Introduction
Aging is associated with a progressive decline in muscle mass and muscle strength,
affecting physical function [1 ]
[2 ]
[3 ]
[4 ]
[5 ]. Low physical function is likely to affect
quality of life and independence and increase the risk of falls, morbidity, and
mortality in older and frail humans [6 ]
[7 ]. The decline in muscle mass with aging is
mainly caused by a reduction in type II muscle fiber size [8 ]
[9 ]
[10 ], with a foreseeable
consequence of decreased muscle strength and power and ultimately muscle function.
However, neural drive is also affected in the elderly compared with the young [11 ], which could be the result of the loss of
spinal motor neurons that occurs with aging [12 ]
[13 ]. The loss of spinal motor
neurons will cause muscle fiber denervation and thereby a decrease in the number of
active muscle fibers, ultimately causing a decrease in functional capacity during
daily living activities [12 ]. A key target in
preventing a decremental decrease in physical function is therefore to preserve fast
type II muscle fiber size as well as the neural drive in older adults [14 ].
Resistance training is often used to either prevent or reverse the age-related loss
of muscle mass, muscle strength, and function. More specifically, heavy resistance
training leads to an increase in muscle strength and muscle hypertrophy in both
moderately old, old, and the oldest old men and women [15 ]
[16 ]
[17 ]
[18 ]
[19 ]
[20 ]. These beneficial effects
of resistance training are also observed when analyzing changes at the muscle fiber
level, and previous studies in the elderly have shown an increase in type II muscle
fiber size as a result of the training [9 ]
[10 ]
[14 ]
[16 ]
[20 ]
[21 ]
[22 ].
A very well-recognized adaptation to resistance training in both young and elderly
individuals is a shift in the relative amount of type IIX and IIA fibers, where a
reduction in the relative amount of type IIX fibers and a corresponding increase in
the relative amount of type IIA fibers are observed [9 ]
[10 ]
[23 ]. This adaptation occurs in the early phase
of commencing resistance training, is detectable before myofiber hypertrophy [24 ], and is considered as favorable for fatigue
resistance of the skeletal muscle [10 ].
Together, these adaptations in muscle fiber characteristics are to some extent the
reason why an increase in muscle strength, muscle power, and physical function is
observed after a period of intense resistance training [25 ]. However, previous studies have primarily
been of shorter duration, and therefore the current knowledge is sparse when it
comes to the responses of human skeletal muscle fibers to a long-term resistance
training intervention.
We hypothesized that muscle function would be improved as a response to the
resistance training intervention and that the size of type II muscle fibers would be
larger in the resistance-trained participants compared to the controls. Secondly, we
also hypothesized that there would be more type IIA fibers and fewer type IIX fibers
in the resistance training group compared to the controls. Thus, the present study
aimed to investigate the effect of one year of heavy resistance training in elderly
adults on muscle mass and muscle strength and relate this to specific differences in
muscle fiber characteristics of the resistance trained group compared with a
non-exercising control group after the intervention.
Methods
Experimental approach to the problem
The present investigation was a sub-study of a larger randomized controlled trial
with the primary aim being to investigate the effect of one year of resistance
training upon muscle mass, strength, and function in 451 participants aged 62–70
years that were randomized to one of three groups: heavy resistance training
(HRT), moderate intensity training (MIT), or control (CON) [26 ]. In the present study, 20 participants
(both men and women) were recruited and gave consent to undergo additional
muscle-specific tests at the end of the intervention. From the beginning of the
original study, the 20 participants included in the present study were allocated
to either one year of heavy resistance training or a non-exercising control
group.
Participants
The original study inclusion criteria were an age between 62–70 years and
independent living. The participants were not enrolled in the study if they
performed more than one hour per week of regular strenuous exercise training,
had severe unstable medical diseases (e. g., active cancer or severe heart
disease), had musculoskeletal diseases that inhibited training ability, were
using medication that may influence the effects of training (e. g., androgens or
antiandrogens), and/or drugs that caused safety concerns in relation to training
[26 ]. The participants in the present
study were recruited at the end of the one-year intervention and were included
only if they had a high training compliance (HRT) or had not changed their
habitual physical activity level (CON) during the intervention.
All participants were informed of the benefits and risks of the investigation
prior to signing the informed consent document to participate in the study. The
study was approved by the regional ethical committee, complied with the
Declaration of Helsinki, and approved by the National Data Protection Agency and
registered on clinicaltrials.gov.
Procedures
Interventions
The heavy resistance training intervention has been described elsewhere [17 ]. In brief, the participants
exercised three times/week for one year with at least 48 hours between
sessions. Experienced physical trainers supervised all sessions. Initially,
the participants were familiarized with the program for 6–8 weeks at low
intensity and loads to reduce the risk of musculoskeletal injury and
familiarize them with the exercises. For the remaining part of the one-year
intervention, the participants performed a progressive whole-body training
program with increasing load. The participants performed three sets of 6–12
repetitions corresponding to an estimated intensity between ≈70–85% of 1
repetition maximum (RM) in a linear periodized regime over 9 weeks. Every
second week the load was increased and after week 9, which was a restitution
week, the participants performed 3×12 repetitions with a higher load than
the first week of the last periodization, and thus the load increased
throughout the entire intervention period. The training program consisted of
leg press, knee extension, leg curl, calf raises, hip abduction, chest
press, seated row, crunches, and back extensions. The control group was not
allowed to perform more than one hour of strenuous physical exercise per
week and were encouraged to continue their habitual physical activity level
during the one-year intervention.
Measurements
Before and after the intervention, all participants went through a
comprehensive assessment battery including a medical examination, physical
testing, body composition measurements, and determination of muscle size. In
the present study, only some of the assessments are included. To determine
maximal muscle strength, an isometric knee extensor strength test was
performed in a Good Strength device (V.3.14 Bluetooth; Metitur, Jyvaskyla,
Finland). Body composition was measured by a dual-energy X-ray
absorptiometry (DEXA) scan, where lean body mass (LBM) and lean leg mass
(LLM) were determined. A magnetic resonance imaging (MRI) scan was used to
determine the cross-sectional area (CSA) of the vastus lateralis muscle.
Unfortunately, the MRI scan from two participants (one from each group)
could not be used for analysis, and the analysis is therefore based on the
remaining 18 participants. A detailed description of all assessments has
been described previously [17 ]
[26 ].
Experimental protocol
Muscle biopsy: On the day of the muscle biopsy sampling, the
participants entered the laboratory facilities in a non-fasted state. A
muscle biopsy was obtained from the non-dominant leg using a 6-mm Bergström
needle using manual suction. Prior to obtaining the biopsy, 1% lidocaine was
applied as local anesthesia and an incision of approximately 6 mm was made
through a skin incision. The biopsy was extracted from the most central
position of m. vastus lateralis in accordance with the procedure by
Bergström [27 ]. After extraction, all
visual fat and connective tissue were removed from the biopsy, which was
then embedded in Tissue-Tek and transferred into liquid nitrogen-cooled
isopentane. Another piece of the biopsy was snap-frozen directly in liquid
nitrogen. Both pieces were stored at –80°C until further analysis. Biopsies
were obtained after the intervention only.
Immunohistochemistry: The Tissue-Tek embedded piece of the muscle
biopsies were cut in 10-µm thick transverse sections at –20°C in a cryostat.
The sections from each participant were placed on glass slides and stored in
boxes at –80°C until further analysis. The investigator was blinded to the
participant’s identity and group allocation.
ATPase staining: Four separate slides containing the cut sections from each
participant were prepared for staining using the ATPase histochemistry
method. The slides were preincubated in solutions with a pH of 4.37, 4.53,
4.57, and 10.30 at room temperature. After preincubation, the slides were
rinsed twice in a pH solution of 9.4 for 15 s and 30 s and then incubated
for 30 min in a pH 9.4 ATP solution at 37°C. Thereafter, the slides were
rinsed in 1% CaCl2 for 1, 2, and 3 min followed by an incubation
in a 2% CoCl2 solution for a period of 3 min. Lastly, the slides
were then washed 25 times in H2 O, incubated with 1% ammonium
sulfide for 1 min, washed 25 times in H2 O again, and finally, the
slides were mounted with polyvinylpyrrolidone [23 ]
[28 ]
[29 ].
Capillary staining: A slide from each participant was prepared for
immunohistochemical staining of capillaries. The double-staining method
combining ulex europaeus lectin 1 (UEA-1) and collagen type IV staining was
used [30 ]. First the sections were
dried, then the slides were fixed in acetone for 30 s, incubated in 1% BSA
for 20 min followed by an incubation of UEA-1 protein for 30 min at room
temperature. Thereafter, the slides were incubated with anti-UEA-I for 15
min and anti-human collagen IV for 30 min. The slides were then incubated
with the secondary antibodies, biotinylated goat anti-rabbit antibody, and a
biotinylated goat anti-mouse antibody for 30 min before a Vector Elite ABC
HRP kit was applied to the slides for an additional 30 min. Lastly, the
slides were incubated with a 3,3’-diaminobezidine substrate for 3–4 min
before being mounted in Aquatex [30 ].
Analysis of capillary and ATPase staining: To evaluate fiber type,
fiber size, and the number of capillaries, the ATPase and capillary
stainings were analyzed by a blinded assessor. Serial sections were
visualized and analyzed using an Olympus BX40 microscope (Olympus Optical
Co., Tokyo, Japan), connected to Sanyo Hi-resolution Color CCD camera (Sanyo
Electronic Co., Osaka, Japan), and an eight-bit Matrox Meteor Frame Grabber
(Matrox Electronic Systems, Quebec, Canada), combined with image-analysis
software (Tema, Scanbeam, Hadsund, Denmark). Using the capillary staining, a
fiber mask was drawn along the cell borders of approximately 200 fibers per
biopsy, and capillaries were marked. Afterwards, images from the ATPase
staining were fitted into the fiber mask and a number was assigned to each
specific fiber. The fibers were then displayed on the screen in multiple
images and the individual fibers could be identified. The fibers were then
assigned to a specific fiber type group in order to determine the relative
proportion of the various fiber types, fiber type areas, and fiber sizes as
well as the number of capillaries associated with each fiber [23 ]
[31 ]. The analysis defined five different fiber types (type I,
I/IIA, IIA, IIAX, and IIX) from which the fibers came. From this overall
classification, the number of fiber types was reduced to three main fiber
types (type I, IIA, and IIX) as described previously by Andersen and Aagaard
[23 ] to provide an easier dataset
to compare with earlier studies. In extension of this, the number of minor
sub-fiber types (I/IIA, IIAX, and IIX) was so small in some individuals that
a reliable statistical comparison of differences in fiber size of these
minor fiber types was impossible. Therefore, calculations of fiber type size
were done only for the two major fiber types (I and II) [23 ].
Statistical analyses
A two-way mixed model with repeated measures was used to evaluate the overall
effects of group and time for all parameters, except data from the muscle
biopsies, including data from pre- and post-intervention. In case of a
significant group×time interaction, Tukey post hoc analysis was used to
evaluate within-group comparisons as well as a one-way ANOVA (a generalized
linear model) to detect any group differences from baseline to one year. If
no significant group×time interaction was observed, the same model but
without interaction was used to evaluate the effect of time. As we have only
post-intervention muscle biopsies, a one-way ANOVA was used to evaluate
whether there were any differences between HRT and CON. In addition, to
evaluate the magnitude of the mean differences, Hedges’ g effect sizes (ESs)
were calculated for comparison groups (HRT vs. CON). The interpretation of
the effect sizes is similar to the scale proposed by Rhea 2004 for untrained
participants [32 ]: trivial <0.50,
small=0.50–1.25, moderate=1.25–1.9, and large >2.0. Further, a two-way
mixed model was used to evaluate any potential group and sex differences in
fiber size. If no significant group×sex interaction was observed, a one-way
ANOVA was used to evaluate sex differences. All data are presented as
mean±SE unless otherwise stated. All missing data were removed for the same
participant at all time points (e. g., if a participant had one missing
value from baseline, data from one year were removed). We chose a
significance level of 0.05 for the mixed model and ANOVA. All statistical
analysis was performed using SAS Enterprise Guide 8.3 (SAS Institute Inc.,
Cary, NC, USA).
Results
Participants
Twenty participants (10 men/10 women) with an average age of 67±2.2 years were
enrolled in the study, all participants from a larger cohort that had concluded
the one-year intervention [17 ]. [Table 1 ] provides baseline characteristics
of the participants. Only age differed between the two groups, where
participants in HRT were younger than CON (p<0.05). For all other parameters,
there was no difference between groups. In the present study, the participants
randomized to the heavy resistance training had a training compliance of 88%±5%
(mean±SD) during the intervention.
Table 1 Participant characteristics at baseline
(mean±SD). The isometric muscle strength test was performed in a
Good Strength device.
Total (n=20)
HRT (n=10)
CON (n=10)
Sample size
Age (years)
67±2
66±2*
68±2
20
Sex (women%)
50
50
50
20
BMI (kg/m2 )
23.5±2.4
23.8±2.8
23.2±2.2
20
Lean body mass (kg)
47.7±7.8
48.5±7.9
46.8±8.0
20
Isometric muscle strength (Nm)
151.7±36.7
151.5±38.2
151.9±37.2
20
30-s chair-stand (reps)
17±4
18±4
17±3
20
Total step count (steps/day)
9992±4462
11254±5398
8729±3058
20
*Significant difference between HRT and CON (p<0.05). BMI: body mass
index.
Fiber type composition, size, and capillarization
On average, the muscle biopsy sample was obtained 6.9±0.3 days after the last
exercise session. The number of fibers analyzed for each group was 207±4 and
207±2 (mean±SE) for HRT and CON, respectively. There was no difference in the
percentage of type I and IIA fibers between groups. However, the percentage of
type IIX fibers was significantly lower in HRT than in CON (4.7%±1.4% and
12.3%±3.0%, respectively) (p<0.05, ES: 0.99) ([Fig. 1a ]). This was also the case when the
type IIX fibers were expressed in percentage of the fiber size (p<0.05).
Fig. 1 Fiber type composition (%) (a ) and fiber size
(µm2 ) (b ) of muscle fibers from the vastus
lateralis muscle after one year of heavy resistance training (HRT, dark
grey bars) or habitual physical activity (CON, light grey bars)
(mean±SE). *Significant difference between groups (p<0.05, ES:
0.99).
The size of the fibers did not differ between groups in either muscle fiber type
I (4725 µm2 ±245 µm2 and 4795 µm2 ±267
µm2 for HRT and CON, respectively) or II (3660 µm2 ±389
µm2 and 3821 µm2 ±584 µm2 for HRT and CON,
respectively) ([Fig. 1b ]). When the fiber
size was analyzed to evaluate any sex differences, we observed that men in
general had a significantly higher fiber size in the type II fibers compared
with women (p<0.01) (data not shown). This was independent of which group the
participants were allocated to as there was no significant group×sex
interaction.
We did not observe any difference between groups in capillarization. The number
of capillaries per fiber was 2.2±0.1 capillaries for both groups and the amount
of capillaries per mm2 was 470.4±27.3 and 486.7±21.1 capillaries for
HRT and CON, respectively.
Muscle strength
Similar to the original study with a much larger number of participants [17 ], participants in the heavy resistance
training group experienced an increase in isometric muscle strength as a
response to the training intervention, resulting in a significant group×time
interaction (p<0.0001). The change from baseline to one year in isometric
muscle strength in HRT was significantly higher than in CON (33.7 Nm±4.3 Nm and
–4.7 Nm±5.2 Nm, respectively) (p<0.0001, ES: 2.43) ([Fig. 2a ]). Additionally, compared with
baseline the isometric muscle strength at one year was higher in HRT
(p<0.0001) and unchanged in CON.
Fig. 2 Changes in muscle strength (Nm) (a ) and lean body
mass (g) (b ) after either one year of heavy resistance training
(HRT, dark grey bars) or habitual physical activity (CON, light grey
bars) (mean±SE). *Significant difference between groups (A: p<0.0001,
ES: 2.43, B: p<0.05, ES: 0.96).
Body composition and muscle size
In line with isometric muscle strength, we observed an overall interaction in LBM
(p<0.05), which was similar to what we found in the original study [17 ]. The change from baseline to one year
in LBM was significantly higher in HRT compared with CON (1086 g±302 g and 177
g±169 g, respectively) (p<0.05, ES: 0.96) ([Fig. 2b ]). In addition, LBM was higher after the one-year
intervention in HRT compared with baseline (p<0.01), whereas it was unchanged
in CON.
For either the CSA of the vastus lateralis muscle or LLM, we could not detect any
difference between groups as a response to the intervention in this study ([Table 2 ]).
Table 2 Lean leg mass and muscle size before (baseline)
and after either one year of heavy resistance training (HRT) or
habitual physical activity (CON) (mean±SE).
HRT
CON
Sample
Baseline
1 yr
Baseline
1 yr
size
Lean leg mass (kg)
17.2±1.1
17.6±1.2
16.5±1.1
16.6±1.0
20
CSA m. vastus lateralis (mm2 )
1494±139
1502±136
1476±85
1493±88
18
CSA: cross-sectional area.
Discussion
The main finding of the study was that one year of organized systematic heavy-load
resistance training improved muscle strength and muscle mass in older adults and
that these adaptations were accompanied by the observation of a significantly lower
relative number of muscle fiber type IIX in the trained group compared to the
control group after the intervention. A decrease in the relative amount of type IIX
fibers is a well-known adaptation to heavy resistance training when carried over a
shorter period [23 ], and here we demonstrate
that this also seems to be the case when training is continued up to one year in
elderly individuals.
Somewhat unexpectedly, we could not detect any differences in muscle fiber size
between the two groups after the intervention. This lack of difference in muscle
fiber size is in contrast to the general hypothesis of resistance training
stimulating an increase in muscle fiber size, especially in type II fibers [9 ]
[10 ]
[14 ]
[21 ]
[22 ]
[31 ]
[33 ]. However, in a study by Ziegler et al. also
using a sub-population (n=25) from the same original study as the present study, no
significant increase in fiber size between the HRT and the control group was
observed [34 ]. In that study, muscle biopsies
from both pre-training and post-training were directly compared.
As we did not have biopsies before the training, it cannot be ruled out that the HRT
group could have had a somewhat lower fiber size at baseline than the CON group and
that we could have missed any true increase in fiber size. Another explanation could
be the relatively small number of participants in the present study, and in fact in
the much larger study from which participants in this study were recruited, it was
in fact demonstrated that training increased both strength and cross-sectional area
of skeletal muscle [17 ]. Further, the
determination of fiber size from muscle biopsy sections is widely used as a reliable
assessment of muscle hypertrophy, but it has also been demonstrated that there is an
increasing variation in fiber size with age [35 ], which potentially could have contributed to our lack of findings in
hypertrophy in muscle fiber size. In addition, it is worth mentioning that it is not
unusual to find a discrepancy between adaptation at fiber level and whole-muscle
[36 ] or whole-body level [37 ].
Our finding of an increase in muscle strength and lean body mass in response to a
resistance training intervention has been observed previously in all age groups
including the oldest old [9 ]
[10 ]
[20 ]
[38 ]
[39 ]. The gains in isometric muscle strength and
lean body mass were ~22% and ~2%, respectively, and are similar to what has been
reported with resistance training interventions in older adults [15 ]
[18 ]
[20 ]
[31 ]
[40 ].
Therefore, it is likely that our training program has provided an appropriate
stimulus to the skeletal muscle. Likewise, the apparent decrease in the percentage
of type IIX fibers and corresponding increase in type IIA fibers found in the
resistance-trained participants compared with the controls is a response that has
been observed in earlier resistance training studies [9 ]
[10 ]
[21 ]
[23 ]
[25 ].
It should be noted that we did not see a difference in the percentage of type IIA
fibers between the two groups.
Even though we could not detect any difference in muscle fiber size, there was a
relatively high increase in muscle strength, which could indicate that the increased
strength could be primarily a consequence of neuromuscular changes in response to
the resistance training intervention rather than changes at the muscle level, in
line with earlier resistance training studies that have found increased neural drive
[11 ]
[41 ] and increased motoneuron firing frequency [41 ]. A combination of these changes would
increase the amount of recruited muscle fibers and thereby the potential to increase
muscle strength.
In conclusion, one year of heavy resistance training increased muscle strength and
lean body mass in elderly individuals, and we observed a lower percentage of type
IIX muscle fibers in the heavy resistance training participants compared with the
non-training controls. The lack of any difference in muscle fiber size in muscle
biopsies between groups obtained after the training intervention indicates that
long-term resistance training in elderly individuals predominantly improves muscle
strength through neuromuscular adaptation rather than morphological changes per
se.
