Clin Genet 2003: 63: 139±144 Copyright # Blackwell Munksgaard 2003

Printed in Denmark. All rights reserved CLINICAL GENETICS

ISSN 0009-9163

Short Report

The I allele of the angiotensin-converting

enzyme gene is associated with an increased
percentage of slow-twitch type I fibers in
human skeletal muscle
a b c
Zhang B, Tanaka H, Shono N, Miura S, Kiyonaga A, Shindo M, Saku B Zhang , H Tanaka , N Shono ,
K. The I allele of the angiotensin-converting enzyme gene is associated S Miuraa, A Kiyonagac,
with an increased percentage of slow-twitch type I fibers in human M Shindoc and K Sakua
skeletal muscle. a
Clin Genet 2003: 63: 139±144. # Blackwell Munksgaard, 2003 Department of Cardiology, Fukuoka
University School of Medicine, Fukuoka,
b
Faculty of Sports and Health Science,
The insertion (I) allele of the human angiotensin-converting enzyme Fukuoka University, Fukuoka, Japan and
(ACE) gene is associated with lower serum and tissue ACE activity, and c
Department of Community Health Science,
with greater endurance performance and enhanced mechanical efficiency Saga Medical School, Saga
of trained muscle. We tested the hypothesis that the ACE-I allele may be
associated with increased slow-twitch fiber, which is more efficient than
fast-twitch fiber in low-velocity contraction, by examining the
association between the ACE genotype and skeletal muscle fiber (SMF)
types in 41 untrained healthy young volunteer subjects (31 males, 10
females, age 24  3 years). Skeletal muscle samples were taken from the
left vastus lateralis using the needle-biopsy method. Slow-twitch type I
fibers and fast-twitch type IIa and IIb fibers were classified
histochemically based on staining for myosin adenosine triphosphatase
(ATPase) activity at different pH values. Amylase-periodic acid-Schiff
staining was used to visualize capillaries around fibers. ACE-II subjects
had significantly (p < 0.01) higher percentages of type I fibers
(50.1  13.9% vs 30.5  13.3%) and lower percentages of type IIb fibers
(16.2  6.6% vs 32.9  7.4%) than ACE-DD subjects. The linear trends
Key words: ACE genotype ^ exercise
for decreases in type I fibers and increases in type IIb fibers from ACE-II endurance ^ muscle biopsy ^ muscle fiber
! ID ! DD genotypes were significant as assessed by an analysis of type ^ skeletal muscle
variance. The ratio of type I:II fibers also differed according to the ACE Corresponding author: Keijiro Saku, MD,
genotype. A multivariate logistic regression analysis showed that the PhD, FACP, FACC, Department of Cardiology,
ACE-I allele had significant additive and recessive (codominant) effects Fukuoka University School of Medicine,
on the increased type I fibers and the ratio of type I:II fibers. No specific 7-45-1Nanakuma Jonan-ku, Fukuoka
814-0180, Japan.Tel.: ‡81 92 8011011;
pattern of capillarization was observed among the three ACE genotypes. Fax: ‡81 92 865 2692;
In conclusion, the ACE-I allele was associated with increased type I e-mail: saku-k@cis.fukuoka-u.ac.jp
SMF, which may be a mechanism for the association between the ACE Received 8 August 2002, revised and
genotype and endurance performance. accepted for publication 9 October 2002

The circulating human renin-angiotensin system
(RAS) (1) plays an important role in circulatory
homeostasis. Angiotensin-converting enzyme (ACE)
Parts of this study were presented at the 72nd American Heart
Association Scientific Sessions, 10 November 1999, in Atlanta.
produces the vasoconstrictor angiotensin II and
metabolizes vasodilatory kinins. A local RAS also
exists in skeletal muscle (2), and may work with other
metabolic tissue to influence the whole-body energy
balance (3). The ACE gene shows a polymorphism
based on the presence [insertion (I)] or absence

139
Zhang et al.
[deletion (D)] of a 287-bp Alu-repeat element
within intron 16 (4). The I allele is associated with
lower serum (5) and tissue ACE activities (6).
The ACE I/D polymorphism has recently
attracted considerable attention with regard to its
association with exercise performance. The I allele
has been associated with superior exercise endur-
ance, as it is found at greater than expected fre-
quency in mountaineers (7), rowers (8), and elite
distance runners (9). Conversely, the D allele has
been associated with power performance, and has
been found at a greater than expected frequency in
sprinters (9) and short-distance swimmers (10).
Several studies have failed to identify any associa-
tion between the ACE I/D polymorphism and elite
endurance performance (11±13) due to the use of
subjects from mixed sporting disciplines (10, 14).
Although the mechanism by which the ACE I/D
polymorphism is associated with endurance per-
formance is not clear, William et al. (15) reported
that the presence of the ACE-I allele confers
enhanced mechanical efficiency (energy used per
unit power output) in aerobically trained skeletal
muscle.
Human skeletal muscle is composed of a hetero-
geneous collection of muscle fibers of different
types with various metabolic and contractile pro-
perties. Muscle fibers are generally classified as being
either slow-twitch (type I) or fast-twitch (type IIa
and IIb) based on their histochemical staining for
myosin adenosine triphosphatase (ATPase) activity
(16). ATPase-based type I, IIa, and IIb fibers cor-
respond to myosin heavy chain (MHC) isoforms
[MHCI, MHCIIa, and MHCIIx/d, respectively
(16)], and to metabolic enzyme-based fiber typing
[slow-twitch oxidative (SO), fast-twitch oxidative-
glycolytic (FOG), fast-twitch glycolytic (FG) fibers,
respectively (17)].
Because muscular efficiency is related to the type
of muscle fiber [slow-twitch type I fibers are more
efficient than type II fibers when contractions are
performed isometrically or at relatively low veloci-
ties (18)], we tested the hypothesis that the ACE-I
allele may be related to an increased percentage
oftype I skeletal muscle fiber (SMF). Because
muscle fibers can be induced to transition from
type I to type II by specific training (17), we
examined the association between the ACE I/D
polymorphism and SMF types in untrained young
healthy subjects.
Methods
Study subjects
The subjects consisted of 41 young healthy Japa-
nese volunteers. They were undergraduate or grad-
140
uate college students, and were not participating in
any specific training. This protocol was approved
by the Ethics Committee of Fukuoka University
School of Medicine and written informed consent
was obtained from all of the subjects.
Muscle biopsy
Skeletal muscle samples were taken from the vastus
lateralis using the needle-biopsy method, immedi-
ately mounted in an embedding medium (OCT
compound) and frozen in isopentane cooled with
liquid nitrogen as described previously (19, 20).
Serial 10 mM cross-sections were cut using a cryo-
stat at 20  C, pre-incubated at pH 4.3, 4.6, and
10.3, and incubated for the histochemical reaction
for myofibrillar ATPase at pH 9.4 to identify dif-
ferent types of fibers. Figure 1 shows serial sections
of human skeletal muscle using histochemical reac-
tions after pre-incubation at pH 4.6 for typical
ACE-II (Fig. 1a) and DD subjects (Fig. 1b). As
shown, pre-incubation at pH 4.6 did not inhibit
the ATPase reaction in type I fibers and inhibited
the ATPase reaction more in type IIa than in IIb
fibers, so that type I, IIa, and IIb fibers could be
easily recognized based on their staining intensity:
type I fibers were stained dark, type IIa fibers were
stained light, and type IIb fibers had intermediate
staining intensity. More than 200 fibers were
counted for each subject, and the cross-sectional
area was calculated. Fiber type was expressed
as the percent distribution area of the fiber type
(types I, IIa, and IIb). Recently, fibers that are
primarily denoted type IIb in human skeletal
muscle have been shown to resemble type IIx
fibers in rat muscle. Capillaries were visualized by
amylase-periodic acid-Schiff staining as described
Fig. 1. Serial sections of human skeletal muscle using
histochemical reactions after pre-incubation at pH 4.6 for
typical ACE-II (a) and DD subjects (b).
 ACE genotype, skeletal muscle fiber types, and exercise
previously (19, 20). Capillary density was expressed Table1. Distribution of the ACE genotype in all of the subjects and
as the number of capillaries around each fiber the characteristics of subjects according to the ACE genotype
type. ACE genotypes
II ID DD
DNA extraction and ACE gene polymorphism
n 15 (39.0%) 17 (46.3%) 6 (14.6%)
Blood for leukocyte isolation and subsequent Age (years) 24.1  3.9 24.4  3.0 22.8  1.2
DNA preparation was collected in ethylenediami- Gender (F/M) 5/11 4/15 1/5
netetraacetic acid (EDTA)-containing tubes at a BMI (kg/m2) 23.3  3.7 22.5  2.6 21.0  1.9
final concentration of 50 mmol/l. Genomic DNA BMI: body mass index; ACE: angiotensin-converting enzyme.
was amplified as previously described using the
polymerase chain reaction (PCR) with primers
flanking the polymorphic region (21, 22). PCR pro- differed among ACE-II, ID, and DD subjects
ducts of 490 and 290 bp were separated on 1.5% (Table 1), as assessed by an analysis of variance.
agarose gels and visualized by ethidium-bromide Figure 2(a) shows the percent distributions of the
staining, as described previously (23). Because the different types of skeletal muscle fiber (types I, IIa,
D allele in heterozygous samples is preferentially and IIb) among ACE-II, ID, and DD subjects. In
amplified, each sample that was found to have the all of the subjects, the average percentage of type
DD genotype was subjected to a second, indepen- I fiber was 43.9  13.6%, similar to that (45±55%)
dent PCR amplification with a primer pair that reported in untrained men and women (17). As
recognizes an insertion-specific sequence to avoid shown in Fig. 2(a), ACE-II subjects had a higher
mistyping the DD genotype (24). percentage of type I fibers and a lower percentage
of type IIb fibers than ACE-DD subjects, as
Statistical analysis assessed by ANOVA and Dunnett's multiple compari-
son test, while there was no significant difference
All of the statistical analyses were performed using in the percentage of type IIa fibers by ACE geno-
the Statistical Analysis System (Version 6.12, SAS type. As shown in Table 2, the linear decrease in
Institute Inc., Cary, NC). Differences in variables type I fibers and increase in type IIb fibers from
among ACE genotypes were examined by an ana- ACE-II ! ID ! DD genotypes were significant
lysis of variance (ANOVA) and multiple comparisons
were made using Scheffe and Dunnett's multiple
comparison tests (25). Linear trends in the percen-
tages of SMF types across ACE genotypes were
also examined by ANOVA. The additive (scores of 0,
1, and 2 for DD, ID, and II, respectively), domi-
nant (scores of 0 for DD and 1 for ID and II
combined), and recessive effects (scores of 0 for
DD and ID combined and 1 for II) of ACE-I allele
on the distributions of the types of skeletal muscle
fibers were assessed by a multiple logistic regression
analysis (25). Dummy variables were produced
using the median values in all of the subjects. A
p-value of <5% was considered significant.

Results
Table 1 shows the frequency distribution of the ACE
genotype in all of the subjects and the characteris-
tics of subjects according to ACE-II, ID, and DD
genotypes. The frequencies of the ACE-II, ID and
DD genotypes among the 41 subjects (39.0%,
46.3%, and 14.6%, respectively) agreed with
those predicted by the Hardy±Weinberg equili-
brium (x2 ˆ 0.0004, p ˆ 0.998), and were similar
Fig. 2. (a) Percentages of the different types of skeletal
to those reported in healthy Japanese individuals muscle fiber (types I, IIa, and IIb) and (b) the ratio of type
(37.3%, 48.1%, and 14.7%) (26). Neither age, I:II fibers in ACE-II, ID, and DD subjects. * p < 0.05, II vs.
gender, nor body mass index (BMI) significantly DD, by ANOVA and multiple comparison of Dunnett.

141
Zhang et al.
Table 2. Linear trend across ACE genotypes, as tested by an
analysis of variance
Variable Type II sum of F value
square
Type I fibers 1837 5.88
Type IIa fibers 111 0.29
Type IIb fibers 1230 6.43
Ratio of type I : II fibers 2.5 5.52
(p <0.01), as assessed by an ANOVA. As shown in
Fig. 2(b) and Table 2, the ratio of the percentages
of type I:II fibers also differed according to the
ACE genotype: ACE-II subjects had a higher
ratio of type I:II fibers than ACE-DD subjects
(Fig. 2b) and the linear trend for a decrease in the
ratio of type I:II fibers from ACE-II ! ID ! DD
genotypes was significant (Table 2) as assessed by
ANOVA, indicating that the distributions of different
types of skeletal muscle fibers differed among
ACE-II, ID, and DD subjects. Capillary densities,
as expressed as the number of capillaries around
each fiber type, were not significantly different
among the three ACE genotypes (data not shown).
Table 3 shows the results of a multiple logistic
regression analysis that tested the association
between the ACE-I allele and the percentages of
type I and IIb skeletal muscle fiber after adjusting
for age, gender, and BMI. As shown, after con-
trolling for age, gender, and BMI, the ACE-I allele
was additively and positively associated with
the percentage of type I SMF and the ratio of
type I:II SMF, and additively but negatively
associated with the percentage of type IIb SMF,
confirming the results in Table 2. As also shown
in Table 3, the recessive (codominant) effects of the
ACE-I allele on the percentages of type I and IIb,
and the ratio of type I:II fibers were also sign-
ificant. The dominant effects of the ACE-I allele
(II and ID vs DD) were not tabulated because the
data may not be reliable due to the large variance
produced by the small number of ACE-DD
subjects (n ˆ 6).
p value
<0.01 Discussion
n.s.
<0.01 Our result that the ACE-II genotype was asso-
<0.01 ciated with an increased percentage of type I
SMF compared with the DD genotype provides a
possible mechanism for the reported association of
the ACE-I allele with greater endurance
performance (7±9), and enhanced mechanical
(15) and metabolic (3) efficiency of trained muscle,
as type I SMF is more efficient than type II SMF in
slow contraction (18). Our finding that the ACE-
DD genotype had a greater percentage of type IIb
SMF than the ACE-II genotype may support the
notion that the ACE-D allele is associated with
power performance (9, 10).
Our findings indicated that the distribution of
type I and II SMF, as reflected by the ratio of
type I:II fibers, differed according to the ACE
genotype. Muscle fibers can adapt to changing
demands by changing size or fiber-type composi-
tion. This adaptive responsiveness is the basis of
fiber-type transition (27). The transition between
FG (IIb) and FOG (IIa) is the most common due
to decreases or increases in the amount and/or
intensity of performance (16), although this meta-
bolic adaptation does not necessarily affect the
myosin ATPase-based fiber-type distribution.
However, the transition between types I and II
needs to be induced by specific training (17):
athletes with the highest aerobic and endurance
capacities, such as distance runners and cross-
country skiers, have the highest percentage of
slow-twitch fibers, often as high as 90±95% in the
leg's gastrocnemius muscle, while weight lifters, ice
hockey players, and sprinters tend to have more
fast-twitch fibers and a relatively low aerobic
capacity. Therefore, because subjects in the present
study were untrained college students, our finding

Table 3. Multiple logistic regression analysis of the additive, recessive, and dominant effects of the ACE-I allele on type I and IIb SMF, and
the ratio of type I:II SMF
SMF Model Effect of the Regression coefficient Wald p-value
types studied I allele  standard error x2
Type I fibers
II vs ID vs DD Additive 1.53  0.61 6.23 0.013
II vs ID and DD Recessive 1.92  0.78 6.09 0.014
Type IIb fibers
II vs ID vs DD Additive 1.78  0.64 7.80 0.005
II vs ID and DD Recessive 1.68  0.74 5.22 0.022
Ratio of type I:II
fibers
II vs ID vs DD Additive 1.53  0.61 6.23 0.013
II vs ID and DD Recessive 1.92  0.78 6.09 0.014

142
 ACE genotype, skeletal muscle fiber types, and exercise
that the distribution of fiber types differed
according to ACE genotype may reflect genetic
determination of the characteristics of fiber-type
distribution (17).
Our result that the ACE-I allele was recessively
associated with the distribution of fiber types
(Table 3) is consistent with the codominant effect
of the ACE- I allele on endurance performance as
reported by Montgomery et al. (7) and on enhanced
mechanical efficiency of trained muscles as reported
by Williams et al. (15). It is not clear why the ACE
genotype is related to muscle fiber-type distribution
or whether or not the ACE I/D polymorphism may
be in linkage disequilibrium with the gene that
controls muscle fiber-type distribution. However,
because muscle fiber adaptations to endurance
exercise depend on the fiber type, although the
oxidative capacity of all fibers is increased (16),
and as type I fibers become faster through the
conversion of a myosin light chain isoform from
slow to fast isoforms (16, 27), it is possible that the
ACE-II genotype can improve the mechanical
efficiency of trained muscle (15) due to an increase
in slow-twitch muscle fibers. Because a person's
fiber composition is not the sole determination of
performance (17), the ACE-I allele may also be
associated with endurance performance through
some as yet unidentified mechanisms.
Because a muscle biopsy is not easy to perform in
normal volunteers, relatively few subjects were
examined, which may have limited the power of
this study. In addition, whether or not the associ-
ation between the ACE genotype and skeletal mus-
cle fiber types is also found in trained athletes will
require further investigation.
Acknowledgements
This work was supported by grants-in-aid from the Ministry of
Education, Science and Culture of Japan (Nos. 10670221,
10670693, 11670724, and 12670712), by research grants from
the Ministry of Health and Welfare, by a grant from the Uehara
Memorial Foundation (2002), and by a research grant
(No. 026001) from the Central Research Institute of Fukuoka
University.
References
1. Kem DC, Brown RD. Renin ± from beginning to end. N Engl
J Med 1990: 323: 1136±1137.
2. Dragovic T, Minshall R, Jackman HL, Wang LX, Erdos EG.
Kininase II-type enzymes. Their putative role in muscle
energy metabolism. Diabetes 1996: 45 (Suppl. 1): S34±S37.
3. Montgomery H, Clarkson P, Barnard M et al. Angioten-
sin-converting-enzyme gene insertion/deletion polymorph-
ism and response to physical training. Lancet 1999: 353:
541±545.
4. Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of
the insertion/deletion polymorphism of human angiotensin
converting enzyme gene (DCP1) (dipeptidyl carboxypepti-
dase 1). Nucleic Acids Res 1992: 20: 1433.
5. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P,
Soubrier F. An insertion/deletion polymorphism in the
angiotensin I-converting enzyme gene accounting for half
the variance of serum enzyme levels. J Clin Invest 1990: 86:
1343±1346.
6. Danser AH, Schalekamp MA, Bax WA et al. Angiotensin-
converting enzyme in the human heart. Effect of the
deletion/insertion polymorphism. Circulation 1995: 92:
1387±1388.
7. Montgomery HE, Marshall R, Hemingway H et al. Human
gene for physical performance. Nature 1998: 393: 221±222.
8. Gayagay G Yu B, Hambly B et al. Elite endurance athletes
and the ACE I allele ± the role of genes in athletic perfor-
mance. Hum Genet 1998: 103: 48±50.
9. Myerson S, Hemingway H, Budget R, Martin J, Humphries S,
Montgomery H. Human angiotensin I-converting enzyme
gene and endurance performance. J Appl Physiol 1999: 87:
1313±1316.
10. Woods D, Hickman M, Jamshidi Y et al. Elite swimmers
and the D allele of the ACE I/D polymorphism. Hum Genet
2001: 108: 230±232.
11. Karjalainen J, Kujala UM, Stolt A et al. Angiotensinogen
gene M235T polymorphism predicts left ventricular hyper-
trophy in endurance athletes. J Am Coll Cardiol 1999: 34:
494±499.
12. Rankinen T, Wolfarth B, Simoneau JA et al. No association
between the angiotensin-converting enzyme ID polymorph-
ism and elite endurance athlete status. J Appl Physiol 2000:
88: 1571±1575.
13. Taylor RR, Mamotte CD, Fallon K, van Bockxmeer FM.
Elite athletes and the gene for angiotensin-converting
enzyme. J Appl Physiol 1999: 87: 1035±1037.
14. Nazarov IB, Woods DR, Montgomery HE et al. The angio-
tensin converting enzyme I/D polymorphism in Russian
athletes. Eur J Hum Genet 2001: 9: 797±801.
15. Williams AG, Rayson MP, Jubb M et al. The ACE gene
and muscle performance. Nature 2000: 403: 614.
16. Scott W, Stevens J, Binder-Macleod SA. Human skeletal
muscle fiber type classifications. Phys Ther 2001: 81:
1810±1816.
17. William D, Frank I, Victor L. Exercise physiology. Phila-
delphia: Lea & Febiger, 1981: 330±332.
18. Coyle EF, Sidossis LS, Horowitz JF, Beltz JD. Cycling
efficiency is related to the percentage of type I muscle fibers.
Med Sci Sports Exerc 1992: 24: 782±788.
19. Shono N, Urata H, Saltin B et al. Effects of low intensity
aerobic training on skeletal muscle capillary and blood
lipoprotein profiles. J Atheroscler Theomb 2001: 9: 78±85.
20. Shono N, Mizuno M, Nishida H et al. Decreased skeletal
muscle capillary density is related to higher serum levels of
low-density lipoprotein cholesterol and apolipoprotein B in
men. Metabolism 1999: 48: 1267±1271.
21. Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P,
Soubrier F. An insertion/deletion polymorphism in the angio-
tensin I-converting enzyme gene accounting for the variance
of serum enzyme levels. J Clin Invest 1990: 86: 1343±1346.
22. Cambien F, Poirier O, Lecerf L et al. Deletion polymorph-
ism in the gene for angiotensin-converting enzyme is a
potent risk factor for myocardial infarction. Nature 1992:
359: 641±644.
23. Takezako T, Saku K, Zhang B et al. Angiotensin I converting
enzyme gene polymorphism and insulin resistance in patients
with angina pectoris. Am J Hypertens 1999: 12: 291±297.
143
Zhang et al.
24. Lindpaintner K, Pfeffer MA, Kreutz R et al. A prospective
evaluation of an angiotensin-converting-enzyme gene poly-
morphism and the risk of ischemic heart disease. N Engl J
Med 1995: 332: 706±711.
25. SAS/STAT user's guide, Version 6, 4th edn, Vol. 2. Cary,
NC: SAS Institute Inc., 1990: 891±1210.
144
26. Zaman MM, Yoshiike N, Date C et al. Angiotensin con-
verting enzyme genetic polymorphism is not associated with
hypertension in a cross-sectional sample of a Japanese
population: the Shibata Study. J Hypertens 2001: 19: 47±53.
27. Petter D, Staron RS. Mammalian skeletal muscle fiber type
transitions. Int Rev Cytol 1997: 170: 143±223.