated with AZA (GMA, DMA) was detected by
immunofluorescence 72 h (A) and 96 h (a) after differentiation. The diameters of MHC-positive myotubes differentiated for 72 h (B) and 96 h (b) were measured
and the average, expressed in cm, were graphically represented. The length of MHC-positive myotubes differentiated for 72 h (C) and 96 h (c) were measured
and the average, expressed in cm, were graphically represented. The data of 3 independent experiments are expressed as means ⫹SD.
970 ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
and synthesis that we demonstrated to be some of the factors
leading to myogenic differentiation stimulation and muscle
mass increase (Fig. 5).
The primary mechanisms involved in muscle mass increase
are the proliferative activation (22, 23) and myogenic differ-
entiation of mononuclear satellite cells that fuse with the
enlarging myofiber, as well as an increased protein synthesis
(10, 14, 30), although some evidence is present that hyperpla-
sia of existing muscle fibers also plays a role in muscle
enlargement along with hypertrophy alone (1).
Several factors besides DNA hypomethylation induce mus-
cle hypertrophy. Skeletal muscle-specific growth factors and
hormones modulate satellite cell activity during muscle growth
and development; however, a single bout of voluntary high-
intensity exercise was shown to increase the number of satellite
cells, and repeated bouts of exercise are sufficient for the
satellite cell to undergo terminal differentiation (5), indicating
in physical exercise a major stimulus in muscle hypertrophy.
Therefore, in elite athletes, two major stimuli inducing muscle
hypertrophy are present, one hereditary (DNA hypomethyla-
tion due to polymorphic variants of enzymes of the DNA
methylation cycle) and one environmental (high intensity and
frequency of physical exercise training).
Studies in human bladder smooth muscle cells (16) have
confirmed that a cyclic stretch-relaxation signal is able to
increase the rate of protein synthesis and accelerate entry into
the S phase, when DNA synthesis or replication occurs, induc-
ing both hypertrophic and hyperplastic responses.
An adaptive response to exercise training is the increment of
active muscle mass achieved by an increase in the volume of
individual myofibers (11). To sustain hypertrophic enlarge-
ment, myofibers need the insertion of new nuclei. Muscle
satellite cell proliferation, differentiation, and fusion with the
existing myofibers are known to be responsible for postnatal
Fig. 5. Proposed skeletal muscle mechanism
derived from the study data. Our results
suggest that in athletes the increased speed
of entry into S phase, and then DNA synthe-
sis, stimulated by physical exercise, should
be facilitated by the reduced enzymatic ac-
tivity due to the polymorphic MTHFR gene,
which increases 5–10-methylenetetrahydro-
folate availability. A larger amount of sub-
strate becomes thus available for the in-
creased speed of entry into S phase and then
DNA synthesis, stimulated by physical ex-
ercise facilitating hypertrophic responses.
Likewise, as a consequence of the less effi-
cient MS and MSR enzymes due to genetic
variants as MTR (AG) and MTRR (AG)
heterozygous genotypes, cellular 5-methyl-
tetrahydrofolate is depleted, inducing DNA
hypomethylation and a consequent increase
of muscle-specific genes (Myf-5, MyoD,
Myf-6 and MHC) expression, crucial in the
myogenic differentiation. The data we ob-
tained support our hypothesis of a functional
role for the studied genetic variants to deter-
mine a role in athletic performance and iden-
tify polymorphisms of these genes as medi-
ators of the myogenic and hypertrophic ef-
fects exerted by physical exercise on skeletal
muscles.
971ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
muscle hypertrophy, by supplying more DNA to the individual
fiber (4). One of the main physiological functions of meth-
ylation cycle can be classified as DNA synthesis by the
activity of MTHFR enzyme responsible for transferring the
methyl group from 5-10-methylenetetrahydrofolate to ura-
cil, converting it to thymine used for DNA synthesis and
repair (26). The reduced MTHFR enzyme activity (7), due
to the C677T gene polymorphism, engenders an accumula-
tion of 5-10-methylenetetrahydrofolate, increasing the avai-
lability of methyl groups.
Our data suggest that in athletes the increased speed of entry
into S phase, and then DNA synthesis, stimulated by physical
exercise, should be facilitated by the presence of MTHFR
polymorphisms, which increase the 5-10-methylenetetrahydro-
folate availability.
Moreover, DNA stability is affected also through another
pathway (see Fig. 1) regulated by MS and MSR enzyme
activity. 5-Methyltetrahydrofolate also serves as methyl donor
in the methylation of specific cytosines in DNA, an epigenetic
mechanism to exert transcriptional control and regulate gene
transcription.
While in vitro models are routinely used to try to elucidate
signaling pathways, the genetic factors influencing muscle
response to exercise training are more difficult to dissect out,
although correlation studies of polymorphic variation associ-
ated with muscle size and strength were performed (33, 24),
and several papers have been published on this topic.
The role of DNA methylation as a locking mechanism for an
important event, such as tissue-specific gene expression during
development, is well established (8, 27, 20). In particular,
several studies of single muscle genes (2, 12, 18, 37) have
demonstrated a role of hypomethylation in the induction of
muscle differentiation (21, 29, 31). Of the several tissue-
specific transcription factors, a family of transcriptional regu-
lators (MyoD, Myf-5, and Myf-6) is known to be closely
involved in the commitment to myogenic fate and in the
induction of muscle-specific genes expression (MHC).
It might be expected that, in athletes, as a consequence of
reduced MTHFR, MS, and MSR enzyme activity due to
genetic variants [namely MTHFR (AC), MTR (AG), and
MTRR (AG) heterozygous genotypes], cellular 5-methyltetra-
hydrofolate is depleted, inducing DNA hypomethylation and a
consequent increase of muscle-specific genes expression. To
verify this hypothesis, we utilized a murine cell line model to
study the effects of DNA hypomethylation by AZA (5 M), a
DNA methyltransferase inhibitor, on the regulation of muscle-
specific genes expression, myoblast differentiation, and hyper-
trophy, using the mouse myoblast C2C12 cell line model
system. This immortalized cell line derived from satellite cells
has routinely been used as a model for skeletal muscle devel-
opment and skeletal muscle differentiation. In these cells, DNA
hypomethylation increased the expression of the myogenic
regulatory factors Myf-5 and MyoD, required for myogenic
determination, and Myf-6, which has a role in terminal differ-
entiation inducing an increment in MHC muscle-specific gene
expression, confirming that the mononucleated cells finally
become muscle cells. The greater capability of hypomethylated
cells, not only to differentiate earlier in mature muscle cell, but
also to support an hypertrophy process that leads treated cells
to reach length and diameter size considerably higher than
unhypomethylated cells is confirmed by the immunofluores-
cence images.
Elite athletes have normally an high insulin sensitivity.
Although several substrates and cofactors involved in the
methylation cycle are shown to modulate insulin action, the
level of homocysteine, involved in this metabolic pathway, is
important for the interpretation of the present findings. Li et al.
(13) showed that high homocysteine concentration impairs
insulin sensitivity via a proinflammatory action and a resistin-
mediated effect. In our athletes bearing the polymorphic vari-
ants, the methylation of homocysteine to methionine is reduced
so that it is very likely that the all cycle flux is down regulated.
In contrast, CBS function was found to be normal in our
athletes, explaining the normal homocysteine levels. More
directly related to insulin sensitivity are probably polymor-
phisms of the respiratory chain enzymes, not under study in
this experimental setting.
In conclusion, the significant increase of MTHFR A1298C,
MTR A2756G, and MTRR A66G polymorphic variant fre-
quency, in athletes with respect to controls, leads us to spec-
ulate that these polymorphisms represent a genetic factor that
determines athletic performance in association with other en-
vironmental factors. Significant progress has been made in
recent years to understand the mechanisms that induce both
skeletal muscle differentiation and hypertrophy, but our data
provide the first evidence of a potential functional role for the
genetic variants studied herein to determine a role in athletic
performance. Therefore, the search for polymorphisms of these
genes as mediators of the myogenic and hypertrophic effects
exerted by exercise on skeletal muscles may become a routine
genetic test in sport medicine.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
REFERENCES
1. Antonio J, Gonyea WJ. Skeletal muscle fiber hyperplasia. Med Sci Sports
Exerc 25: 1333–1345, 1993.
2. Brunk BP, Goldhamer DJ, Emerson CP Jr. Regulated demethylation of
the myoD distal enhancer during skeletal myogenesis. Dev Biol 77:
490 –503, 1996.
3. Camp VM, Chipponi J, Faraj BA. Radioenzymatic assay for direct
measurement of plasma pyridoxal 5=-phosphate. Clin Chem 29: 642–644,
1983.
4. Chung KY, Johnson BJ. Application of cellular mechanisms to growth
and development of food producing animals. J Anim Sci 86: E226 –E235,
2008.
5. Crameri RM, Langberg H, Magnusson P, Jensen CH, Schrøder HD,
Olesen JL, Suetta C, Teisner B, Kjaer M. Changes in satellite cells in
human skeletal muscle after a single bout of high intensity exercise. J
Physiol 558: 333–340, 2004.
6. Fermo I, Arcelloni C, Mazzola G, D’Angelo A, Paroni R. High-
performance liquid chromatographic method for measuring total plasma
homocysteine levels. J Chromatogr B Biomed Sci Appl 719: 31–36, 1998.
7. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG,
Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, Rozen
R. A candidate genetic risk factor for vascular disease: a common
mutation in methylenetetrahydrofolate reductase. Nat Genet 10: 111–113,
1995.
8. Geiman TM, Muegge K. DNA methylation in early development. Mol
Reprod Dev 77: 105–113, 2010.
9. Giaccaglia V, Nicklas B, Kritchevsky S, Mychalecky J, Messier S,
Bleecker E, Pahor M. Interaction between angiotensin converting en-
zyme insertion/deletion genotype and exercise training on knee extensor
strength in older individuals. Int J Sports Med 29: 40 –44, 2008.
972 ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
10. Goldspink DF, Garlick PJ, McNurlan MA. Protein turnover measured
in vivo and in vitro in muscles undergoing compensatory growth and
subsequent denervation atrophy. Biochem J 210: 89 –98, 1983.
11. Green H, Goreham C, Ouyang J, Ball-Burnett M, Ranney D. Regu-
lation of fiber size, oxidative potential, and capillarization in human
muscle by resistance exercise. Am J Physiol Regul Integr Comp Physiol
276: R591–R596, 1999.
12. Jones PA, Wolkowicz MJ, Rideout 3rd WM, Gonzales FA, Marziasz
CM, Coetzee GA, Tapscott SJ. De novo methylation of the MyoD1 CpG
island during the establishment of immortal cell lines. Proc Natl Acad Sci
USA 87: 6117–6121, 1990.
13. Li Y, Jiang C, Xu G, Wang N, Zhu Y, Tang C, Wang X. Homocysteine
upregulates resistin production from adipocytes in vivo and in vitro.
Diabetes 57: 817–27, 2008.
14. MacDougall JD, Sale DG, Alway SE, Sutton JR. Muscle fiber number
in biceps brachii in bodybuilders and control subjects. J Appl Physiol 57:
1399 –1403, 1984.
15. Olteanu H, Munson T, Banerjee R. Differences in the efficiency of
reductive activation of methionine synthase and exogenous electron ac-
ceptors between the common polymorphic variants of human methionine
synthase reductase. Biochemistry 41: 13378 –13385, 2002.
16. Orsola A, Adam RM, Peters CA, Freeman MR. The decision to
undergo DNA or protein synthesis is determined by the degree of me-
chanical deformation in human bladder muscle cells. Urology 59: 779 –
783, 2002.
17. Ott N, Geddert H, Sarbia MJ. Polymorphisms in methionine synthase
(A2756G) and cystathionine beta-synthase (844ins68) and susceptibility
to carcinomas of the upper gastrointestinal tract. Cancer Res Clin Oncol
134: 405–410, 2008.
18. Paroush Z, Keshet I, Yisraeli J, Cedar H. Dynamics of demethylation
and activation of the alpha-actin gene in myoblasts. Cell 63: 1229 –1237,
1990.
19. Paz MF, Avila S, Fraga MF. Germ-line variants in methyl-group me-
tabolism genes and susceptibility to DNA methylation in normal tissues
and human primary tumors. Cancer Res 62: 4519 –4524, 2002.
20. Razin A, Cedar H. DNA methylation, and gene expression. Microbiol
Rev 55: 451–458, 1991.
21. Scarpa S, Lucarelli M, Palitti F, Carotti D, Strom R. Simultaneous
myogenin expression and overall DNA hypomethylation promote in vitro
myoblast differentiation. Cell Growth Differ 7: 1051–1058, 1996.
22. Seale P, Asakura A, Rudnicki MA. The potential of muscle stem cells.
Dev Cell 1: 333–342, 2001.
23. Seale P, Rudnicki MA. A new look at the origin, function, and “stem-
cell” status of muscle satellite cells. Dev Biol 218: 115–124, 2000.
24. Seibert MJ, Xue QL, Fried LP, Walston JD. Polymorphic variation in
the human myostatin (GDF-8) gene and association with strength mea-
sures in the Women’s Health and Aging Study II cohort. J Am Geriatr Soc
49: 1093–1096, 2001.
25. Selhub J. Homocysteine metabolism. Annu Rev Nutr 19: 217–246, 1999.
26. Smulders YM, Stehouwer CD. Folate metabolism and cardiovascular
disease. Semin Vasc Med 5: 87–97, 2005.
27. Song F, Mahmood S, Ghosh S, Liang P, Smiraglia DJ, Nagase H, Held
WA. Tissue specific differentially methylated regions (TDMR): Changes
in DNA methylation during development. Genomics 93: 130 –139, 2009.
28. Sunden SL, Renduchintala MS, Park EI, Miklasz SD, Garrow TA.
Betaine-homocysteine methyltransferase expression in porcine and human
tissues and chromosomal localization of the human gene. Arch Biochem
Biophys 345: 171–174, 1997.
29. Szyf M, Rouleau J, Theberge J, Bozovic V. Induction of myogenic
differentiation by an expression vector encoding the DNA methyltrans-
ferase cDNA sequence in the antisense orientation. J Biol Chem 267:
12831–12836, 1992.
30. Taylor NA, Wilkinson JG. Exercise-induced skeletal muscle growth.
Hypertrophy or hyperplasia? Sports Med 3: 190 –200, 1986.
31. Taylor SM, Jones PA. Multiple new phenotypes induced in 10T1/2 and
3T3 cells treated with 5-azacytidine. Cell 17: 771–779, 1979.
32. Terruzzi I, Pellegatta F, Luzi L. Differential p70S6k and 4E-BP1
regulation by insulin and amino acids in vascular endothelial and smooth
muscle cells. Acta Diabetol 42: 139 –146, 2005.
33. Thompson PD, Moyna N, Seip R, Price T, Clarkson P, Angelopoulos
T, Gordon P, Pescatello L, Visich P, Zoeller R, Devaney JM, Gordish
H, Bilbie S, Hoffman EP. Functional polymorphisms associated with
human muscle size and strength. Med Sci Sports Exerc 36: 1132–1139,
2004.
34. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins
from polyacrylamide gels to nitrocellulose sheets: procedure and some
applications. Proc Natl Acad Sci USA 76: 4350 –4354, 1979.
35. Ulrey CL, Liu L, Andrews LG, Tollefsbol TO. The impact of metabo-
lism on DNA methylation. Hum Mol Genet 14, Suppl 1: R139 –R14, 2005.
36. Van der Put NM, Gabreels F, Stevens EM. A second common mutation
in the methylenetetrahydrofolate reductase gene: an additional risk factor
for neural-tube defects? Am J Hum Genet 62: 1044 –1051, 1998.
37. Warnecke PM, Clark SJ. DNA methylation profile of the mouse skeletal
alpha-actin promoter during development and differentiation. Mol Cell
Biol 19: 164 –172, 1999.
38. Weisberg IS, Park E, Ballman KV. Investigations of a common genetic
variant in betaine-homocysteine methyltransferase (BHMT) in coronary
artery disease. Atherosclerosis 167: 205–214, 2003.
39. Weisberg I, Tran P, Christensen B, Sibani S, Rozen R. A second
genetic polymorphism in methylenetetrahydrofolate reductase (MTHFR)
associated with decreased enzyme activity. Mol Genet Metab 64: 169 –
172, 1998.
40. Wilson A, Platt R, Wu Q. A common variant in methionine synthase
reductase combined with low cobalamin (vitamin B12) increases risk for
spina bifida. Mol Genet Metab 67: 317–323, 1999.
41. Yang N, MacArthur DG, Gulbin JP, Hahn AG, Beggs AH, Easteal S,
North K. ACTN3 genotype is associated with human elite athletic
performance. Am J Hum Genet 73: 627–631, 2003.
42. Zhang G, Dai C. Gene polymorphisms of homocysteine metabolism-
related enzymes in Chinese patients with occlusive coronary artery or
cerebral vascular diseases. Thromb Res 104: 187–195, 2001.
973ATHLETES AND METHYL CYCLE ENZYMES POLYMORPHISMS
Physiol Genomics •VOL 43 •www.physiolgenomics.org
on December 28, 2011physiolgenomics.physiology.orgDownloaded from
