The Muscle Fiber Profiles, Mitochondrial Content, and Enzyme Activities of the Exceptionally Well-Trained Arm and Leg Muscles of Elite Cross-Country Skiers

Article (PDF Available)inFrontiers in Physiology 9:1031 · August 2018with 290 Reads
DOI: 10.3389/fphys.2018.01031
Abstract
As one of the most physically demanding sports in the Olympic Games, cross-country skiing poses considerable challenges with respect to both force generation and endurance during the combined upper- and lower-body effort of varying intensity and duration. The isoforms of myosin in skeletal muscle have long been considered not only to define the contractile properties, but also to determine metabolic capacities. The current investigation was designed to explore the relationship between these isoforms and metabolic profiles in the arms (triceps brachii) and legs (vastus lateralis) as well as the range of training responses in the muscle fibers of elite cross-country skiers with equally and exceptionally well-trained upper and lower bodies. The proportion of myosin heavy chain (MHC)-1 was higher in the leg (58 ± 2% [34–69%]) than arm (40 ± 3% [24–57%]), although the mitochondrial volume percentages [8.6 ± 1.6 (leg) and 9.0 ± 2.0 (arm)], and average number of capillaries per fiber [5.8 ± 0.8 (leg) and 6.3 ± 0.3 (arm)] were the same. In these comparable highly trained leg and arm muscles, the maximal citrate synthase (CS) activity was the same. Still, 3-hydroxy-acyl-CoA-dehydrogenase (HAD) capacity was 52% higher (P < 0.05) in the leg compared to arm muscles, suggesting a relatively higher capacity for lipid oxidation in leg muscle, which cannot be explained by the different fiber type distributions. For both limbs combined, HAD activity was correlated with the content of MHC-1 (r2 = 0.32, P = 0.011), whereas CS activity was not. Thus, in these highly trained cross-country skiers capillarization of and mitochondrial volume in type 2 fiber can be at least as high as in type 1 fibers, indicating a divergence between fiber type pattern and aerobic metabolic capacity. The considerable variability in oxidative metabolism with similar MHC profiles provides a new perspective on exercise training. Furthermore, the clear differences between equally well-trained arm and leg muscles regarding HAD activity cannot be explained by training status or MHC distribution, thereby indicating an intrinsic metabolic difference between the upper and lower body. Moreover, trained type 1 and type 2A muscle fibers exhibited similar aerobic capacity regardless of whether they were located in an arm or leg muscle.
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ORIGINAL RESEARCH
published: 02 August 2018
doi: 10.3389/fphys.2018.01031
Edited by:
Gregoire P. Millet,
Université de Lausanne, Switzerland
Reviewed by:
Stefanos Volianitis,
Aalborg University, Denmark
Louise Deldicque,
Université catholique de Louvain,
Belgium
*Correspondence:
Niels Ørtenblad
nortenblad@health.sdu.dk
Deceased
Specialty section:
This article was submitted to
Exercise Physiology,
a section of the journal
Frontiers in Physiology
Received: 09 May 2018
Accepted: 11 July 2018
Published: 02 August 2018
Citation:
Ørtenblad N, Nielsen J, Boushel R,
Söderlund K, Saltin B and
Holmberg H-C (2018) The Muscle
Fiber Profiles, Mitochondrial Content,
and Enzyme Activities of the
Exceptionally Well-Trained Arm
and Leg Muscles of Elite
Cross-Country Skiers.
Front. Physiol. 9:1031.
doi: 10.3389/fphys.2018.01031
The Muscle Fiber Profiles,
Mitochondrial Content, and Enzyme
Activities of the Exceptionally
Well-Trained Arm and Leg Muscles of
Elite Cross-Country Skiers
Niels Ørtenblad1,2*, Joachim Nielsen1, Robert Boushel2, Karin Söderlund3, Bengt Saltin4
and Hans-Christer Holmberg5,6
1Department of Sports Science and Clinical Biomechanics, SDU Muscle Research Cluster, University of Southern Denmark,
Odense, Denmark, 2School of Kinesiology, University of British Columbia, Vancouver, BC, Canada, 3Åstrand Laboratory,
The Swedish School of Sport and Health Sciences, Stockholm, Sweden, 4Copenhagen Muscle Research Centre,
Copenhagen, Denmark, 5Swedish Winter Sports Research Centre, Mid Sweden University, Östersund, Sweden, 6School
of Sport Sciences, UiT The Arctic University of Norway, Tromsø, Norway
As one of the most physically demanding sports in the Olympic Games, cross-
country skiing poses considerable challenges with respect to both force generation and
endurance during the combined upper- and lower-body effort of varying intensity and
duration. The isoforms of myosin in skeletal muscle have long been considered not
only to define the contractile properties, but also to determine metabolic capacities. The
current investigation was designed to explore the relationship between these isoforms
and metabolic profiles in the arms (triceps brachii) and legs (vastus lateralis) as well
as the range of training responses in the muscle fibers of elite cross-country skiers with
equally and exceptionally well-trained upper and lower bodies. The proportion of myosin
heavy chain (MHC)-1 was higher in the leg (58 ±2% [34–69%]) than arm (40 ±3% [24–
57%]), although the mitochondrial volume percentages [8.6 ±1.6 (leg) and 9.0 ±2.0
(arm)], and average number of capillaries per fiber [5.8 ±0.8 (leg) and 6.3 ±0.3 (arm)]
were the same. In these comparable highly trained leg and arm muscles, the maximal
citrate synthase (CS) activity was the same. Still, 3-hydroxy-acyl-CoA-dehydrogenase
(HAD) capacity was 52% higher (P<0.05) in the leg compared to arm muscles,
suggesting a relatively higher capacity for lipid oxidation in leg muscle, which cannot
be explained by the different fiber type distributions. For both limbs combined, HAD
activity was correlated with the content of MHC-1 (r2= 0.32, P= 0.011), whereas
CS activity was not. Thus, in these highly trained cross-country skiers capillarization of
and mitochondrial volume in type 2 fiber can be at least as high as in type 1 fibers,
indicating a divergence between fiber type pattern and aerobic metabolic capacity. The
considerable variability in oxidative metabolism with similar MHC profiles provides a new
perspective on exercise training. Furthermore, the clear differences between equally
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Ørtenblad et al. Limb Muscle Profiles of Well-Trained
well-trained arm and leg muscles regarding HAD activity cannot be explained by training
status or MHC distribution, thereby indicating an intrinsic metabolic difference between
the upper and lower body. Moreover, trained type 1 and type 2A muscle fibers exhibited
similar aerobic capacity regardless of whether they were located in an arm or leg muscle.
Keywords: limb muscles, fiber plasticity, training, capillarization, mitochondria, IMCL, cross-country skiing
INTRODUCTION
Among the most demanding of Olympic sports, cross-country
skiing competitions on varying terrain require the use of a variety
of skiing techniques that involve the upper and/or lower body
to different extents. In recent decades, this primarily endurance
sport has changed to include novel events such as pursuit,
mass-start, and sprint races, with head-to-head competitions
and a wider range of speeds. Improved track preparation,
equipment, and skiing techniques, in combination with more
effective training (especially of upper-body strength/power and
endurance), have elevated racing speeds in general (Holmberg,
2015).
The necessity for today’s elite cross-country skier to combine
considerable endurance with rapid generation of high forces
during short contacts with the ground has enhanced focus on
optimizing related morphological and metabolic adaptations in
the skeletal muscles of the upper and lower body (Holmberg
et al., 2005). The relatively unique situation that both the leg
and arm muscles of elite cross-country skiers are highly trained
has allowed important comparisons that have helped provide
novel insights into the limits of physiological regulation and
performance, thereby helping to improve training routines.
Skeletal muscles are composed of motor units, containing
muscle fibers with the same specific characteristics (Canepari
et al., 2010). In general, muscle fibers are distinguished from
one another on the basis of (1) the contractile apparatus
[myosin heavy chain (MHC) or ATPase isoforms]; (2) contractile
characteristics (fast vs. slow twitch); (3) Ca2+handling properties
and metabolic profile (oxidative or glycolytic), with the golden
standard being the MHC-isoform (Schiaffino and Reggiani,
2011). The functional significance of the MHC isoform for
its contractile characteristics is well established (Schiaffino and
Reggiani, 2011), even for hybrid fibers co-expressing MHC
isoforms. The metabolic capacity of the muscle fiber is dependent
on the degree of capillarization, substrate availability and
mitochondrial content, while the Ca2+handling properties
are dependent on sarcoplasmic reticulum (SR) content and
property (Stephenson et al., 1998;Ørtenblad et al., 2000b;Gejl
et al., 2014). The metabolic and Ca2+handling properties
are generally considered as being linked with contractile fiber
type characteristics. Human muscle fibers expressing MHC-
1 have the highest oxidative capacity while having slow
shortening velocity (incl. excitation–contraction coupling) and
slower Ca2+handling, whereas MHC-2 fibers have the opposite
characteristics. However, metabolic variation within each fiber
type and fibers in arm and leg muscle is less well explored, both
with regard to extent and influence on the metabolic response of
the fiber.
Most Olympic disciplines involve mainly the legs and lower
body, with fewer combining upper and lower body as in cross-
country skiing. Despite the importance of the arms in sports such
as swimming, rowing, and cross-country skiing, our knowledge
of arm muscle physiology is considerably less than in the
case of the legs and warrants more attention. The few direct
comparisons of arm and leg muscles indicate that arm muscles
are less oxidative and less capable of extracting oxygen from the
circulation, irrespective of training status, with greater variability
in blood flow during exercise (Van Hall et al., 2003;Calbet et al.,
2005). Furthermore, exercising arm muscle has evidently a lower
fat oxidation compared to leg muscle (Calbet et al., 2005;Helge,
2010). However, the physiological comparison of arms and legs is
hampered by an often-unequal training status of the limbs. Thus,
direct comparisons of the highly trained arm and leg muscles of
elite cross-country skiers can be made unequivocally.
Accordingly, the current investigation assessed further the
metabolic capacity in the upper and lower body of such skiers,
as well the potential relationship between the various isoforms
of MHCs and metabolic profile. For this purpose, we examined
type 1 and type 2 fibers from leg (vastus lateralis) muscle
and arm muscle (triceps brachii) from successful cross-country
skiers with exceptionally well-trained lower and upper body. Our
hypotheses were that (1) there are intrinsic metabolic differences
between equally well-trained arm and leg muscles and (2) type1
and type 2 muscle fibers possess similarly metabolic capacity,
regardless of their location in an arm or leg muscle and that this
possible adaptation is not linked to the isoform of the muscle
fibers.
MATERIALS AND METHODS
Subjects
Ten elite male Norwegian cross-country skiers participated in
the study, as part of a larger project and related data from
the project has already been published (Nielsen et al., 2011;
Ørtenblad et al., 2011;Koh et al., 2017). Their mean (±SD) age,
height, weight, and ˙
VO2max were 22 ±1 yr, 181 ±2 cm, 79 ±8 kg,
and 5.37 ±0.46 L·min1(69 ±5 ml·kg1·min1), respectively
(Table 1) and a hematocrit of 47 ±1% and hemoglobin of
155 ±2 mmol/l. These skiers had trained systematically for
an average of 11 years; six had competed as members of the
Norwegian national team; and eight competed in the FIS World
Cup the year after this study, with one winning a World Cup
race (Table 1). All subjects were informed of the test procedures
and potential risks prior to providing their written informed
consent to participate. The research procedures and experimental
protocol were pre-approved by the Human Ethics Committee
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TABLE 1 | Characteristics of the 10 elite male cross-country skiers who participated in this study.
Subject Age
(years)
Weight
(kg)
Height
(cm)
VO2max
(L·min1)
VO2max
(mL·kg1·min1)
Performance
1 22 81.4 190 5.82 71.5 12th in WC 50-km C (2012)
2 21 77.2 182 5.10 66.1 among the top 30/15 in NOR Tr
and Sp, respectively
3 22 87.3 188 6.08 69.6 among the top 30 in NOR Tr
4 19 76.0 178 5.21 68.6 12th in NNC Sp (2009)
5 21 77.2 178 5.16 66.8 40th in NNC 15F (2011)
6 23 66.8 172 5.30 79.3 9th in WC 15-km F (2008)
7 23 92.4 193 6.05 65.5 14th in WC Sp (2011)
8 23 87.1 179 5.34 61.3 Among the top 50 and 30 in NOR
Tr and Sp, respectively
9 24 69.9 175 4.82 69.0 Among the top 30 in NOR Sp
10 22 72.5 173 4.85 66.9 Among the top 60 in NOR
Mean ±SD 22 ±1 78.8 ±8.2 181 ±7 5.37 ±0.46 68.5 ±4.7
WC, World Cup; NC, Norwegian National Championship; Tr, traditional/longer distances; Sp, sprint distances; C, classical technique; F, free technique.
of Umeå University, Sweden (#07-076M), and performed in
accordance with the Declaration of Helsinki.
Procedures
Laboratory Tests
˙
VO2max was determined during diagonal skiing with roller
skis on a treadmill (Rodby, Södertälje, Sweden; Calbet et al.,
2005), starting at 11 km·h1on a treadmill inclination of 4
and increasing the incline by 1each minute until exhaustion.
During the tests, each subject was secured with a safety harness
suspended from the ceiling. For the subjects, roller skiing on the
treadmill was a regular part of their training.
Respiratory variables were determined with the mixed expired
gas procedure, employing an ergo-spirometry system (AMIS
2001 model C, Innovision A/S, Glamsbjerg, Denmark) equipped
with an inspiratory flowmeter. The gas analyzers were calibrated
with a high-precision mixture of 16.0% O2and 4.0% CO2(Air
Liquide, Kungsängen, Sweden) and the flowmeter calibrated at
low, medium, and high flow rates with a 3-l air syringe (Hans
Rudolph, Kansas City, MO, United States). Ambient conditions
were monitored with an external apparatus (Vaisala PTU 200,
Vaisala OY, Helsinki, Finland). Expired O2and CO2and the
inspired minute ventilation (V ˙
E) were monitored continuously
and VO2values averaged during the final 30 s at each workload.
Heart rate was recorded continuously by the Polar S610 monitor
(Polar Electro Oy, Kempele, Finland).
Muscle Biopsy Preparation and Analysis
Muscle biopsies were taken from leg and arm muscles and
standardization of the location on the muscle and muscle depth
was ensured. After local anesthesia (2–3 ml 2% lidocaine), an
incision was made through the skin and fascia and the muscle
biopsy was taken from the vastus lateralis (leg) and triceps brachii
(distal part of the lateral head, arm), using a modified Bergström
needle with suction. These muscles were selected because they
are very active during cross-country skiing (Komi and Norman,
1987;Holmberg et al., 2005). The skiers had four biopsies
taken from both arm and leg muscle. The muscle specimen
was dried on filter paper and placed on a glass plate cooled
on ice. After the removal of visible connective tissue and fat,
each muscle specimen was divided into four specimens then
handled in the following ways: (1) frozen directly in liquid N2
and stored for later analyses of enzyme activity and glycogen
content; (2) fixed for transmission electron microscopy (TEM)
analysis; (3) 10–20 mg was mounted in an embedding medium
(OCT compound), frozen rapidly in isopentane pre-cooled with
liquid N2, and stored at 80C for later histochemical analysis;
or (4) a segment was weighed and homogenized in 10 volumes
(wt/vol) of ice-cold buffer (300 mM sucrose, 1 mM EDTA, 10 mM
NaN3, 40 mM Tris-base, and 40 mM histidine at pH 7.8) at 0C
in a 1-ml glass homogenizer with a glass pestle (Kontes Glass
Industry, Vineland, NJ, United States). Prior to homogenization,
the muscle sample was rinsed free of contaminating blood by
washing it in an ice-cold buffer. The homogenate was analyzed
for protein content and MHC composition. All in all 40 biopsies
were obtained from the leg and arm muscles, and in one biopsy
from arm, the sample portion was not large enough to obtain CS
activity.
Myosin Heavy Chain Composition
Myosin heavy chain composition was analyzed using gel
electrophoresis. Briefly, muscle homogenate (80 µl) was mixed
with 200 µl sample buffer (10% glycerol, 5% 2-mercaptoethanol,
2.3% SDS, 62.5 mM Tris-base, and 0.2% bromophenolblue at pH
6.8), boiled in a water bath at 100C for 3 min, and loaded with
three different amounts of protein (10–40 µl) on an SDS-PAGE
gel [6% polyacrylamide (100:1, acrylamide:bis-acrylamide), 30%
glycerol, 67.5 mM Tris-base, 0.4% SDS, and 0.1 mM glycine]. Gels
were run at 80 V for at least 42 h at 4C and MHC bands made
visible by staining with Coomassie and three separate bands could
be detected and characterized as MHC-1, MHC-2A, and MHC-
2X. The gels were scanned (Linoscan 1400 scanner, Heidelberg,
Germany) and the MHC bands were quantified densitometrically
(Phoretix 1D, nonlinear, Newcastle, United Kingdom). MHC-
2 was identified with Western blot using monoclonal antibody
(Sigma M 4276) with the Xcell IITM protocol (Invitrogen,
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Carlsbad, CA, United States). All values presented are the means
of three biopsies (two from one leg/arm and one from the other
leg/arm), utilizing three different concentrations of protein from
each biopsy.
Enzyme Activity
The maximal activities of 3-hydroxy-acyl-CoA-dehydrogenase
(HAD) and citrate synthase (CS), were determined
fluorometrically at 25C (Lowry and Passonneau, 1972) in
freeze-dried muscle dissected free of non-muscle constituents.
CS activity was determined by the addition of oxaloacetate to
a buffer solution containing muscle homogenate, DTNB buffer,
acetyl-CoA. HAD activity was measured after the addition
of acetoacetyl-CoA to a buffer solution containing imidazole,
NADH and EDTA. Absorbance of CS and HAD was recorded
for 600 s, converted into enzyme activity rates, and expressed as
µmol·g1dw·min1.
Histochemical Analysis of Capillarization and ATPase
Fiber Typing
Histochemical analysis of ATPase (Brooke and Kaiser, 1970)
was used to determine the fiber type composition (type 1, 2a,
2x) and fiber cross-sectional area (CSA), while the amylase
periodic acid-Schiff reaction (Andersen, 1975) was applied for
staining of capillaries (TEMA image analysis system; Scanbeam
a/s, Hadsund, Denmark). In brief, serial sections (10 µm) of the
muscle biopsies samples were cut in a cryostat at 20C, and fiber
type distribution was obtained by ATPase histochemistry analysis
performed after pre-incubation at pH 4.37, 4.60, and 10.30. An
average of 85 ±16 fibers was analyzed in each biopsy. The serial
sections of the various ATPases were visualized and analyzed for
fiber type, using a TEMA image analyzing system (Scanbeam,
Hadsund, Denmark).
Transmission Electron Microscopy
To examine the content and subcellular localization of
mitochondria and lipids, muscle biopsy specimens were prepared
for TEM as described previously (Nielsen et al., 2010a,b). In
the prepared sections, all longitudinal-oriented fibers (9
per biopsy) were photographed at x40,000 magnification in a
randomized, systematic order to ensure unbiased results. From
each fiber, 12 images both from the myofibrillar (six from the
superficial and central region, respectively) and subsarcolemmal
(SS) regions were obtained as previously described (Nielsen
et al., 2010a,b). Fibers were identified as type 1 or type 2 based
on a combination of mitochondrial volume fraction and z-line
width as described elsewhere (Nielsen et al., 2011). In order to
identify the two main fiber types, all intermediate fibers were
discarded and only distinct type 1 and 2 fibers were included,
respectively (n= 2–3 fibers of each type per biopsy). The contents
of mitochondria in the intermyofibrillar (IMF) and SS regions
were estimated by point counting (Weibel, 1980,Figure 1). IMF
mitochondria is expressed as volume fractions of the myofibrillar
space and the values for the superficial region were weighted
three times higher than those for the central region, to account
for the cylindrical shape of the fibers, in which the superficial
region (outermost half of the diameter) occupies three-quarters
FIGURE 1 | TEM images showing the subcellular localization of skeletal
muscle mitochondria. All images are from leg muscle (vastus lateralis).
(A) Overview of a part of fiber showing the myofibrillar (Myo) space and
subsarcolemmal (SS) space. (B) The typical localization of SS mitochondria
(mit) in skeletal muscle, also showing a intramyocellular lipid (IMCL). (C) In the
Myo space, intermyofibrillar mitochondria are wrapped around the myofibrils,
mainly in the I-band and often connected to an adjacent mitochondrion
through the A-band. There is less marked connection between neighboring
mitochondria in the I-band. (D) Intermyofibrillar mitochondria in the I-band on
each side of the Z-line, with the t-tubular system (t-system) and mitochondria
intertwined. (E) Overview demonstrating the IMF mitochondria are mainly
located in the I-band on each side of the z-line and often connected to an
adjacent mitochondrion in the same sarcomere through the A-band. All the
gray structures in the fiber are mitochondria with slightly visible inner cristae.
Glycogen granules can be seen as black dots. Z, Z-line; A, A-band; IMCL,
intramyocellular lipid; T-tubule, transverse tubular system. Scale bar: A,
10 µm; B,C,1µm; D, 0.5 µm; E,1µm. Original magnification: A, x1,600;
B,C, x20,000; D, x50,000; E, x13,000.
of the volume. The SS mitochondria are expressed as volume per
surface area of the muscle fiber. The estimated coefficient of error
(estCE; see Howard and Reed, 2005) was 0.18 and 0.24 for IMF
and SS mitochondria, respectively, with no difference between
legs and arms. Total volume fractions of mitochondria and
lipids, respectively (IMF +SS), were obtained by recalculating
the SS subfractions relative to myofibrillar volume density,
assuming a cylindrical shape of the fibers and a radius of 40 µm,
as previously described (Nielsen et al., 2010a).
Statistical Analyses
All values presented are means ±standard error of the mean
(SEM) and were subjected to ANOVA test, with significant
differences between means identified using the Bonferroni
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Ørtenblad et al. Limb Muscle Profiles of Well-Trained
post hoc test (GraphPad Prism 6.07). All interactions or main
effects were examined using a linear mixed-effects model, with
the subject, limb, fiber type, and fiber as random effects and
limb, fiber type, and location as fixed effects, using the Stata
10.1 software (StataCorp. 2007; Stata Statistical Software: Release
10; StataCorp LP, College Station, TX, United States). Variables
exhibiting skewed distributions were log-transformed prior to
analysis. The level of significance was set at α= 0.05.
RESULTS
Fiber Type Distribution
The MHC distribution in the vastus lateralis and triceps brachii
was the same on the left- and right-hand sides, with a significantly
higher proportion of MHC-1 in the legs (58 ±2%, range [34–
69%]) than the arms (40 ±3%, range [24–57%]) (P<0.01,
Table 2). Accordingly, the proportion of MHC-2A in the legs
was lower (41 ±2 vs. 60 ±3%). The average MHC distribution
showed a considerable variation between the skiers with MHC-1
ranging between 34–69% (leg) and 24–57% (arm) (Table 2).
Notably, the two skiers with the highest proportion of MHC-2A
in arms (70 and 72%) were successful sprint skiers.
Enzyme Activities
The maximal CS activity of well-trained arm and leg muscles was
the same (Table 2), despite the higher MHC-1 content of the legs,
thus demonstrating a non-MHC-dependency in the CS activity.
In contrast, the maximal activity of the key enzyme in the ß-
oxidation, HAD, was 52% higher (P<0.05) in the leg compared
to arm muscles. Accordingly, the ratio between the HAD and
CS activity was 45% higher in leg than arm (1.22 in the leg and
0.86 in arm, P<0.01), suggesting a relatively higher capacity for
lipid oxidation in leg muscle. Further, there was no association
between CS activity and MHC distribution (Figure 2A). Thus,
CS activity in these highly trained muscles is not associated with
the MHC distribution. In contrast, MHC-1 content was a robust
predictor of HAD capacity (P= 0.011, r2= 0.32, Figure 2B).
In line with this, there was also a strong correlation between
HAD/CS ratio and the MHC-1 content (P= 0.021, r2= 0.27), with
no association in trained leg (Figure 2C). Taken together, in these
highly trained skiers, there is a close association between MHC
distribution and both absolute (HAD) and relative (HAD/CS)
capacity to oxidize fat, with no association between CS capacity
and MHC distribution.
Fiber Capillarization and Size
The total number of capillaries per total number of fibers and the
number of capillaries per fiber area were not different between
leg and arm muscle, averaging 2.9 ±0.1 capillaries per fiber
and 417 ±14 capillaries/mm2(Table 3). The average number
of capillaries around each fiber was 5.8 ±0.8 for the leg and
6.3 ±0.3 for the arm. When considering capillaries around each
fiber type, there were significantly fewer capillaries in type 2x
fibers as compared with type 1 and 2a in leg muscle (P<0.05,
Table 3). There were no fiber type differences in arm muscle;
however, there was a tendency toward a higher capillarization in
type 2a fibers (P= 0.078). Further, there was a clear difference in
capillarization between leg and arm muscle in type 2a fibers, with
14% more capillaries per fiber in arm muscle (Table 3).
The average fiber size for each fiber type and hybrid fibers, in
arm and leg muscles, is shown in Table 4. There was no significant
difference in mean fiber size between fiber types in leg muscle.
However, in arm muscle, type 2a fibers were significantly larger
than type 1 fibers (P<0.05).
Estimation of the number of capillaries per individual fiber
area in trained muscles demonstrated that type 1 fibers in both
leg and arm muscles had, on average, 27% higher capillarization
than type 2 fibers (P<0.05), with no difference between limbs.
Thus, a higher number of capillaries per fiber type 2a fibers of the
arm are linked with a larger fiber size.
Mitochondrial Content and Subcellular
Localization
Transmission electron microscopy images showing the
subcellular localization of skeletal muscle mitochondria in
the highly trained cross-country skiers are shown in Figure 1,
clearly demonstrating a very high mitochondrial volume in these
trained muscles. The SS mitochondria were unevenly distributed
below the sarcolemma, with a higher volume located near the
capillaries and around the nuclei. The IMF mitochondria are
wrapped around the myofibrils, mainly located on each side of
the z-line. These mitochondria in the I-band are often connected
to an adjacent mitochondrion in the same sarcomere through the
A-band. Individual values for the total volume of mitochondria
per volume of myofiber are given in Table 5. The total volume
of mitochondria is a volume-weighted average of the superficial
region and the central region of the myofiber as well as the
SS space. The individual values are based on 8–12 myofibers
from two different biopsies. The total mitochondrial volume
averaged 8.6 ±1.6 and 9.0 ±2.0 µm3·µm3, for the arm and
leg, respectively. The relative distribution of the mitochondrial
subcellular regions was estimated in a total of 29 or 30 fibers from
the 10 participants. In these highly endurance-trained athletes,
the skeletal muscle mitochondria had similar relative distribution
between IMF and SS localizations in both leg and arm muscles
and in type 1 and 2 fibers. Thus, 83–86% of the mitochondria are
localized in the IMF region and 11–14% in the SS region. The
mitochondrial content and subcellular localization in distinct
fiber types and at the whole-muscle level of leg and arm muscles
is shown in Figure 3. Intriguingly, there was a tendency toward
(10–20%) a lower mitochondrial content in the IMF and SS
regions of leg muscle fibers compared with arm muscle fibers
(Figure 3A,P= 0.095). This is also apparent when calculating
a total (IMF +SS) mitochondrial content (Figure 3B). By
taking the different MHC composition of leg and arm muscles
into account, the average fiber type-mitochondrial volume can
be estimated, given a fiber type distribution of 57 and 37%
MHC-1 in leg and arm, respectively. Weighting the fiber type
distribution, the whole-muscle mitochondrial volume in leg and
arm muscle was similar (Figure 3C). Thus, at the whole-muscle
level, the non-significantly higher mitochondrial content in the
arms mediated, despite a relatively higher number of MHC-2
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TABLE 2 | The profile of myosin heavy chains and enzyme activities in the arm (triceps brachii) and leg (vastus lateralis) muscles of elite cross-country skiers (n= 10).
Fiber type distribution (% of total) Enzyme activity
MHC-1 MHC-2A MHC-2X CS HAD HAD/CS
Leg 58 ±2 41 ±2 1.0 ±0.4 118 ±6 144 ±12 1.22
Arm 40 ±360 ±30.4 ±0.2 111 ±10 95 ±120.84
The maximal activities of 3-hydroxy-acyl-CoA-dehydrogenase (HAD) and citrate synthase (CS) are given in µmol/g dw/min. Significantly different from the leg muscle.
FIGURE 2 | The relationships between the percentage of MHC-1 and 3-hydroxyacyl-CoA dehydrogenase activity (HAD, n= 10) (A); citrate synthase activity (CS,
n= 9) (B); and relative capacity to oxidize fat (HAD/CS ratio) (C). The open circles depict data for the arms and the leg black squares for the legs. For the arm and leg
combined, there was a significant correlation between the MHC-1 content and HAD activity (r2= 0.32, P= 0.011), as well as the HAD/CS ratio (r2= 0.27, P= 0.021).
TABLE 3 | Capillary density in the arm (triceps brachii) and leg (vastus lateralis) muscles of elite cross-country skiers (n= 10).
#cap/fiber cap/mm2Type 1 Type 2a Type 2x Average
Leg 2.8 ±0.1 437 ±22 5.9 ±0.3 5.9 ±0.2 5.1 ±0.3#5.8 ±0.8
Arm 3.0 ±0.2 394 ±14 5.8 ±0.3 6.7 ±0.36.0 ±2.06.3 ±0.3
Capillary density was assessed immunohistochemically. Number of capillaries is given in: total number of capillaries per total number of fibers (#cap/fiber); total number
of capillaries per muscle area (cap/mm2), and number of capillaries around each fiber for each fiber type and average for all fibers. Significantly different from the
corresponding value for leg muscle; #significantly different from the corresponding values for the other fiber types.
TABLE 4 | Fiber size in the arm (triceps brachii) and leg (vastus lateralis) muscles
of elite cross-country skiers (n= 10).
Type 1 Type 2a Type 2x Type 2a/x
Leg 5423 ±272 6811 ±297 6590 ±363 5840 ±518
Arm 5356 ±200U8105 ±3946125 ±960U4576 ±176U
Fiber size (in µm2) was assessed immunohistochemically. Significantly different
from the corresponding value for leg muscle; U
significantly different from type 2a
fibers; significantly different from type 2x fiber.
fibers, an equal whole-muscle mitochondrial content in the
legs and arms (Figure 3C). There was a significant correlation
(P= 0.02) between the total mitochondrial content in arm muscle
and whole body VO2 max (L·min1), which was not apparent in
leg muscle.
DISCUSSION
Here we compare equally trained limb muscles from elite cross-
country skiers. A key finding here was that the mitochondrial
volume percentage and CS activity is equal in legs and arms,
despite the presence of a higher proportion of MHC-2 fibers
in the arms. Furthermore, we demonstrate that well-trained
type 1 and type 2 muscle fibers can have similar capillarization,
regardless of whether they are located in arm or leg muscle
and that the capillarization is not linked with the muscle fiber
type, indicating a divergence between fiber type pattern and
aerobic metabolic capacity. Also, comparable highly trained leg
and arm muscles exhibited clear difference in their enzyme-
linked ability to oxidize fatty acids (HAD capacity) and combined
with previous data on a fourfold higher intramyocellular lipid
(IMCL) volume contents in leg muscles; this points to a clear
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TABLE 5 | The volume of mitochondria – total and in the superficial and central intermyofibrillar space (IMF) and the sarcolemmal space (SS) – in the arm and leg
muscles of elite cross-country skiers (n= 10).
Participant Arm (triceps brachii) Leg (vastus lateralis)
Total IMFSuperficial IMFCentral SS Total IMFSuperficial IMFCentral SS
1 9.7 10.2 4.1 0.21 –
2 10.1 9.8 5.2 0.28 7.1 6.6 4.4 0.20
3 12.7 11.5 5.8 0.53 9.4 9.2 4.3 0.29
4 9.4 9.0 2.9 0.38 9.1 7.9 4.6 0.41
5 8.1 6.7 5.3 0.34 8.1 7.9 4.4 0.23
6 5.6 5.8 2.4 0.13 6.2 5.7 3.6 0.21
7 7.4 6.8 5.5 0.19 9.3 9.2 4.7 0.25
8 10.6 10.1 4.7 0.36 11.8 11.0 7.0 0.37
9 8.6 7.6 5.9 0.29 8.8 8.9 3.9 0.24
10 8.0 7.7 2.4 0.33 7.6 6.9 4.5 0.25
Mean 9.0 8.5 4.4 0.31 8.6 8.1 4.6 0.27
SD 2.0 1.9 1.4 0.11 1.6 1.6 1.0 0.07
Total, total volume of mitochondria per volume of myofiber; IMFsuperficial, intermyofibrillar mitochondrial volume per volume of myofibrillar space in the superficial region of
the myofiber; IMFcentral, intermyofibrillar mitochondrial volume per volume of myofibrillar space in the central region of the myofiber; SS, subsarcolemmal mitochondrial
volume per area of myofiber surface. The individual values presented are the means for 8–12 myofibers in biopsies taken before and 22 h after the race. All data are given
in volume densities (µm3µm3), except in the case of SS, where they are volume per SS area (µm3·µm2).
FIGURE 3 | Mitochondria content and subcellular localization in distinct fiber
types and at whole-muscle level of leg and arm muscles. There was a
tendency (P= 0.095) toward a higher mitochondrial content in the
intermyofibrillar (IMF) and subsarcolemmal (SS) regions, of arm muscle (open
bars) compared with leg muscle (filled bars) (A). This tendency is also
apparent when calculating total mitochondrial content (IMF + SS) (B).
(C) Weighted mitochondrial volumes in the arm and leg muscle, estimated
from a fiber type distribution of 57 and 37% MHC-I for the leg and arm (n= 9),
respectively. These MHC weighted values of whole-muscle mitochondrial
content in arm and leg muscles are similar. Values are means ±SE (n= 29–30
fibers from 10 subjects).
limb difference in fat metabolism between the leg and the
arm, which cannot be explained by the different fiber type
distributions.
Fiber Type Malleability
In order to fulfill various functional needs, different skeletal
muscle fiber types express different molecular isoforms of
myosin. The contractile characteristics of the given muscle fiber
type are generally considered as being linked with metabolic
and Ca2+handling properties, with fibers expressing MHC-
1 having the highest oxidative capacity while being slow to
shorten and having slower Ca2+handling, with MHC-2 fibers
having the opposite characteristics. This was demonstrated
very clearly in the early studies by Burke et al. (1971), who
showed a phenotypic characterization of quite strict links
between contractile function and metabolic profile in that
type 2 fibers are glycolytic, while type 1 fibers are oxidative.
Despite several reports indicative of plasticity in this relationship,
this long-held concept is still the reigning dogma. In later
studies on humans, more evidence has been provided on
the large plasticity of all fiber types with respect to their
aerobic potential despite no or only a small transformation
of the type 2a to the type 1 isoform (Holloszy, 1967;
Hoppeler and Fluck, 2003;Schiaffino and Reggiani, 2011).
In line with this, Essén et al. (1975) reported an equally
high SDH activity in the type 2 and type 1 muscle fibers
in top endurance runners [with a maximal oxygen uptake
(˙
VO2max)>72 ml·kg1·min1], with untrained having a clear
fiber type difference with only half the SDH activity in their
type 2 muscle fibers. Also, the mitochondrial volume density
is generally considered to be strongly fiber type-dependent. In
untrained humans, the mitochondrial volume varies from 6%
in type I fibers to 4.5% in type 2a and 2.3% in type 2x fibers
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(Howald et al., 1985), with a more pronounced difference in
animal studies of oxidative and glycolytic muscle, i.e., 2.7 times
higher in rabbits and 4.5 times higher in rats (Saltin and Gollnick,
1983;Jackman and Willis, 1996). In the current study, we
compared equally trained arm and leg muscle based on the
same CS activity (Table 2), the same average capillarization
(Table 3), and no difference in the mitochondrial content at
whole-muscle level. Based on this, we state that arm and leg
muscle are equally trained. In these endurance-trained humans,
there is a twofold higher mitochondrial volume density between
type 1 and 2 fibers (Figure 3). Furthermore, the volume density
of the type 2 fibers from trained is equal to (Howald et al.,
1985) or higher (Nielsen et al., 2010a) than in type 1 fibers
from untrained individuals. Thus, fiber type mitochondrial
content is extremely malleable with muscle activity and inactivity
(Hoppeler, 1986;Nielsen et al., 2010b). These changes in fiber
metabolic characteristics are clearly not fiber-type-dependent,
and a considerable variation exists within each fiber type with a
clear overlay between fiber types. In line with this, a recent study
indicated that type 2a fibers can possess equally high or even
higher mitochondrial respiration as type 1 fibers (Boushel et al.,
2014). The equal volume density of mitochondria and CS activity
in different types of fibers suggest that the intrinsic characteristics
of mitochondria are variable and not determined solely by fiber
type.
Here we report that the metabolic profile of muscle fibers
varies with no change in the myosin isoform they express. Thus,
in highly trained humans, the mitochondrial volume percentage
is equal in the arms and legs, despite a relatively higher number
of MHC-2 fibers in arms, and type 2A fibers from the arm
being larger, with the same number of capillaries per fiber
area. In these highly trained skiers, the type 2 fibers have an
equally high oxidative capacity as type 1 fibers, demonstrating
that the metabolic profile of a given fiber isoform displays
considerable plasticity. Interestingly, we have previously reported
in the same subjects, an approximate 4.6-fold higher SR Ca2+
release rate in MHC 2 fibers compared to MHC 1 (Ørtenblad
et al., 2011). As SR Ca2+handling is a key component in
the development of fatigue during most types of exercise, it
is physiologically crucial for these skiers to possess a high SR
Ca2+uptake and release rate (Ørtenblad et al., 2000a;Gejl et al.,
2014). These data on trained skiers suggest a new perspective
on fiber types, indicating a divergence between MHC isoform
pattern and aerobic metabolic capacity, with a high variability
in the metabolic profile, closely related to the usage of the
muscle fiber, within the various MHC isoforms. Thus, these
highly trained skiers possess a type 2 fiber which is highly
oxidative, has an equal CS activity as type 1 fibers, has a larger
CSA, with the same capillarization per CSA, while having a
near fivefold higher SR Ca2+handling capacity than type 1
fibers. In all, these findings represent a muscle fiber with high
force and power properties, while having a highly developed
endurance capacity to fulfill the demands of today’s elite cross-
country skier requiring the combined ability to generate and
sustain rapid, prolonged high force production during short
contacts with the ground (Holmberg, 2015;Andersson et al.,
2016).
Mitochondrial Subcellular Distribution
and Volume Fraction
The current data from arm and leg muscles drawn from the elite
endurance-trained subjects revealed that type 1 and 2 fibers have
the same relative subcellular distribution of mitochondria. Thus,
around 85% of the muscle mitochondria are located in the IMF
region and the remainder in the SS region, regardless of fiber type
and limb. This is in line with a training study showing that type
1 and 2 fibers have similar relative distribution of mitochondria
after training (Howald et al., 1985).
The mitochondrial volume fraction was not different between
limbs, averaging 9.5%. The reported mitochondrial volume
fraction is 20–30% higher than found in previously reported
short-term training studies (Howald et al., 1985;Nielsen et al.,
2010a) as well as in endurance-trained athletes (Hoppeler, 1986).
However, a mitochondrial volume percentage of 11.4% in vastus
lateralis for a similar group of highly trained athletes, i.e.,
professional cyclists (n= 3), has been reported (Hoppeler, 1986).
In these athletes, vastus lateralis played a more primary role
in performance than in cross-country skiing, explaining the
greater necessity for mitochondria in that particular muscle.
The mitochondrial volume fraction of 9.5% in trained skiers is
about two times larger than previously reported in untrained
individuals using the same method (Nielsen et al., 2010a), and
is in line with data showing a two to two-and-a-half fold higher
activity of key mitochondrial enzyme (SDH, CS, and HAD)
activity in trained cross-country skiers than observed in sedentary
individuals (Gollnick et al., 1972;Saltin, 1996). In addition to
mitochondrial distribution and volume percentage of the cell
and mitochondrial enzymes, there may be other differences in
mitochondria network, shape, topology, or function between
fiber types, limbs, and human populations (Nielsen et al., 2017).
Mitochondrial Content and Distribution
in Leg Versus Arm
Weighing the different fiber type distribution in leg and arm
muscle, the mitochondrial volume fraction was equal in both
(Figure 3D). This suggests that arm muscles, despite lower fat
oxidation capacity (Helge, 2010), HAD activity (present data),
lower IMCL content (Koh et al., 2017), and higher lactate release
during exercise (Van Hall et al., 2003), still require a high
mitochondrial oxidative capacity. Indeed, there was a tendency
(P= 0.095) toward a 10% higher mitochondrial volume fraction
in the fibers from the arms compared with the legs (Figure 3C),
predominantly due to a tendency to higher volume fraction in
type 2 fibers in the arms (Figure 3C). Thus, differences in leg
and arm whole-muscle metabolic characteristics may not solely
be explained by the dissimilar fiber type distribution in the
limbs. The high mitochondrial content in type 2 fibers in arm
could either be a consequence of the high metabolic demand
in the upper body of these trained subjects or, possibly, due to
a high demand for glycolytic flux in type 2 fibers. Thus, there
is a clear necessity for being able to convert lactate to pyruvate
within the mitochondrial intermembrane space with pyruvate
subsequently taken into the mitochondrial matrix where it enters
the TCA cycle and is ultimately oxidized (Brooks et al., 1999;
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Hashimoto et al., 2006;Jacobs et al., 2013). Furthermore, peak
arm blood flow and O2delivery per unit muscle mass during
arm exercise is higher than that to leg muscle during leg
cycling reflecting the proportional matching of oxygen delivery
to oxidative capacity (Boushel et al., 2011).
The present study design involved a pair-wise comparison of
equally highly trained muscles from the same individual subjects,
who had trained systematically for 11 years on average and whose
muscle mitochondrial volume fractions are among the highest
ever reported. A cross-sectional comparison of, e.g., kayakers or
cyclists who train their upper or lower bodies specifically, would
have allowed characterization of more highly trained muscles, for
instance, with more extensive local blood flow during exercise.
However, higher mitochondrial volume fractions have not been
reported in larger groups and a cross-sectional design would
limit direct comparisons between limbs. At the same time, it is
important to note that our present observations and conclusions
are relevant only for equally well-trained arm and leg muscles.
Intramyocellular Lipid (IMCL) Content
and Subcellular Localization
We have recently reported in a companion paper (Koh et al.,
2017) that, in these subjects with highly trained upper and lower
body, the IMCL volume fraction was fourfold higher in leg
muscle than in the arm muscle. The higher content of IMCL
content was apparent in both the IMF and the SS regions.
Additionally, there was a fiber type specific difference in IMCL
volume fractions, with a threefold higher IMF (P= 0.0002)
and total (P= 0.0003) lipid droplet volume fractions in type
1 fibers than in type 2 fibers, while no difference was found
between the fiber types (P= 0.6) in the SS lipid droplet volume
fraction. The fourfold lower IMCL content of the arms compared
to the leg cannot solely be explained by the higher proportion
of MHC-2 fibers of arm, so a true intrinsic limb difference in
fat metabolism must exist. The higher IMCL content of the leg
muscle compared with the arm muscle is in accordance with the
lower fat oxidation capacity of arm muscle (Helge, 2010) and
the notion that exercising arm muscle evidently has a lower fat
oxidation compared to leg muscle (Calbet et al., 2005;Helge,
2010).
The high content of IMCL in skeletal muscle of trained
subjects and obese type 2 diabetics has been described as
“the athlete paradox” (Goodpaster et al., 2001). However, the
current data are in accordance with a new perspective on this
apparent paradox, suggesting that the elevated IMCL content
found in both type 2 diabetic patients (Nielsen et al., 2010a)
and endurance-trained athletes (Koh et al., 2017) is an average
of differential subcellular distribution of IMCL, where athletes
have elevated IMF and type 2 diabetes patients elevated SS IMCL
(Nielsen et al., 2010a). Thus, the roles of IMF and SS IMCL in
skeletal muscle glucose regulation are most likely fiber type and
training status specific.
Enzyme Activities
In our highly trained cross-country skiers, with equally and
exceptionally well-trained leg and arm muscle, there was a 52%
higher HAD capacity in the leg compared to arm muscles and
the ratio between the HAD and CS activity was 1.22 in the leg
and 0.86 in arm, suggesting a relatively higher capacity for lipid
oxidation in leg muscle. These enzyme activity data demonstrate
a very good agreement with the four-fold higher IMCL in the
leg compared to the arm muscle. Notably, the MHC-1 content
was a very strong predictor of HAD activity in trained arm
(r2= 0.69), with no association in leg muscle. This clear limb
difference in the association between fiber MHC distribution
and HAD capacity was not apparent with CS activity, depending
rather on other factors as, i.e., training-induced adaptations.
Taken together, there is a clear limb difference in HAD and
HAD/CS ratio, with legs having a non-MHC dependent capacity
to oxidize fat, while in arm muscle, there is a very close
association between MHC distribution and both absolute (HAD
activity) and relative capacity to oxidize fat (HAD/CS). This
supports the notion that the upper body has a lower capacity to
oxidize fat and a lower fat oxidation during the same relative
intensity compared to leg muscle (Calbet et al., 2005;Helge,
2010). This lower capacity to oxidize fat and higher reliance on
CHO oxidation in the upper body compared to the lower body is
irrespective of limb training status, and persistent also in highly
trained cross country skiers with equally trained arm and leg
muscle.
CONCLUSION AND PERSPECTIVES
Here, we show that in highly trained muscles of elite cross-
country skiers, the mitochondrial volume percentage as well as
the number of capillaries per fiber area are the same in the
arms and legs, despite the presence of relatively more MHC-
2 fibers and larger type 2A fibers in the arms. Thus, the
metabolic profile of muscle fibers can vary without any change
in the myosin isoform they express. These findings provide
a new perspective, with a divergence between fiber type and
aerobic metabolic capacity, and considerable variability in the
metabolic profile of the various MHC-isoforms which is closely
related to the usage of the muscle fiber. Our well-trained cross-
country skiers have developed highly oxidative type 2 muscle
fibers capable of producing great force and power in order to
meet today’s need for pronounced endurance in combination
with rapid generation of large forces during short contact
periods.
We also demonstrate that leg and arm muscles exhibit a clear
difference in their IMCL content and distribution, as well as
in the ability to oxidize fatty acids. The observed difference in
IMCL content in the upper and lower body cannot be explained
by training status of the involved muscles or the different fiber
type distribution in the limbs. This implies that the capacity
to oxidize and store IMCL is clearly higher in leg compared
with arm muscle, even though limbs are equally highly trained
and express similar mitochondrial content and capillarization.
In line with this, the HAD activity and the HAD/CS ratio
were significantly higher in leg muscle. Thus, it is evident that
limbs have different lipid metabolism independent of fiber type
differences.
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AUTHOR CONTRIBUTIONS
NØ, JN, H-CH, and BS were involved in the study
design. NØ, JN, H-CH, KS, and BS have collected
the data. All authors contributed to interpretation
of data and drafting of the manuscript and all
but BS have reviewed the final version of the
submitted manuscript. BS passed away before the final
approval of the manuscript. Transmission electron
microscopy measurements were performed by JN at the
Department of Pathology, Odense University Hospital,
Denmark.
FUNDING
This work was supported by funds from the Danish Ministry of
Culture Committee on Sports Research, Swedish National Centre
for Sports Research and Copenhagen Muscle Research Centre,
and the Natural Science and Engineering Council of Canada.
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Conflict of Interest Statement: The authors declare that the research was
conducted in the absence of any commercial or financial relationships that could
be construed as a potential conflict of interest.
Copyright © 2018 Ørtenblad, Nielsen, Boushel, Söderlund, Saltin and Holmberg.
This is an open-access article distributed under the terms of the Creative Commons
Attribution License (CC BY). The use, distribution or reproduction in other forums
is permitted, provided the original author(s) and the copyright owner(s) are credited
and that the original publication in this journal is cited, in accordance with accepted
academic practice. No use, distribution or reproduction is permitted which does not
comply with these terms.
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