Skeletal muscle morphology in sarcopenia defined using the EWGSOP criteria: findings from the Hertfordshire Sarcopenia Study (HSS)
Patel et al. BMC Geriatrics
Skeletal muscle morphology in sarcopenia defined using the EWGSOP criteria: findings from the Hertfordshire Sarcopenia Study (HSS)
H. P. Patel 0 1 2
M. C. White 1
L. Westbury 0
H. E. Syddall 0
P. J. Stephens 1
G. F. Clough 6
C. Cooper 0 2 4
A. A. Sayer 0 1 2 3 5
0 Medical Research Council Lifecourse Epidemiology Unit, University of Southampton , UHSFT, Tremona Road, Southampton SO16 6YD , UK
1 Academic Geriatric Medicine, University of Southampton, University Hospital Southampton FoundationTrust (UHSFT) , Tremona Road, Southampton SO16 6YD , UK
2 National Institute for Health Research Southampton Biomedical Research Centre, University of Southampton and UHSFT , Tremona Road, Southampton SO16 6YD , UK
3 Newcastle University Institute for Ageing and Institute of Health & Society, Newcastle University , Newcastle , UK
4 National Institute for Health Research Musculoskeletal Biomedical Research Unit, University of Oxford , Oxford , UK
5 National Institute for Health Research Collaboration for Leadership in Applied Health Research and Care: Wessex, Academic Geriatric Medicine, University of Southampton , UHSFT, Tremona Road, Southampton SO16 6YD , UK
6 Institute for Developmental Sciences, University of Southampton , UHSFT, Tremona Road, Southampton SO16 6YD , UK
Background: Sarcopenia is defined as the loss of muscle mass and function with age and is associated with decline in mobility, frailty, falls and mortality. There is considerable interest in understanding the underlying mechanisms. Our aim was to characterise muscle morphology changes associated with sarcopenia among community dwelling older men. Methods: One hundred and five men aged 68-76 years were recruited to the Hertfordshire Sarcopenia Study (HSS) for detailed characterisation of muscle including measures of muscle mass, strength and function. Muscle tissue was obtained from a biopsy of the vastus lateralis for 99 men and was processed for immunohistochemical studies to determine myofibre distribution and area, capillarisation and satellite cell (SC) density. Results: Six (6 %) men had sarcopenia as defined by the European Working Group on Sarcopenia in Older People (EWGSOP) criteria. These men had lower SC density (1.7 cells/mm2 vs 3.8 cells/mm2, p = 0.06) and lower SC/fibre ratio (0.02 vs 0.06, p = 0.06) than men without sarcopenia. Although men with sarcopenia tended to have smaller myofibres and lower capillary to fibre ratio, these relationships were not statistically significant. Conclusion: We have shown that there may be altered muscle morphology parameters in older men with sarcopenia. These results have the potential to help identify cell and molecular targets for therapeutic intervention. This work now requires extension to larger studies which also include women.
Sarcopenia; Muscle morphology; Community dwelling older men
Sarcopenia is associated with adverse health outcomes
and incurs a substantial health care cost [
]. Defined as
the loss of skeletal muscle mass and function, sarcopenia
is common in both men and women over the age of 65
across a range of healthcare settings [
]. For example,
among community dwelling older people in the UK,
prevalence rates for sarcopenia have been estimated at
4.6 % for men and 7.9 % for women [
]. Sarcopenia has
been defined based on lean mass indices i.e., total lean
mass, appendicular lean mass and muscle function i.e.,
grip strength or physical performance. Notable diagnostic
algorithms include The European Working Group on
Sarcopenia in Older People (EWGSOP) [
Foundation for the National Health Institutes of Health (FNIH)
Sarcopenia Project [
], and the Asian Working Group for
Sarcopenia (AWGS) [
], the latter definition to account
for ethnic variations in muscle mass and muscle function.
There is considerable interest in understanding the
mechanisms driving sarcopenia and there have been a
number of small studies investigating morphological
changes in skeletal muscle with increasing age. For
example, it has been reported that between the sixth and
ninth decades, myofibre size and number decrease in
both men and women [
]. In addition there is type I and
type II myofibre atrophy, as well as other changes in
muscle morphology including the appearance of hybrid
fibres, the presence of hypertrophied fibres and fibre
type grouping [
]. Although not completely
understood, several factors contribute to these morphological
changes including, inflammation, denervation, oxidative
stress, imbalance in protein synthesis and reduced
satellite cell number and or function [
Studies of muscle morphology have rarely been
population based and how these morphological changes relate to
altered skeletal muscle mass, function and sarcopenia is
unclear. The objective of this study was therefore to
determine the relationship between muscle morphology and
sarcopenia as defined using the European Working Group
Sarcopenia in Older People (EWGSOP) criteria in a
population based study of community dwelling older men.
One hundred and five community dwelling older men
aged 68–76 years who had participated in the UK
Hertfordshire Cohort Study (HCS) [
] were involved in the
Hertfordshire Sarcopenia Study (HSS) [
characterised their muscle morphology and functional
parameters and applied the European Working Group on
Sarcopenia in Older People (EWGSOP) diagnostic
algorithm to identify sarcopenia [
]. Inclusion and exclusion
criteria and study methods have been previously
described in detail [
]. The study received ethical
approval from the Hertfordshire Research Ethics
Committee, number 07/Q0204/68. Each participant gave
written informed consent.
Percutaneous muscle biopsies of the vastus lateralis
were conducted under local anaesthetic using a
Weil-Blakesley conchotome [
]. One hundred and
two participants were eligible for the procedure; three were
ineligible as they were taking medication that might
influence subsequent wound healing (n = 2) or predispose to
haematoma formation (n = 1). Biopsies from a further three
participants were not suitable for analysis. Thus, the final
muscle biopsy analysis sample comprised 99 participants.
Muscle tissue was fixed overnight at −20 °C before
being embedded in glycol methacrylate resin [
Serial cross-sections at 7 μm were cut and stained
for type II fast-twitch myofibres using the
monoclonal anti-myosin fast antibody at a dilution of 1:6000
(clone MY-32; Sigma-Aldrich, Dorset, UK) (Fig. 1).
Capillaries were stained by incubating separate slides
with biotinylated Lectin Ulex Europeaus Agglutinin 1
(UEA-1, Vector Laboratories, Peterborough, UK) at a
dilution of 1:200 for two hours (Fig. 2). Stained
sections were examined under a photomicroscope
(Zeiss Axioskop II, Carl Ziess Ltd, Welwyn Garden
City, UK) coupled to KS 400 image analysis software
(Image Associates, Bicester, UK). Sections were
viewed at a × 5 magnification and digitized to obtain tissue
area, myofibre number (type I, slow fibre vs type II, fast
fibre) and myofibre cross-sectional areas (μm2). Slow and
fast fibre proportions were expressed as a percentage of
total fibres. For capillaries, a digital image at a
magnification of ×40 was taken of the section. The total number of
muscle fibres and capillaries was quantified from the
whole tissue area manually from the digital image. For
each section, capillary density (capillaries per mm2) and
Fig. 2 A serial cross section showing capillary staining. Capillaries are
stained brown and are located at the peripheries of the myocyte.
(Capillaries have been stained with Ulex Europeaus Agglutinin 1, 1:200,
Vector laboratories, UK, visualised at magnification × 40 and are arrowed)
capillary: fibre ratio was calculated. Satellite cells (SCs)
were identified in separate tissue sections using a similar
immunohistochemistry protocol with the primary
antibody PAX-7 (Paired-box transcription-factor 7,
Developmental Studies Hybridoma bank, University of Iowa) [
(Fig. 3). SCs were identified by light microscopy and
quantified (SC density [cells/mm2] and SC to fibre ratio) using
image analysis techniques as described above. Muscle
morphology parameters were analysed in all samples by a
Defining sarcopenia using the EWGSOP criteria
For a diagnosis of sarcopenia, the EWGSOP recommend
the presence of both low muscle mass and low muscle
function as measured by strength and or muscle
]. We use this definition based on our previous
published work detailing the prevalence of sarcopenia in
this cohort [
]. In our study, lean muscle mass was
determined by dual-energy x-ray absorptiometry scanning
(DXA; Hologic Discovery, auto whole body software
version 12.5). Isometric maximum grip strength was
identified from three measurements in each hand using a
standardised protocol and Jamar dynamometer [
validated battery of physical performance tests was
administered which included time to complete 5 chair rises and
measurement of customary gait speed over 3 m [
used values in the lowest third of the distributions of DXA
derived lean mass and gait speed, and grip strength values
of <20 kg for women and <30 kg for men, within the
EWGSOP diagnostic algorithm for sarcopenia [
Normally distributed variables were summarised using
means and standard deviations (SD). Skewed variables
were loge transformed to normal distributions as
necessary; for these variables, means and SDs on the loge scale
were back transformed to geometric means and SDs on
the original scale of measurement. Student’s t test was
used to compare age, body size, physical performance,
cardiovascular fitness and fibre morphology variables
between those without, and those with sarcopenia. All
analyses were carried out using Stata release 13
(StataCorp, Texas, USA). A p value of <0.05 was
considered statistically significant.
Six men had sarcopenia (6.2 % of the 97 men with
complete data for items comprising the EWGSOP
definition). Descriptive statistics for participant characteristics
and fibre morphology in terms of satellite cell density
(cell/mm2), satellite to fibre ratio type I and type II
fibre distribution (%), size/area (μm2), capillary density
(capillaries/mm2) and capillary to fibre ratio are
presented in Table 1. The total number of fibres counted
to determine the capillary and satellite cell indices is
also presented in Table 1.
Relationships between fibre morphology and sarcopenia
We have previously reported on the relationships
between sarcopenia and age, anthropometry and muscle
]. Men with sarcopenia tended to have lower
SC density (1.7 cells/mm2 vs 3.8 cells/mm , p = 0.06)
and lower SC/fibre ratio (0.02 vs 0.06, p = 0.06) than
men without sarcopenia (Table 2). Men with sarcopenia
also tended to have, on average, smaller slow and fast
fibre areas and lower capillary to fibre ratios. However,
these morphological relationships were not statistically
significant (Table 2).
We have investigated the association between skeletal
muscle morphology and sarcopenia defined by the
EWGSOP criteria in a population based study of
community dwelling older men. In this study, there was a
suggestion that the six HSS men with sarcopenia had
lower average SC density and SC/fibre ratio in
comparison with those without sarcopenia but the relationship
was not significant at the 5 % level.
Satellite cells (SC) are undifferentiated stem cells
responsible for myofibre maintenance and are central
to the growth and repair of muscle and have the
ability to enter the cell cycle, proliferate and
]. SC number fluctuate in younger and
older adults in response to both intrinsic and
extrinsic regulatory cues . For example, physical
] as well as with drug treatment [
niche surrounding the SC is also a critical regulator
of function and is governed by growth factors,
signalling molecules as well as innervation [
In support of our results, satellite cell content of fibres
has been reported to decrease in the muscles of older
humans. For example, Verdijk et al. showed that in older
humans, SC content, specifically in type II myofibres,
were lower when compared to younger controls [
a study by Kadi et al. SC per fibre ratio was significantly
lower in healthy older men and women compared to
their younger counterparts [
]. In the later study, no
inference was made on whether there was a fibre specific
reduction as was reported by Verdijk et al. Therefore it
appears that a reduction in satellite cells may mediate
the observed muscle atrophy, specifically of type II
fibres, with age. However, a detailed morphological study
conducted by Purves-Smith et al. suggested that in very
old individuals who may have severe muscle atrophy,
both myofibre types show atrophic changes [
The exact reasons for a decrease in SC content or a
decline in SC function with age are unknown.
Contributing factors include alterations in the surrounding
environment including denervation and oxidative damage
] as well as decrease in activity of crucial myogenic
regulatory factors coupled with an increase in negative
regulators of muscle growth [
]. For example,
myostatin appears to suppress certain myogenic
regulatory factors crucial for proliferation and differentiation
and therefore has been postulated to impair function as
well as self-renewal of satellite cells . Taken together,
it appears that loss of SC or reduced SC function
diminishes the ability of ageing muscle to both hypertrophy in
response to stimulus or repair and self-renew in
response to injury thereby contributing to sarcopenia [
However, a recent study in older mice who were SC
deplete suggested that neither force generation nor single
fibre cross sectional area was affected by a reduction in
aGeometric mean (SD)
** p value for t-test between non sarcopenic and sarcopenic individuals as defined by the EWGSOP criteria
Sarcopenia status could not to be determined for two participants because of missing walking speed data hence maximum sample size of 97
SC number [
]. Clearly, further studies are needed in
both male and female ageing cohorts to investigate the
role of SC in sarcopenia.
We observed a non-significant trend (p > 0.05) for
men with sarcopenia to have, on average, smaller slow
and fast fibre areas and lower capillary to fibre ratios.
Whereas myofibre morphology measurements in
relation to exercise, immobilisation and ageing have been
described in a number of studies [
], this is one of the
first studies to describe muscle morphology in relation
to sarcopenia in community dwelling older men. Muscle
mass and cross sectional area (CSA) are functions of
myofibre size and number [
] and we speculate that
smaller fibres seen in our study are associated with the
decrease in total lean mass in men who were sarcopenic.
The results of a longitudinal study by Frontera et al. [
which revealed age related reductions in muscle cross
sectional area as well as capillary to fibre ratio in men is
in partial support of our cross sectional findings.
Our study had several limitations. First, the sample
size was modest which will have limited statistical
power. Second, the immunohistochemical methodology
used to quantify the morphology parameters was open
to observer error although consistent and rigorous
methods were applied throughout the study in order to
limit this possibility. Also, we did not evaluate SC
content per specific fibre type or other indices of muscle
]. Finally, the parameters measured may
not accurately reflect the morphological changes
occurring in muscle. Longitudinal studies would be helpful to
more fully characterise the morphological changes that
occur over time in the muscle of people with sarcopenia.
However, our study has a number of strengths. First,
we have shown that it is feasible to obtain tissue from
community dwelling older men in the context of an
epidemiological birth cohort. The advantage of this is that
morphological data can be combined with the extensive
phenotypic data that has already been collected. Second,
the immunohistochemical methodologies employed were
based on tested protocols and can be applied to future
large scale studies. For example, whereas SC have
typically been studied through electron microscopy and
], few studies have employed
simple immunohistochemistry techniques to quantify
satellite cells in healthy individuals [
]. Our study
shows that quantification of several morphological
variables is possible using these methods. However,
measurements of specific fibre type/SC content as well as
other indices of capillarisation will need to be considered
when applied to future studies.
Our results suggest that men with sarcopenia may have
decreased satellite cell content and perhaps also smaller
fibres and lower capillary to fibre ratios in comparison
with men without sarcopenia. We can only speculate,
given the sample size and cross sectional nature of the
study, that the morphological results seen not only have
an effect on muscle mass but also on muscle quality
]. These morphological changes impact on muscle
performance in men with sarcopenia, who by definition
have grip strengths of less than 30 kg, walking speed less
than 0.8 m per second and low muscle mass [
The importance of this work is that the identification
of morphological changes in older people with
sarcopenia has the potential to identify cellular and molecular
targets for therapeutic intervention. Methodological
findings from this study now need to be applied to large
scale studies that also include women.
The authors declare that they have no competing interests.
HPP, HES, AAS, CC, MW, PJS, GC participated in the conception, design
and conduct of the study. LW and HES conducted statistical analyses.
HPP drafted the first version of the manuscript. All authors read and
approved the final manuscript.
We wish to acknowledge Katy Gould and Henry James on their contribution
to this work and thank the study participants as well as the staff at the
Wellcome Trust Clinical Research Facility, University Hospital Southampton
for making this work possible. This study was funded by the Medical
Research Council UK and the University of Southampton. The British
Geriatrics Society provided additional financial support to HPP.
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