Skeletal muscle characteristics and physical activity patterns in COPD
To my family
Örebro Studies in Sport Sciences 10
GABRIELLA ELIASON
Skeletal muscle characteristics
and physical activity patterns in COPD
© Gabriella Eliason 2010
Title: Skeletal muscle characteristics and physical activity patterns in COPD.
Publisher: Örebro University 2010
www.publications.oru.se
[email protected]
Print: Intellecta Infolog, Kållered 11/2010
ISSN 1654-7535
ISBN 978-91-7668-768-0
Abstract
Gabriella Eliason (2010): Skeletal muscle characteristics and physical
activity patterns in COPD. Örebro Studies in Sport Sciences 10, 65 pp.
Chronic obstructive pulmonary disease (COPD) is one of the leading causes
of morbidity and mortality worldwide. Besides abnormities within the respiratory system COPD is also associated with effects outside the lungs, so
called systemic effects. One systemic effect that has been highlighted is
skeletal muscle dysfunction, which has also been associated with reduced
exercise capacity. Apart from changes in muscle morphology, low levels of
physical activity has also been suggested as a plausible mediator of reduced exercise capacity in COPD. The aim of this thesis was to study muscle morphology and physical activity patterns in patients with different
degrees of COPD and to examine the associations between muscle morphology, physical activity and exercise capacity in these patients.
Skeletal muscle morphology was found to shift towards a more glycolytic muscle profile in COPD patients and changes in muscle morphology
were found to be correlated to disease severity and to exercise capacity.
Muscle capillarization was also found to be lower in COPD compared with
healthy subjects and to be correlated to disease severity and exercise capacity. When studying signalling pathways involved in muscle capillarization,
an overexpression of VHL was observed in patients with mild and moderate COPD compared with healthy subjects. Furthermore, COPD patients
were found to be less physically active compared with healthy subjects and
the level of physical activity was associated with exercise capacity.
In conclusion, changes in skeletal muscle morphology and low levels of
physical activity are present in COPD patients and may partly explain the
lower exercise capacity observed in these patients. The more glycolytic
muscle profile observed in COPD is suggested to be mediated by hypoxia
and low levels of physical activity in this patient group. Furthermore, increased levels of VHL may lead to impaired transduction of the hypoxic
signalling pathway, which may contribute to the decreased muscle capillarization observed in COPD.
Keywords: COPD, muscle morphology, muscle fibre distribution, muscle
capillarization, physical activity, von Hippel-Lindau protein, exercise capacity.
Gabriella Eliason, School of Medical Sciences,
Örebro University, SE-701 82 Örebro, Sweden, [email protected]
LIST OF PUBLICATIONS
This thesis is based on the following original papers, which will be referred
to in the text by their roman numerals:
I.
Eliason G, Abdel-Halim S, Arvidsson B, Kadi F, Piehl-Aulin K.
Physical performance and muscular characteristics in different
stages of COPD. Scand J Med Sci Sports 2009; 19(6):865-70.
II.
Jatta K, Eliason G, Portela-Gomes G M, Grimelius L, Caro O,
Nilholm L, Sirsjö A, Piehl-Aulin K, Abdel-Halim S M. Overexpression of von Hipple-Lindau protein in skeletal muscles of
patients with chronic obstructive pulmonary disease. J Clin
Pathol 2009; 62:70-76.
III.
Eliason G, Abdel-Halim S M, Piehl-Aulin K, Kadi F. Alterations in the muscle-to-capillary interface in patients with different degrees of chronic obstructive pulmonary disease. Respiratory Research 2010; 11:97
IV.
Eliason G, Zakrisson A, Piehl-Aulin K, Hurtig-Wennlöf A.
Physical activity patterns in patients with different degrees of
chronic obstructive pulmonary disease. Submitted
TABLE OF CONTENTS
INTRODUCTION ................................................................................... 13 Skeletal muscle ......................................................................................... 13 Muscle fibre types ................................................................................ 14 Muscle regeneration ............................................................................. 15 Muscle capillarization .......................................................................... 16 Angiogenesis ........................................................................................ 16 Physical activity........................................................................................ 18 Exercise capacity ...................................................................................... 19 COPD ...................................................................................................... 20 Skeletal muscle in COPD ..................................................................... 22 Physical activity in COPD .................................................................... 23 Exercise capacity in COPD .................................................................. 24 AIM OF THE THESIS ............................................................................. 25 MATERIALS ........................................................................................... 27 Subjects .................................................................................................... 27 METHODS .............................................................................................. 31 Muscle biopsy sampling and analysis ....................................................... 31 Fibre type distribution.......................................................................... 31 Fibre area and perimeter ...................................................................... 32 Satellite cells ......................................................................................... 33 Capillary network ................................................................................ 33 Capillary parameters ............................................................................ 34 VHL-identification ............................................................................... 35 HIF and VEGF identification ............................................................... 35 Exercise capacity ...................................................................................... 35 Body composition .................................................................................... 35 Physical activity........................................................................................ 36 Statistical analyses .................................................................................... 37 RESULTS ................................................................................................. 39 Study I ...................................................................................................... 39 Aim ...................................................................................................... 39 Results ................................................................................................. 39 Study II .................................................................................................... 40 Aim ...................................................................................................... 40 Results ................................................................................................. 40 Study III ................................................................................................... 42 Aim ...................................................................................................... 42 Results ................................................................................................. 42 Study IV ................................................................................................... 43 Aim ...................................................................................................... 43 Results ................................................................................................. 43 DISCUSSION ........................................................................................... 45 Muscle fibre type and fibre area ............................................................... 45 Muscle regenerative capacity .................................................................... 46 Muscle capillarization .............................................................................. 46 Physical activity ........................................................................................ 48 CONCLUSIONS ...................................................................................... 51 FUTURE PERSPECTIVES........................................................................ 53 ACKNOWLEDGEMENTS ...................................................................... 55 REFERENCES ......................................................................................... 57 LIST OF ABBREVIATIONS
ATP
BMI
CAF
CAFA
cDNA
C:Fi
CFPEindex
COPD
DXA
FFM
FFMI
FEV1,0
FVC
GOLD
HIF
LC/PFindex
mAb
MVPA
MyHC
N-CAM
NO
PaCO2
PaO2
PCR
pVHL
RNA
SF
VEGF
VO2max
adenosine triphosphate
body mass index
number of capillaries around a single muscle fibre
number of capillaries around a fibre in relation to fibre area
complementary deoxyribonucleic acid
capillary-to-fibre ratio
capillary-to-fibre perimeter exchange index
chronic obstructive pulmonary disease
dual x-ray absorptiometry
fat free mass
fat free mass index
forced expiratory volume in one second
forced vital capacity
Global Initiative for Chronic Obstructive Lung Disease
hypoxia-inducible factor
length of capillary/perimeter of the fibre
monoclonal antibody
moderately or vigorously physically active
myosin heavy chain
neural cell adhesion molecule
nitric oxide
partial arterial pressure for carbon dioxide
partial arterial pressure for oxygen
polymerase chain reaction
von Hippel-Lindau tumour suppressor protein
ribonucleic acid
sharing factor
vascular endothelial growth factor
maximal oxygen consumption
INTRODUCTION
Chronic obstructive pulmonary disease (COPD) is one of the leading
causes of morbidity and mortality worldwide [43, 72, 112]. The disease is
characterised by irreversible progressive airflow limitation with breathingrelated symptoms such as coughing, phlegm, and dyspnea [72, 112]. Besides structural and functional abnormities within the respiratory system,
COPD is also associated with effects outside the lungs, so called systemic
effects. One systemic effect that has been highlighted is skeletal muscle
dysfunction. Both morphological and functional changes of the muscle
have been described [22, 39, 60, 68]. Skeletal muscle dysfunction has also
been suggested to play an important role in reducing exercise capacity in
patients with COPD [68]. The mechanisms leading to changes in skeletal
muscle are not fully understood; however, systemic inflammation, oxidative stress, use of oral corticosteroids, malnutrition, tissue hypoxia and low
levels of physical activity have been suggested as potential mediators [4,
22, 60, 68]. The present thesis elucidates changes in skeletal muscle of
COPD patients and highlights physical activity and hypoxia as plausible
mechanisms involved in these changes.
Skeletal muscle
Skeletal muscle consists of a number of contractile muscle cells (fibres) that
join into a tendon at each end. The muscle is surrounded by a sheath of
connective tissue, the epimysium, which is also referred to as the fascia. In
the muscle, smaller fascicles are made up of bundles of thousands of muscle fibres surrounded by connective tissue, the perimysium. Each individual
muscle fibre is also surrounded by a thin layer of connective tissue, the
endomysium, which contains a capillary network and nerve fibres [115]
(Figure 1).
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F
Figure 1. Overview of skeletal muscle tissue organization. Illustration from
Fundamentals of Anatomy & Physiology by Martini (2008) with permission.
Muscle fibre types
In human muscle, three major muscle fibres types have been distinguished.
The different fibre types express different myosin heavy chain (MyHC)
isoforms (I, IIa, IIx) which give the fibres different contractile properties.
Type I fibres are referred to as slow twitch fibres, while type IIx are referred to as fast twitch fibres. The different fibre types also have different
metabolic properties. Type I fibres are the most oxidative type of fibres and
have a rich mitochondria supply and high concentrations of oxidative enzymes. Fast type IIx fibres are the most glycolytic fibre types, relying almost entirely on anaerobic energy metabolism and containing high concentrations of glycolytic enzymes. Type IIa fibres are considered to have both
glycolytic and oxidative properties [115]. In addition to the three main
fibre groups, there are also so called hybrid fibres expressing more than
one MyHC isoform, usually MyHC I/IIa or MyHC IIa/IIx [7, 91-92]. Classification of muscle fibres can be done according to their morphological or
metabolic characteristics. Histochemical staining of myosin adenosine
triphosphate (ATPase) made it possible to delineate slow type I and fast
type II fibres [87]. More recently, type I, type IIa and type IIb (now a days
referred to as type IIx) fibres have been distinguished using this method by
pre-incubation at different pH values [15]. Immunohistochemical staining,
based on the expression of MyHC isoforms, has made it possible to distinguish both major fibre types and hybrid fibres [92].
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Skeletal muscle tissue is extremely heterogeneous in regards to fibre type
composition. Moreover, muscle fibres are dynamic structures that are capable of changing phenotype under various conditions such as changes in
neuromuscular activity, mechanical load, changes in hormonal profile and
aging [91]. Regarding the metabolic properties of skeletal muscle, the oxidative capacity of the muscle has been shown to be affected by environmental changes such as hypoxia. It has been suggested that longterm exposure to hypoxia leads to markedly lowered oxidative capacity of the muscle
due to decreased mitochondrial volume and reduced oxidative enzyme
activity [54, 73].
Muscle regeneration
Muscle fibres are postmitotic cells unable to re-enter the cell cycle. In spite
of this, the skeletal muscle possesses a remarkable ability to repair and
regenerate itself under extraordinary conditions such as extreme physical
activity, trauma or injury. The majority of this regeneration is carried out
by activation, proliferation and differentiation of satellite cells.
Satellite cells, which were first described by Mauro in 1961 [76], represent undifferentiated myogenic precursor cells that lie between the external
lamina and sarcolemma of skeletal muscle fibres (Figure 1). Under normal
conditions satellite cells are quiescent, but with appropriate environmental
signals they become activated and re-enter the cell cycle to either generate
new muscle fibres or provide new myonuclei to the parent fibre [46, 51,
114].
Quiescent satellite cells are present throughout the muscle, but the distribution of satellite cells has been shown to vary between different individuals and between different muscle groups and muscle fibre types [51,
58]. In rodents it has been shown that slow type I fibres have a higher
number of satellite cells than fast type II fibres. Thus, muscles containing
mainly slow type I fibres tend to contain more satellite cells than muscles
containing a large proportion of type II fibres [51]. However, in a study by
Kadi et al [57], the satellite cell content in the tibial anterior muscle of
healthy untrained humans did not differ between type I and type II muscle
fibres. These findings have more recently been confirmed by Verdijk et al
[110].
The satellite cell population within the muscle is not static. The proportion of satellite cells has been shown to increase following exercise [58], as
a result of low-frequency stimulation [98] and in neuromuscular disorders
such as Duchenne muscular dystrophy and neurogenic atrophy [69], while
a lower proportion of satellite cells has been reported in elderly men and
women compared with in a younger population [57].
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The signalling pathways for satellite cell activation are not fully understood, but several mediators of satellite cell activation such as growth factors, nitric oxide (NO), mechanical stimulation and physiological stimuli
through exercise have been suggested [114].
Identification of satellite cells has commonly been done using electron
microscopy. However, the development of antibodies raised against specific proteins expressed by satellite cells has made immunohistochemical
identification of satellite cells possible. The membrane-bound neural cell
adhesion molecule (N-CAM) is a cell-surface glycoprotein localised on
satellite cells and the N-CAM antibody has been demonstrated to be a
successful marker for both quiescent and activated satellite cells in human
skeletal muscle [58]. Likewise, it has been shown that antibodies against
the transcription factor Pax-7, which is located in the nucleus of the satellite cell, can also be used for identification of satellite cells in sections of
human skeletal muscle biopsies [67].
Muscle capillarization
The capillary bed in skeletal muscle mainly serves to supply the muscle
with oxygen but also to remove different metabolites and heat produced
during muscle contraction [55]. The main arteries that supply a muscle
with blood lie outside the muscle and it is a secondary artery that enters
into the muscle. Inside the muscle, the secondary artery divides into smaller
arterioles, which subdivide into metaarterioles. From the metaarterioles,
blood moves into the capillaries, which can be identified by their size and
cross section. Vessels in the endomysium that have a diameter < 15μm are
most commonly capillaries. The capillaries are longitudinally oriented and
run between the muscle fibres [17, 29]. The histological structure of the
capillary, with the capillary wall being about 1μm thick and consisting of a
single layer of endothelial cells, allows a two-way exchange of gases and
substances. Capillaries within the muscle are organized as an interconnected network where the entrance to each capillary is guarded by a capillary sphincter. Contraction of the sphincter leads to reduced or altered
blood flow in the specific capillary while relaxation of the sphincter results
in increased blood flow in the capillary [10]. It is known that highly oxidative muscle fibres have a denser capillary network than glycolytic muscle
fibres [55].
Angiogenesis
Blood vessel growth is of great importance to match the needs of blood
supply to the tissue. Capillary growth through sprouting of capillaries from
an already established capillary network is referred to as angiogenesis or
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sprouting angiogenesis [41]. This process is carried out by activated endothelial cells that branch out from existing capillaries and assemble into
tubes that re-enter the capillary bed [14]. The process of angiogenesis is
controlled by both growth promoting and growth inhibitory factors. One
of the most central growth factors in the process of initiating skeletal muscle angiogenesis is vascular endothelial growth factor (VEGF), which
stimulates proliferation, migration and survival of endothelial cells [85].
Adequate levels of VEGF in skeletal muscle are also essential for maintaining muscle capillarization; it has been shown that lowered levels of VEGF
decreases capillary density and capillary-to-fibre ratio [14].
Several factors are involved in the regulation of VEGF. These include
growth factors, tumour suppressor factors, exercise and hypoxia [85, 97].
The responses to local hypoxia within the tissue are largely considered to
be mediated by the widely-expressed hypoxia-inducible factor (HIF) of
which three isoforms (HIF-1α, HIF-2α and HIF-3α) are known to date [47,
77]. Intracellular levels of HIF are tightly regulated by the von HippelLindau tumour suppressor protein (pVHL). pVHL is a component of an E3
ubiquitin ligase that mediates ubiqiutylation of the α subunits of the HIF
protein, resulting in rapid degradation of the protein by the ubiquitinproteasome pathway in the presence of oxygen [6, 61, 101]. As a response
to local hypoxia within the tissue, intracellular HIF levels are stabilized and
increased through decreased degradation. The HIF signal is then transduced to regulate the transcription of a multitude of gene responses including VEGF [101] (Figure 2).
Whether or not systemic hypoxia induces capillary growth in skeletal
muscle is controversial. Studies on mice have suggested an induced angiogenesis due to systemic hypoxia [24-25] while increased capillarity as a
response to systemic hypoxia in humans has been explained by a parallel
reduction in fibre area [53]. Other studies show no change in capillary
supply during systemic hypoxia in human skeletal muscle [66, 96] and
longterm exposure to hypoxia has even been shown to decrease skeletal
muscle capillarization [23, 54]. Taken together, these conflicting results
indicate that the degree of hypoxia and the duration of exposure to hypoxic conditions are important factors for angiogenesis in skeletal muscle.
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Figure 2. Regulation of VEGF transcription by HIF 1-α during hypoxic and
normoxic conditions.
Endurance exercise has been reported to induce capillary growth in
skeletal muscle [14, 55, 97]. Furthermore, VEGF and HIF 1-α have been
shown to be up-regulated following exercise training [6, 14, 42]. The
mechanisms resulting in exercise-induced angiogenesis are not fully understood. However, increased blood flow within the muscle, local hypoxia in
the muscle tissue and mechanical stretching of the muscle have been suggested as plausible mediators [55, 97].
Physical activity
Physical activity is defined as any bodily movement produced by skeletal
muscles resulting in energy expenditure beyond resting energy expenditure
[16]. It has been shown that the level of physical activity is an important
factor related to a number of health outcomes such as reduced risk of cardiovascular disease, thromboembolic stroke, hypertension, type 2 diabetes
mellitus, osteoporosis, obesity, colon cancer, breast cancer, anxiety and
depression [84]. In addition to these benefits, physical activity in older
adults has been suggested as effective therapy for many chronic diseases
such as coronary heart disease, peripheral vascular disease, elevated cholesterol, osteoarthritis, claudication and CODP [84]. Health recommendations state that healthy adults aged 18–65 years should perform 30 minutes
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of at least moderate intensity physical activity most preferably on all weekdays, but at least five days a week, to promote and maintain health [45,
89]. These recommendations have also been shown to be applicable in
older adults (>65 years) and in adults aged 50–64 years with clinically
significant chronic conditions [84].
Three different dimensions are included when describing a physical activity: frequency, duration and intensity. The frequency refers to how often
the activity occurs over a specific time period, the duration refers to how
long time the activity is sustained and the intensity refers to how strenuous
the activity is. Intensity levels of physical activity can be expressed in terms
of absolute or relative intensity. Absolute intensity level is commonly defined as the rate of oxygen required per time unit (l x min-1 or ml-1x min x
kg body weight-1) while relative intensity level often is defined in terms of
relative load on physiological markers in relation to maximal capacity (e.g.
percentage of maximal oxygen consumption or percentage of maximal
heart rate) [78].
Various methods for assessment of physical activity are available. These
methods can be broadly divided into two groups: subjective and objective
methods. Subjective measurements are also referred to as self-report methods and include questionnaires, activity diaries and interviews, while the
most common objective measurements are the double labelled water
method, heart rate monitoring and motion sensors such as pedometers and
accelerometers [79].
Exercise capacity
Exercise is defined as planned, structured and repetitive bodily movement
done to improve or maintain components of physical activity and is not
synonymous with physical activity [16]. Exercise capacity refers to the
ability to perform exercise and is often divided into anaerobic and aerobic
capacity. Anaerobic work capacity is dependent on energy-yielding processes that do not require oxygen and can contribute to physical work for a
limited period of time. Long-term exercise is mainly dependent on aerobic
energy production. Therefore the single most important component in
endurance exercise capacity is the ability to provide oxygen for energy
supply. The maximum rate at which an individual is able to consume oxygen is referred to as maximal oxygen consumption (VO2-max) and is an
important determinant of exercise capacity. Endurance exercise capacity is
often determined by measuring VO2-max in laboratory settings or by estimating VO2-max using standardized submaximal tests [78].
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COPD
Chronic obstructive pulmonary disease (COPD) is one of the most important causes of morbidity and mortality worldwide [72]. COPD is defined as
a preventable and treatable disease characterised by progressive, not fully
reversible airflow limitation [2]. The two major components of COPD are
chronic bronchitis and emphysema. Chronic bronchitis is clinically defined
as chronic coughing for three months in each of two successive years were
other causes of coughing have been ruled out. Chronic bronchitis is also
associated with inflammation and nonspecific bronchial hyperreactivity
causing airway obstruction [72, 112]. Emphysema is characterised by an
enlargement of the lower airways and by destruction of their walls without
obvious fibrosis, which will cause airflow limitation [103, 112].
Estimating the prevalence of COPD has been shown to be complicated,
as it can vary depending on many factors such as diagnostic criteria, age
and survey methods. In a systematic review by Halbert et al [43], the
prevalence of COPD defined as not fully reversible airflow limitation in
adults aged ≥ 40 years was estimated to be 9–10% in European and northern American countries, while the disease prevalence in persons > 45 years
in northern Sweden was estimated to be around 14% in a study by Lundbäck et al [65]. In addition, the study by Lundbäck et al reported no gender differences in prevalence of CODP, but showed that prevalence increases rapidly with increased age [65].
The major risk factor for developing COPD is tobacco smoking. Cigarette smokers have a higher prevalence of abnormal lung function and
respiratory symptoms as well as a greater rate of annual increase in airflow
obstruction than non-smokers [90, 112]. Not all smokers develop COPD
suggesting that other factors, such as genetic factors, may modify the individual risk for disease development. The genetic risk factor that is best
described is deficiency of α-1-antitrypsin, which generates premature and
accelerated development of emphysema and decline in lung function. However, only about 1% of COPD patients have an inherited form of α-1antitrypsin deficiency [90, 103, 112]. Other known risk factors for development of COPD are air pollution and occupational exposure to dusts and
chemicals [90].
Typical symptoms of COPD are coughing, phlegm, and dyspnea. However, there is a weak relationship between symptoms and lung function.
Thus, lung function measurements with spirometry are necessary for diagnosing and grading COPD [112]. The presence of a post-bronchodilator
forced expiratory volume in one second (FEV1,0) value less than 80% of
predicted, together with a quotient between FEV1,0 and forced vital capacity (FVC) lower than 0.70 confirms the presence of not fully reversible
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airflow limitation [2, 72, 90, 112]. Commonly, disease severity in COPD is
classified into different stages mainly based on FEV1,0 values in relation to
predicted FEV1,0. The most widely used classification recommendations are
the “global strategy for diagnosis, management and prevention of COPD”
(GOLD) criteria [2, 90]. However, these criteria have been updated in
2006 and differences in classification may occur in the literature [2]. The
GOLD criteria for classification of COPD before and after 2006 are presented in Table 1.
Table 1. Classification of COPD disease severity according to “global
strategy for diagnosis, management and prevention of COPD” (GOLD)
criteria from 2001 and 2006.
Stage
GOLD 2001
Normal spirometry
Chronic symptoms
(coughing, sputum production)
0: At risk
I: Mild COPD
II: Moderate COPD
III: Severe COPD
IV: Very severe
COPD
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GOLD 2006
-
FEV1,0/FVC < 0.70
FEV1,0 ≥ 80% of predicted
FEV1,0/FVC < 0.70
FEV1,0 ≥ 80% of predicted
FEV1,0/FVC < 0.70
30% ≤ FEV1,0 < 80%
of predicted
FEV1,0/FVC < 0.70
50% ≤ FEV1,0 < 80%
of predicted
FEV1,0/FVC < 0.70
FEV1,0 < 30% of predicted or the presence of
respiratory failure or
clinical signs of right
heart failure
FEV1,0/FVC < 0.70
30% ≤ FEV1,0 < 50%
-
FEV1,0/FVC < 0.70
FEV1,0 < 30% of predicted or
FEV1,0 < 50% of predicted plus chronic respiratory failure
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Besides the pulmonary abnormities of COPD, the disease is associated
with effects outside the lungs, so called systemic effects. Systemic inflammation, nutritional abnormities and weight loss, cardiovascular effects,
effects on the nervous system, osteoskeletal effects and skeletal muscle
dysfunction have been reported as potential systemic effects of COPD [4,
22, 40]. The mechanisms mediating these systemic effects are not fully
understood; however, systemic inflammation, oxidative stress, use of oral
corticosteroids, hypoxia and low levels of physical activity have been suggested as plausible mediators [3, 40].
Skeletal muscle in COPD
Several studies have addressed the fact that skeletal muscle is affected in
patients with COPD. Skeletal muscle dysfunction has been reported to be
characterised by reduction in muscle strength (defined as the capacity of
the muscle to generate force) and muscle endurance (defined as the capacity of muscle to maintain a certain force over time) [60]. Furthermore,
several structural changes in peripheral skeletal muscle have been described. The cross sectional area of skeletal muscle fibres has been shown
to be lower in COPD patients compared with in healthy subjects and this
finding has been associated with muscle atrophy, which is commonly described in this patient group [37, 39, 111]. When studying fibre type distribution in the vastus lateralis muscle, several studies have reported a low
proportion of type I fibres in COPD patients compared with in healthy
subjects [38, 56, 71, 81, 111] while there are some discrepancies regarding
the distribution of type II fibres in this muscle. Maltais et al [71] report a
higher proportion of type IIa fibres in COPD patients while Gosker et al
[38] as well as Whittom et al [111] report a higher proportion of type IIx
fibres but no changes in the proportion of type IIa fibres in COPD patients
compared with in healthy subjects. In addition to the reported changes in
fibre type distribution, a lower oxidative enzyme activity [38, 70] and
lower mitochondrial density [93] have been shown in the vastus lateralis
muscle of COPD patients when comparing with healthy subjects. Taken
together, changes in fibre type distribution, low levels of oxidative enzyme
activities and a low mitochondrial density speak in favour of a reduced
oxidative capacity of skeletal muscle in COPD.
Studies on muscle capillarization have reported a lower capillary to
muscle fibre ratio in the vastus lateralis muscle of COPD patients compared with healthy subjects [56, 111]. However, when studying capillarization in relation to fibre area, no significant differences were shown between
COPD patients and healthy subjects [56, 111]. In a study by Barreiro et al
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[11], VEGF protein levels in the vastus lateralis muscle of COPD patients
were reported to be lower than those of healthy subjects.
To our knowledge no studies on satellite cell proportion or function
have been carried out in a population of COPD patients to investigate the
regenerative potential of skeletal muscle. However, it has been suggested
that inflammatory mediators, which are increased in COPD due to systemic inflammation, may affect satellite cells and thereby the regenerative
capacity of the muscle in COPD [44].
It is also important to highlight the fact that changes in skeletal muscle
of COPD patients are not homogenous between various muscle groups.
The most pronounced reductions in oxidative properties have been reported in the lower limb muscles [13]. When studying the diaphragm and
other inspiratory muscles of COPD patients a higher proportion of type I
fibres, increased mitochondrial density and increased oxidative capacity
have been reported [20, 63, 105]. These changes are thought to be caused
by overload of the ventilatory muscles due to increased work of breathing
brought on by airflow obstruction and hyperinflation in COPD [20].
What causes the observed changes in skeletal muscle of COPD patients
is poorly understood. Muscle morphology and function have been suggested to be influenced by several factors such as systemic inflammation,
oxidative stress, nutritional abnormalities, use of corticosteroids, hypercapnia, hypoxia and low levels of physical activity [13, 36, 40, 44].
Physical activity in COPD
Physical activity is an important clinical parameter related to lung function
decline, hospitalisation and mortality in COPD [32, 34]. Likewise, patients
with higher levels of physical activity have been shown to report a better
health-related quality of life compared with more inactive patients [83].
Furthermore, low levels of physical activity have been associated with extrapulmonary effects such as skeletal muscle dysfunction and systemic inflammation in COPD patients [108, 113].
The most widely used research methods for measuring physical activity
in COPD are subjective measurements such as questionnaires and activity
diaries. The advantages with these types of measurement are the low costs
and easy applications while the disadvantages are low accuracy and large
individual variability [94]. Objective measurements have been recommended for more accurate and detailed measurements [12, 94] and motion
sensors such as accelerometers have been shown to provide objective
measurements of duration, intensity and frequency of physical activity in
daily life in COPD patients [94].
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In a previous study, addressing objectively measured free-living physical
activity in CODP patients, it was reported that COPD patients in GOLD
stage II–IV are significantly less physically active compared with control
subjects who were considered at risk for developing COPD (GOLD stage
0) [109]. Furthermore, Pitta et al [95] have found that COPD patients
spend less time walking and standing but more time sitting and lying compared with healthy subjects.
Exercise capacity in COPD
Exercise intolerance has been suggested as one of the most common symptoms in COPD [5]. It has also been shown that exercise capacity is correlated to mortality and reduced exercise capacity has been associated with
reduced quality of life in this patient group [68, 86]. Lung function impairment shows only a weak relation to exercise capacity and it has been
suggested that other factors play an important role in reducing exercise
capacity in COPD [104]. It has been shown that skeletal muscle function
and fat free mass are associated with exercise capacity in COPD [5, 9, 30,
100], raising speculations that muscle weakness and wasting may play
important roles in exercise capacity in these patients. These speculations
are strengthened by studies that have shown improved muscle function and
increased exercise tolerance as a response to exercise training in COPD
patients [5, 104]. It has also been suggested that de-conditioning due to
inactivity is an important determinant to low exercise capacity [104] and
increased levels of physical activity have been shown to have positive effects on exercise tolerance in COPD patients [33].
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AIM OF THE THESIS
The overall aim of this thesis was to study muscular characteristics of the
tibial anterior muscle and physical activity patterns in patients with different degrees of COPD. Furthermore, this thesis aimed to study the relationships between muscle characteristics and exercise capacity as well as between physical activity and exercise capacity in patients with different degrees of COPD.
The specific aims of this thesis were to investigate:
• fibre type distribution, fibre area, satellite cell content and muscle
capillarization of the tibial anterior muscle in patients with different
degrees of COPD (studies I, II and III).
• exercise capacity in patients with different degrees of CODP (studies I, III and IV).
• the relationship between muscle characteristics and exercise capacity in different degrees of COPD (studies I and III).
• the expression of VHL, VEGF and HIF in the tibial anterior muscle
of patients with different degrees of COPD (study II).
• physical activity patterns and their relationship to exercise capacity
in patients with different degrees of COPD (study IV).
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MATERIALS
Subjects
Patients included in study I, II and III were recruited from the Department
of Respiratory medicine at Örebro University Hospital. Twenty-three patients (13 women and 10 men) who were selected in a stable condition and
were not suffering from any respiratory tract infections or exacerbations of
their disease four weeks prior to the sampling date participated. Exclusion
criteria were malignancy, cardiac failure and severe endocrine, hepatic or
renal disorder. Twelve age-matched, healthy, non-smoking subjects (6
women and 6 men) were recruited as a control group. Test subject characteristics are given in Table 2.
In study IV, patients were recruited from primary health care centres in
central Sweden through the “Diagnosdatabasregistret”. Fifty patients (33
women and 17 men) who were selected in a stable condition and were not
suffering from any respiratory tract infections or exacerbations of their
disease four weeks prior to the sampling date participated in the study.
Exclusion criteria were malignancy, cardiac failure and severe endocrine,
hepatic or renal disorder. Nineteen age-matched, healthy, non-smoking
subjects (13 women and 6 men) were recruited as a control group. Of the
recruited subjects, six patients and two healthy subjects were excluded
from the statistical analysis due to incomplete data sampling. Test subject
characteristics are given in Table 3.
All patients and age-matched healthy subjects underwent spirometry to
determine forced expiratory volume in one second (FEV1.0) with the highest
value from at least three technically acceptable assessments being used. In
studies I–III, both patients and controls underwent spirometry with reversibility tests, while in study IV the patients underwent postbronchodilatory spirometry and the healthy subjects underwent spirometry
without administration of bronchodilators. FEV1.0 was then used to divide
patients into subgroup based on the “Global Initiative for Chronic Obstructive Lung Disease (GOLD)” criteria [2, 90].
In study I, patients were divided into two subgroups according to GOLD
criteria from 2006 [2]. Group one (n=12) was considered to have mild or
moderate COPD (FEV1.0 ≥ 50% of predicted) and group two (n=11) was
considered to have severe or very severe COPD (FEV1.0 < 50% of predicted).
In study II and III, patients were divided into three subgroups according
to GOLD criteria from 2001 [90]. Group one (n=8) was considered to have
mild COPD (FEV1.0 > 80% of predicted), group two (n=9) was considered
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to have moderate COPD (FEV1.0 30-80% of predicted) and group three
(n=6) was considered to have severe COPD (FEV1.0 < 30 of predicted). The
healthy subjects had no airway obstruction (mean FEV1.0 115% of predicted).
In study IV, patients were divided into three subgroups according to
GOLD criteria from 2006 [2]. Group one (n=11) was considered to have
mild COPD (FEV1.0 ≥ 80% of predicted), group two (n=21) was considered
to have moderate COPD (80% > FEV1,0 ≥ 50% of predicted) and group
three (n=12) was considered to have severe COPD (50% > FEV1,0 ≥ 30% of
predicted). The healthy subjects had no airway obstruction (mean FEV1.0
103 % of predicted).
For all test subjects, height and weight were measured and body mass
index (BMI) was calculated as weight (kg)/(height (m))2 (Table 2 and Table
3). All test subjects included in studies I–III were sampled for arterial blood
gases from the radial artery at rest. The samples were analysed for partial
arterial pressure for oxygen (PaO2) and carbon dioxide (PaCO2) using the
Radiometer ABL 500 blood gas analyser (Table 2).
All studies were approved by the regional ethical review board in Uppsala, Sweden (dnr 2004:M-355).
Table 2. Characteristics of test subjects included in studies I–III.
Age (years)
Height (cm)
Weight (kg)
BMI (kg/m2)
FEV1,0 (% of predicted)
PaO2 (kPa)
PaCO2 (kPa)
Healthy subjects
(n=12, 6 women
and 6 men)
COPD patients
(n=23, 13 women
and 10 men)
60 ± 7
172 ± 10
79 ± 13
26.7 ± 3.7
113 ± 12
62 ± 6
169 ± 8
76 ± 18
26.3 ± 5.0
56 ± 25*
11.1 ± 1.3
5.2 ± 0.3
9.7 ± 1.3**
5.1 ± 0.6
*= significantly lower compared with healthy subjects, p<0.001
**=significantly lower compared with healthy subjects, p=0.005
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Table 3. Characteristics of test subjects included in study IV.
Age (years)
Height (cm)
Weight (kg)
BMI (kg/m2)
FEV1,0 (% of predicted)
Healthy subjects
(n=17, 11 women
and 6 men)
COPD patients
(n=44, 28 women
and 16 men)
58 ± 5
172 ± 8
75 ± 10
25.2 ± 1.8
103 ± 18
63 ± 7
169 ± 8
78 ± 19
27.0 ± 5.5
65 ± 20*
*= significantly lower compared with healthy subjects, p<0.001
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METHODS
Muscle biopsy sampling and analysis
Muscle biopsies were taken from the bulk of the tibial anterior muscle,
which is an important postural muscle active daily for long periods and
involved in balance control and foot stability during walking [64]. The
biopsies were obtained under local anaesthesia (Xylocaine® 2%) using the
semi-open biopsy technique described by Henriksson [48] (Figure 3). The
biopsies were frozen in isopentane cooled to its freezing point in liquid
nitrogen, and stored at -80°C until analyses were performed. Biopsies were
then studied further using immunohistochemistry and polymerase chain
reaction (PCR) techniques. Biopsies analysed using immunohistochemistry
were prepared by cutting serial transverse section, 5μm thick, at -22°C
using a microtome (Leica CM1850, Leica Microsystems, Germany) and
mounted on glass slides. Biopsies used for PCR analyses were prepared by
isolating total RNA using the RNeasy Fibrous Tissue Mini Kit (Qiagen,
USA) according to the manufacturers’ instructions. The integrity and quantity of the isolated RNA was evaluated with the Agilent 2100 bioanalyzer
using the RNA 6000 Nano Assay Kit (Agilent Technologies) and all isolated RNA was stored at -80°C until use.
Figure 3. Muscle biopsy sampling from the tibial anterior muscle using the
semi-open biopsy technique.
Fibre type distribution
For determination of fibre types immunohistochemistry was used. Primary
monoclonal antibodies (mAb) against myosin heavy chains (MyHC) that
were used were mAb A4.951 and mAb N2.261 (Developmental Studies
Hybridoma Bank, University of Iowa). Using mAb A4.951, type I fibres are
strongly stained while type IIa, IIxa and type IIx fibres are unstained.
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Using mAb N2.261, type IIa fibres are strongly stained, type IIx fibres are
unstained while type I and type IIxa fibres are slightly stained. Type I–IIa
fibres are stained with both antibodies [59] (Figure 4). Analyses were performed using a light microscope (Nikon Eclipse E400) connected to a
computerised image system (SPOT Insight: Diagnostic Instrument, Sterling
Heights, Michigan). Fibre type distribution was analysed on the whole
cross section and a mean of 401 fibres was counted for each subject. The
fibres were designated as type I, type IIa, type IIx, type I–IIa or type IIxa.
Figure 4. Muscle biopsy from the tibial anterior muscle stained using mAb N2.261
(A) and mAb A4.951 (B) for determination of fibre type.
Fibre area and perimeter
For each test subject four to ten randomly selected areas on the cross section were selected and photographed at a magnitude of x20. Fibre area and
fibre perimeter for type I and type IIa fibres were then determined for a
mean of 49 type I and 31 type IIa fibres. Due to the low number of type
IIx, type IIxa and type I–IIa fibres, areas and perimeters for these fibres
types were not included in the statistical analyses. Obliquity in fibre sectioning was assessed using the form factor that represents: (4π x fibre
area)/(fibre perimeter)2. There were no differences in form factor between
the different study groups (p=0.31).
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Satellite cells
For visualization of satellite cells, mAb CD56 (Novocastra Laboratories),
which is similar to N-CAM, was used [57] (Figure 5). Following staining,
the sections were visualized using a light microscope (Nikon Eclipse E400)
and analyses were performed at a magnitude of x40–x60. In each cross
section, the number of satellite cells and number of muscle fibres was
counted on the whole muscle biopsy. The ratio between number of satellite
cells and number of muscle fibres was then calculated.
Figure 5. Muscle biopsy from the tibial anterior muscle immunohistochemically
stained using mAb CD56 for visualization of satellite cells.
Capillary network
The identification of capillaries was done using the mAb CD31 (Dako,
Glostrup, Denmark; MO823), an antibody that recognizes PECAM-1, a
transmembranous glycoprotein that is strongly expressed in vascular endothelial cells. Sections were also counterstained with eosin [17] (Figure 6).
Analyses were performed using a light microscope (Nikon Eclipse E400)
connected to a computerised image system (SPOT Insight: Diagnostic Instrument, Sterling Heights, Michigan). Four to ten randomly selected crosssectional areas corresponding to a mean of 80 fibres were photographed at
a magnitude of x20 and used for determination of the capillary network.
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Figure 6. Muscle biopsy from the tibial anterior muscle immunohistochemically
stained using mAb CD31 and counterstained with eosin for
identification of capillaries.
Capillary parameters
Muscle capillary parameters were in the present thesis calculated as previously described by Charifi et al [17]. The capillary supply in skeletal muscle can be measured by counting the number of capillaries around each
muscle fibre (CAF) or by assessing CAF in relation to fibre area (CAFA).
CAFA is a parameter based on the diffusion distance between the capillary
and the centre of the fibre. Capillary parameters determining the diffusion
distance may not detect actual disturbances in muscle capillarization. It has
previously been suggested that the muscle fibre-to-capillary interface is an
important factor involved in oxygen supply to the muscle [17-18, 35, 4950, 52] and may thereby be used as a more sensitive marker for changes in
the capillary bed compared with CAF and CAFA. Muscle fibre-to-capillary
interface can be assessed using the capillary to fibre perimeter exchange
index (CFPE-index) or the index of tortousity (LC/PF-index). CFPE-index
represents the quotient between the capillary-to-fibre ratio (C:Fi) and the
fibre perimeter, where C:Fi is calculated by counting the number of capillaries around the fibre in question followed by determination of the sharing
factor (SF) for each capillary and thereafter taking the sum of the fractional
contributions of all capillary contacts around the fibre [49]. LC/PF-index is
calculated as the ratio between the length of capillary in contact with the
muscle fibre membrane and the fibre perimeter and represents the percentage of muscle fibre perimeter in contact with the capillary wall. CAF and
CAFA were assessed in study II while CFPE-index and LC/PF-index were
assessed in study III.
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I GABRIELLA ELIASON Skeletal muscle characteristics and physical activity patterns…
VHL-identification
In study II, identification of VHL at protein level was done immunohistochemically using the von Hippel-Lindau protein mAb (GTX11189, GeneTex, Inc, Texas, USA). The number of VHL-immunoreactive cells was
quantitatively scored by counting the number of VHL-positive cells from
six randomly selected cross sectional areas photographed at a magnitude of
x40. Only cells containing visible nuclei were included.
TaqMan PCR was used for gene expression analyses. Reverse transcription of RNA was performed for synthesis of complementary DNA (cDNA)
and the final cDNA product was stored at -20°C until use. The VHL
(Hs00184451_m1) gene designed and premixed by Applied Biosystems
(Foster City, California, USA) was then used for expression analysis.
HIF and VEGF identification
In study II, gene expression of HIF and VEGF was studied using TaqMan
PCR. Reverse transcription of RNA was performed for synthesis of cDNA
and the final cDNA product was stored at -20°C until use. Genes included
for expression analysis were HIF-1α (Hs00936368_m1), HIF-3α
(Hs00541709_m1), VEGF (Hs00900054_m1), VEGFB/VEGFC (Hs 00).
All genes were designed and premixed by Applied Biosystems (Foster City,
California, USA).
Exercise capacity
To determine exercise capacity, the six-minute walk test was used. This test
has been described as a submaximal test that can be performed by patients
who do not tolerate maximal exercise testing [80]. Furthermore, the test is
widely used in clinical practice and is generally recommended as a functional assessment for patients suffering from COPD [1, 28]. The test was
performed on a 25m (studies I and III) or a 20m (study IV) court were the
test subject walked back and forth as fast as possible for six minutes and
the distance walked after six minutes was measured. The test subjects did
not use any bronchodilators or oxygen supplementation before or during
the test.
Body composition
In study I, fat free mass (FFM) and fat free mass index (FFMI) were determined using dual x-ray absorptiometry (DXA) where a double photon
beam generated by an x-ray source is used to distinguish between different
body tissues. A whole body scan was conducted in a Lunar DPX-L scanner
(Lunar cooperation, Madison, WI, USA) with the subjects in a supine posi-
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tion. Lunar software (DPX-L v 4.7E) was used to calculate body composition.
In study IV, body composition was established by measuring skinfold
thickness with a Harpenden® skinfold calliper at four sites; the triceps, the
biceps, the subscapula and the suprailiaca. Body fat content was estimated
from the sum of the skinfolds using the body density equation by Durnin
and Womersley [26] and the Siri equation for body fat [102].
Physical activity
To assess physical activity in study IV, the uniaxial accelerometer ActiGraph, model GT1 M (Manufactoring Technology IC, Fort Walton Beach,
Fl, USA) was used (Figure 7a). The accelerometer consists of a piezoelectric
sensor that is sensitive to vertical accelerations and samples voltage signals
in proportion to detected accelerations (range 0.05–2.0 g with a frequency
rate of 0.25–2.5 Hz) with a sample rate of 10 measures per second. The
signal first becomes filtered to eliminate artefacts such as vibrations and
then becomes converted into a digital set of numbers, so called “counts”.
Finally, all counts are summarised over a user-specified time frame (epoch).
The accelerometer measures both the magnitude and frequency of body
movement, which makes quantification of the intensity and duration of
physical activity possible [19].
The test subjects wore the accelerometer on an elastic belt around the
waist (Figure 7b) all waking hours except during water activities, for seven
days and data were sampled in 60s epochs. Data were reduced using the
ActiGraph analysis software MAHUffe (available from http://www.mrcepid.cam.ac.uk/Research/PA/Downloads.html). Continuous periods of zero
values exceeding 20 min were regarded as “accelerometer not worn” and
were not included in the calculation of total registered time. 500 minutes of
registration per day were required for a day to be considered as valid and
at least three valid days of which at least one was a Saturday or Sunday
were required for the registration to be included in the statistical analysis.
Based on previously reported validation studies, activity count cut-off
points applied to assign accelerometer outcomes to physical activity categories were as followed:
Time spent sedentary was defined as where counts/min were < 100 [75],
light activity was defined as activity resulting in 100–2019 counts/min [74],
moderate activity was defined as activity resulting in 2020–4944
counts/min [107] and vigorous activity was defined as activity resulting in
more than 4944 counts/min [106].
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b
a
Figure 7. The uniaxial accelerometer ActiGraph, model GT1 M (a) used for registration of physical activity and a test subject wearing the accelerometer on an elastic belt around the waist (b).
Statistical analyses
Statistic analyses were performed using Statistix ®8 (Analytic Software,
Tallahassee, Florida, USA) (studies I–III) and PASW Statistics 18.0 (former
SPSS) (study IV). In studies I–III p < 0.05 was considered significant while
in study IV p ≤ 0.05 was considered significant. In studies I and III, all data
were presented as mean ± standard deviation. Comparison between groups
was done using Kruskal-Wallis one way ANOVA followed by the KruskalWallis all pairwise comparison post-hoc test when significance was found
and relationship between variables was studied using Spearman’s rank
correlation test. In study II, capillary variables were presented as mean ±
standard deviation. Comparison between groups was done using KruskalWallis one way ANOVA followed by the Kruskal-Wallis all pairwise comparison post-hoc test when significance was found. To estimate differences
between groups regarding gene expression of VHL, HIF and VEGF and
quantitative analysis of VHL immunohistochemistry regression analysis
was applied. In study IV, variables were checked for normality using
Shapiro-Wiik´s test for normality. All values except time spent MVPA
showed a satisfactory pattern and after logarithm transformation time
spent MVPA also fitted within the normal distribution and parametrical
tests were applied. Data were presented as mean ± standard deviation and
comparison between groups was done using analysis of variance (ANOVA)
followed by Tukey´s post-hoc test when significance was found. To examine associations between variables, linear regression analysis was applied.
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RESULTS
Study I
Physical performance and muscular characteristics in different stages of
COPD
Aim
The aim was to study exercise capacity as well as muscle fibre type distribution, fibre area and satellite cell content in the tibial anterior muscle of
patients with different degrees of COPD and to investigate the existence of
a relationship between exercise capacity and muscle morphology.
Results
Significant differences in exercise capacity expressed as distance walked in
six minutes were observed between the study groups (p < 0.001). Patients
with mild and moderate COPD on average walked a 21% shorter distance
compared with the healthy subjects and patients with severe and very severe COPD on average walked a 41% shorter distance compared with the
healthy subjects. Furthermore, a significant correlation between exercise
capacity and degree of airflow obstruction expressed as percent of predicted FEV1,0 was shown (r=0.89, p < 0.001).
Patients with severe and very severe COPD had a significantly lower
proportion of type I fibres (p=0.01) and a significantly higher proportion
of type IIa fibres (p=0.01) compared with the healthy subjects, while the
proportion of type IIx, type IIxa and type I–IIa fibres did not differ between the study groups.
The fibre area of type IIa fibres was significantly lower in the patients
with severe and very severe COPD compared with the healthy subjects
(p=0.007), while no differences in fibre area of type I fibres were observed
between the study groups. A correlation between degree of airflow obstruction and area of type IIa fibres was also observed (r=0.55, p < 0.001). Furthermore, the area of type IIa fibres was found to significantly correlate to
exercise capacity (r=0.63, p < 0.001).
Body composition expressed as fat free mass index (FFMI) was not
found to differ between the study groups. However, FFMI was significantly
correlated to the fibre area of both type I fibres (r=0.48, p=0.005) and type
IIa fibres (r=0.71, p < 0.001).
The number of satellite cells per muscle fibre did not differ between the
study groups, indicating an intact regenerative capacity of the muscle.
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Study II
Overexpression of von Hipple-Lindau protein in skeletal muscles of patients with chronic obstructive pulmonary disease
Aim
The aim was to investigate the hypothesis that impaired coupling of the
hypoxic-angiogenic signalling cascades mediates decreased skeletal muscle
capillarization in patients with COPD.
Results
Significant differences in the number of capillaries around a single muscle
fibre (CAF) for both type I (p=0.006) and type IIa fibres (p=0.002) were
observed between the study groups with patients with moderate and severe
COPD having a significantly lower number of capillaries/fibre compared
with the healthy subjects. However, when CAF was adjusted for fibre area
(CAFA) the significant differences did not remain.
A significant overexpression of VEGF-A, VEGF-B and HIF 3-α was observed in the patients with moderate CODP compared with the healthy
subjects, while a trend towards overexpression of HIF 1-α, HIF 3-α, VEGFA, VEGF-B and VEGF-C was observed in mild COPD compared with the
healthy subjects. Likewise, a trend towards overexpression of HIF 1-α and
VEGF-C was observed in moderate CODP compared with the healthy
subjects. No significant differences in gene expression of HIF or VEGF
were observed between the patients with severe COPD and the healthy
subjects (Table 4).
The mRNA levels of VHL were significantly higher in the patients with
mild COPD compared with the healthy subjects (p=0.008). In the patients
with moderate CODP, a trend towards overexpression of mRNA VHL was
observed (p=0.077), while mRNA levels of VHL in severe COPD were
similar to the levels in the healthy subjects (Table 4). On a protein level,
the number of cells displaying pVHL immunoreactivity was significantly
higher in the patients with mild (p=0.001) and moderate COPD (p=0.001)
compared with the healthy subjects, while the number of cells displaying
pVHL immunoreactivity in severe COPD was lower, although not significantly, than in the healthy subjects.
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Table 4. mRNA expression of VEGF, HIF and VHL in COPD patients
compared with healthy subjects analysed using regression analysis.
Mild COPD vs
Moderate COPD vs
healthy subjects
healthy subjects
ß
p
(95% CI)
VEGF-A
2.8
7.4
0.533
1.5
0.573
5.0
0.245
14.7
0.303
11.9*
7.0
29.9*
7.1
-10.6
0.312
-15.7
0.239
(-42.2 to11.0)
0.138
-3.1
0.305
(-9.3 to 3.0)
0.081
-2.1
0.665
(-11.7 to 7.6)
0.028
(3.4 to 56.5)
0.008
(3.3 to 20.6)
0.043
(-0.9 to 15.0)
(-13.9 to 43.4)
VHL
3.8
p
(-31.6 to 10.4)
(-4.0 to 7.0)
(-3.6 to 13.6)
HIF 3- α
22.7*
ß
(95% CI)
0.044
(0.7 to 44.7)
(-4.0 to 7.0)
HIF 1- α
17.9*
(0.5 to 35.2)
(-16.4 to 31.2)
VEGF-C
healthy subjects
p
(95% CI)
0.764
(-15.9 to 21.5)
VEGF-B
ß
Severe COPD vs
-5.1
0.746
(-37.3 to 27.0)
0.077
(-0.8 to 15.1)
-1.6
0.741
(-11.2 to 8.1)
*= significant difference compared with healthy subjects (p<0.05)
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Study III
Alterations in the muscle-to-capillary interface in patients with different
degrees of chronic obstructive pulmonary disease.
Aim
The aim was to investigate the muscle fibre-to-capillary interface in patients with different degrees of COPD and its correlation to the degree of
airflow obstruction and exercise capacity.
Results
The C:Fi for type I fibres was significantly lower in the patients with moderate and severe COPD compared with the healthy subjects (p=0.007) and
the C:Fi for type IIa fibres was significantly lower in the patients with severe COPD compared with the healthy subjects (p=0.002), indicating that
each capillary is shared by more fibres in these patient groups. Furthermore, the CFPE-index for type I fibres was significantly lower in the patients with moderate and severe COPD compared with the healthy subjects
(p=0.002).
No significant differences in LC/PF were observed between the study
groups.
The degree of airflow obstruction expressed as percent of predicted
FEV1,0 was found to correlate positively to CFPE-index for both type I fibres (r=0.61, p < 0.001) and type IIa fibres (r=0.37, p=0.04). Furthermore,
a positive correlation was observed between exercise capacity expressed as
distance walked in six minutes and CFPE-index for both type I fibres
(r=0.67, p < 0.001) and type IIa fibres (r=0.40, p=0.02), indicating a parallel reduction in exercise capacity and muscle capillarization.
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Study IV
Physical activity patterns in patients with different degrees of chronic obstructive pulmonary disease.
Aim
The aim was to assess exercise capacity, body composition and physical
activity in daily life in patients with different degrees of COPD and to use
physical activity data to estimate how much time patients spend at least
moderately physically active. Furthermore, the study aimed to examine the
associations between exercise capacity, pulmonary function and physical
activity.
Results
Exercise capacity expressed as distance walked in six minutes was found to
differ significantly between the study groups (p < 0.001) with all patient
groups walking a significantly shorter distance than the healthy subjects.
No differences in fat free mass were observed between the different
study groups.
Regarding physical activity, time spent moderately of vigorously physically active (MVPA), as well as mean physical activity level, were found to
differ between the study groups with patients having moderate and severe
COPD spending on average less time MVPA (p=0.02) and having on average lower mean physical activity level (p=0.01) than the healthy subjects.
Time spent sedentary did not differ between the study groups. The recommendation of 30 minutes of at least moderate physical activity/day was
reached by 47% of the healthy subjects, 27% of the subjects with mild
COPD, 10% of the subjects with moderate COPD and 17% of the subjects
with severe COPD.
When studying only the patients, a significant association between exercise capacity and percent of predicted FEV1,0 was observed (ß=0.363, R2
adjusted=0.110, p=0.02), while when studying only the healthy subjects no
association between exercise capacity and percent of predicted FEV1,0 was
shown.
In the patients, significant associations between exercise capacity and
time spent MVPA (ß=0.335, R2 adjusted=0.090, p=0.03) as well as between exercise capacity and mean physical activity level (ß=0.336, R2 adjusted=0.091, p=0.03) were observed, while in the healthy subjects a significant association between exercise capacity and mean physical activity level
(ß=0.487, R2 adjusted=0.187, p=0.05) but not between exercise capacity
and time spent MVPA was shown. No associations between exercise capacity and time spent sedentary were observed in the patients or in the
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healthy subjects. The significant association between exercise capacity and
time spent MVPA in the patients was found to remain after controlling for
percent of predicted FEV1,0.
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DISCUSSION
Muscle fibre type and fibre area
Previous studies have suggested a more glycolytic muscle profile and a fibre
type shift with a high number of type II fibres and low number of type I
fibres in the vastus lateralis of COPD patients [38, 56, 70-71, 81, 93, 111].
This is in line with the findings in the present thesis, where a lower number
of oxidative type I fibres and a higher number of more glycolytic type IIa
fibres were found in the tibial anterior muscle of patients with COPD
compared with healthy subjects. The changes in fibre type composition
within the tibial anterior muscle appear to be more pronounced in severe
and very severe COPD than in mild and moderate CODP. A plausible explanation to these findings is that changes in muscle characteristics towards
a more glycolytic muscle profile is an adaptive response to hypoxia. As
patients with severe and very severe COPD were found to have a significantly lower PaO2 compared with healthy subjects, these patients most
probably suffer from a chronic state of hypoxia. Likewise, a reduced crosssectional fibre area has been associated with chronic hypoxia [53-54, 73]
and in study I the area of the type IIa fibres was found to be lower in the
patients with severe and very severe CODP compared with the healthy
subjects. The cross sectional area of the muscle fibre has also been associated with muscle volume and reduced fibre area has been suggested to be a
marker for muscle atrophy in COPD [37, 39, 111]. The finding of a reduced fibre area of type IIa fibres together with the significant correlation
shown between fibre area and FFMI indicates the presence of muscle atrophy in the studied patient group. However, FFMI was not found to differ
significantly between the study groups, probably due to the small number
of patients recruited to each group, and therefore muscle atrophy could not
be confirmed in the patients in the present thesis.
The shift towards a more glycolytic muscle profile and a low muscle
mass, reflected by reduction in muscle fibre area, may partly account for
the low exercise capacity expressed as distance walked in six minutes
shown in the patient groups. However, as the morphological changes appear to occur mainly in the later stages of disease while exercise capacity
begins to be reduced in mild and moderate CODP, other mechanisms such
as low levels of physical activity and lung function impairment most likely
also contribute to the decline in exercise capacity.
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Muscle regenerative capacity
The majority of skeletal muscle regeneration is carried out by activation,
proliferation and differentiation of satellite cells. As no differences in the
number of satellite cells/muscle fibre in the tibial anterior muscle were
found between COPD patients and healthy subjects, the regenerative capacity of this muscle appears to be intact in COPD patients. Increasing
muscle mass and thereby fat free mass is of importance for COPD patients
suffering from muscle wasting as low fat free mass has been identified as
an independent predictor of mortality and has been suggested to have an
adverse effect on muscle function, exercise capacity and health status in
this patient group [9, 27, 82, 88, 99]. Satellite cells have been suggested to
be activated in response to stimuli such as physical activity [114] and it can
be speculated that increasing the level of physical activity may result in
increased muscle regeneration and thereby increased muscle mass in COPD
patients. However, as the biopsies were not taken following any form of
stimulation the ability of the satellite cells to re-enter the cell cycle and
contribute to muscle regeneration is not clarified in the present thesis and
needs further investigation.
Muscle capillarization
The finding of a lower number of capillaries/muscle fibre (CAF) in the
tibial anterior muscle of COPD patients compared with healthy subjects
indicates a decreased muscle capillarization in COPD and is in line with
previous studies examining muscle capillarization in the vastus lateralis
muscle of COPD patients [56, 111]. However, as the ratio between the
number of capillaries around a fibre and the area of the muscle fibre
(CAFA) did not differ between the study groups, the lower number of capillaries/muscle fibre may be explained by the observed reduction in fibre
area and not reflect an actual change in muscle capillarization. This is also
in line with a study by Whittom et al [111], where a parallel reduction in
the number of capillaries around a muscle fibre and the fibre area was
observed. CAF and CAFA are variables based on the diffusion distance
between the capillary and the centre of the muscle fibre and may not detect
actual disturbances in muscle capillarization. It has been suggested that the
muscle fibre-to-capillary interface, which has been shown to be a factor
involved in oxygen supply to the muscle [17-18, 35, 49-50, 52], may be a
more sensitive marker for changes in the capillary bed compared with CAF
and CAFA. To assess muscle fibre-to-capillary interface in the present thesis, the capillary-to-fibre perimeter exchange index (CFPE-index) and the
index of tortousity (LC/PF-index) were used. Patients with moderate and
46
I GABRIELLA ELIASON Skeletal muscle characteristics and physical activity patterns…
severe COPD were found to have a lower CFPE-index compared with
healthy subjects indicating that each capillary is shared by more muscle
fibres, which may impair oxygen delivery to the muscle fibre. Taken together, the findings in the present thesis speak in favour of a disturbed
muscle capillarization in the tibial anterior muscle of patients with moderate and severe COPD.
As a positive correlation was observed between CFPE-index and exercise
capacity, impaired muscle capillarization and subsequently impaired oxygen delivery to the muscle appears to have a contributing role in the development of reduced exercise capacity in COPD. The changes in muscle
capillarization are more pronounced in type I fibres and as these fibres are
the most oxygen dependent and also the main fibre type involved in endurance exercise, it is likely that disturbed capillarization within these fibres
will affect endurance exercise capacity negatively.
The mechanisms mediating decreased muscle capillarization in COPD
have not previously been clarified. However, it is known that physiological
stimuli, such as exercise, as well as environmental stimuli, such as hypoxia,
affect muscle capillarization [14, 23, 54-55, 97]. It has previously been
suggested that chronic hypoxia has negative effects on muscle capillarization [23, 54] and one plausible explanation to the lower capillarization
observed in COPD may be the presence of systemic hypoxia. The underlying mechanisms by which hypoxia impairs muscle capillarization have not
yet been fully understood. Contrarily, local hypoxia within the muscle has
been suggested to enhance muscle capillarization by increasing VEGF expression due to increased levels of intracellular HIF through decreased
degradation [101]. However, the increased levels of VHL found in mild
and moderate COPD may affect the transduction of the hypoxic signalling
pathway negatively and may therefore contribute to decreased capillarization in COPD. The correlation between CFPE-index and disease severity
expressed as percent of predicted FEV1,0 indicates a gradual decline in tissue capillarization with increased disease severity. It can be speculated that
overexpression of VHL does not lead to complete blockage of the HIF
signalling pathway but, more likely, a partial blockage leading to expression of VEGF at levels that are inadequate to maintain normal capillarization over a long period of time. This could explain the gradual decrease in
muscle capillarization.
It has been suggested that physical activity enhances muscle capillarization through increased blood flow within the muscle, mechanical stretching
of the muscle and local hypoxia in the muscle tissue [55, 97]. Thus, another plausible explanation to the decreased muscle capillarization in
GABRIELLA ELIASON
Skeletal muscle characteristics and physical activity patterns... I
47
COPD may be the low levels of physical activity shown in these patients in
study IV.
Physical activity
In the present thesis, COPD patients with moderate and severe COPD were
found to be significantly less physically active compared with healthy subjects. Patients were found to have a lower mean physical activity level and
to spend less time at least moderately physically active. Furthermore, the
observed association between degree of airflow obstruction, expressed as
percent of predicted FEV1,0, and physical activity indicates that increased
disease severity can be associated with decreased levels of physical activity.
These findings are in line with previous studies, suggesting that COPD
patients are less active compared with age-matched healthy subjects [95,
109].
It has previously been discussed that decreased exercise capacity in
COPD patients may partly be caused by inactivity [4]. Interestingly, no
significant differences in time spent sedentary were observed between the
study groups in the present thesis. However, it has to be clarified that this
result applies to time spent sedentary during registered time. It cannot be
ruled out that patients wear the accelerometer shorter periods/day and
spend more time sedentary when not wearing the accelerometer. Yet, as
physical activity was positively associated with exercise capacity in both
patients and healthy subjects while no associations between time spent
sedentary and exercise capacity were observed in any of the study groups,
it appears as if physical activity plays a more important role than time
spent sedentary in the low exercise capacity observed in COPD patients.
As previously mentioned, low exercise capacity was found to correlate
to changes in skeletal muscle in COPD patients and taken together with the
observed association between exercise capacity and time spent physically
active, it can be suggested that physical activity partly contributes to skeletal muscle changes observed in COPD. However, as it appears that changes
in muscle morphology occur mainly in the later stages of disease, i.e. in
patients with low PaO2, it is most likely that a combination of hypoxia and
low levels of physical activity strongly contributes to the more glycolytic
muscle profile shown in these patients. Likewise, physical activity has been
shown to have positive effects on skeletal muscle. Endurance training is
known to promote a more aerobic muscle profile with increased number of
type I fibres, increased mitochondrial density, increased oxidative enzyme
activity and increased muscle capillarization [8, 14, 18, 21, 31, 55, 92]. As
changes in muscle fibre type composition and capillarization are most pro-
48
I GABRIELLA ELIASON Skeletal muscle characteristics and physical activity patterns…
nounced in the later stages of disease, it is important to promote physical
activity in the early stages of disease to inhibit these changes.
Health recommendations for healthy adults aged 18–65 years, stating
that 30 minutes of at least moderate physical activity/day is preferable for
promoting and maintain health [45, 89], have been suggested to be applicable also for older adults (<65 years) and adults aged 50–64 years with
clinically significant chronic conditions [84]. As it is shown in the present
thesis that the intensity of physical activity affects exercise capacity, it can
be suggested that these health recommendations can be applied to COPD
patients. However, it appears as if few COPD patients reach the recommended levels, which highlights the need of focusing on moderate and high
intensity physical activity on a regular basis in rehabilitation of CODP
patients.
The low levels of physical activity recorded in study IV may also be a result of seasonal variations in physical activity. It has previously been discussed that seasonal variations may affect physical activity patterns [62]
and as the present study was carried out during the winter season it is possible that test subjects were less active than they would have been during
the summer season. However, data sampling was carried out during the
same season for all subjects. Therefore, seasonal variations cannot explain
differences in physical activity patterns shown between the different study
groups.
GABRIELLA ELIASON
Skeletal muscle characteristics and physical activity patterns... I
49
CONCLUSIONS
From the work of this thesis it was concluded that:
•
•
•
•
•
•
•
•
Exercise capacity expressed as distance walked in six minutes is
lower in COPD patients compared with healthy subjects and is associated with disease severity.
Skeletal muscle morphology shifts towards a more glycolytic muscle
profile in COPD and changes in muscle morphology are correlated
to disease severity.
The number of satellite cells/muscle fibre does not differ between
COPD patients and healthy subjects.
Skeletal muscle capillarization is affected in COPD and the number
of capillaries/muscle fibre, as well as the CFPE-index is lower in
COPD patients compared with healthy subjects. CFPE-index is also
correlated to disease severity.
VHL is overexpressed in the tibial anterior muscle of patients with
mild and moderate COPD compared with healthy subjects.
Changes in skeletal muscle morphology are associated with decreased exercise capacity in COPD.
Patients with moderate and severe COPD are less physically active
compared with healthy subjects.
Physical activity of at least moderate intensity is positively associated with exercise capacity while time spent sedentary is not associated with exercise capacity in COPD patients.
GABRIELLA ELIASON
Skeletal muscle characteristics and physical activity patterns... I
51
FUTURE PERSPECTIVES
In the present thesis it is shown that morphological changes within the
tibial anterior muscle are most pronounced in the later stages of COPD, i.e.
in patients with low PaO2 and low physical activity levels. Systemic hypoxia and inadequate levels of physical activity are therefore suggested as
plausible mediators of changes in skeletal muscle morphology in patients
with COPD. However, the underlying mechanisms by which hypoxia influences skeletal muscle in COPD are not fully understood and need further investigation. Increased levels of pVHL are in study II suggested to
impair hypoxic signalling pathways and thereby reduce muscle capillarization. Whether the overexpression of VHL is a consequence of systemic
hypoxia or has other causes is not clarified in the present thesis and needs
to be addressed further.
In the present thesis it is speculated that increasing the amount of physical activity of at least moderate intensity may generate a more aerobic
muscle profile and may also increase exercise capacity in patients with
COPD. Therefore, investigating the effects of specific exercise protocols on
skeletal muscle morphology and exercise capacity in COPD would be of
great interest.
As the satellite cell population was not found to differ between CODP
patients and healthy subjects it is suggested that the regenerative capacity
of the muscle is intact in COPD patients. However, the ability of the satellite cells to be activated and re-enter the cell cycle has not been addressed
in this thesis and needs further investigation.
GABRIELLA ELIASON
Skeletal muscle characteristics and physical activity patterns... I
53
ACKNOWLEDGEMENTS
This study was performed at the School of Health and Medical Sciences at
Örebro University and all support from the division of Clinical Medicine
and the division of Sports Sciences is gratefully acknowledged.
I wish to express my sincere gratitude to everyone who has made this thesis
possible by supporting me in different ways, especially:
Karin Piehl-Aulin, my supervisor, for giving me the opportunity to be a
PhD student. Your enthusiasm, great knowledge, encouragement and always positive mind has been invaluable for the work of this thesis.
Fawzi Kadi, my co-supervisor, for sharing your amazing knowledge and
for supporting me throughout the work of this thesis in an enthusiastic and
inspiring way.
Samy Abdel-Halim, for giving me the opportunity to carry out data sampling at the Department of Respiratory Medicine at Örebro University
Hospital and for guidance in the work of this thesis.
Anita Hurtig-Wennlöf, for an inspiring collaboration, fruitful discussions
and good friendship.
Eva Oskarsson and Britta Wåhlin-Larsson, for always being there listening,
supporting and advising. Working together with you has made the work of
this thesis easier and more enjoyable.
Birgitta Olsen, for sharing your knowledge on the PCR technique and for
patiently answering all my questions.
Brita, Margareta and Yvonne at the Department of Respiratory Medicine
at Örebro University Hospital, for great assistance during data collection in
the first studies.
Sigrid Odencrants, for your enthusiasm and support during the last study.
Ann-Britt Zakrisson, for great help and quick response during the recruitment of test subjects for the last study.
GABRIELLA ELIASON
Skeletal muscle characteristics and physical activity patterns... I
55
All of you in “the muscle group” for interesting discussions and for giving
valuable feedback on my work.
All my colleagues, past and present, at the School of Health and Medical
Sciences, especially Siw Lunander for believing in me and involving me in
the teaching work at the division of Clinical medicine.
All patients and healthy volunteers participating in the different studies
included in this thesis. Without you this thesis would never have been accomplished.
Friends and relatives for being there and for reminding me that there is so
much more to life than working with this thesis.
My parents, Håkan and Madeleine, my grand-parents Ingrid, Bo and
Ingrid and my late grand-father Ryno for always believing in me and supporting me no matter what.
My family. My precious daughters Filippa and Linnea, for showing me
what is really important in life and for putting things in a new perspective
and Andreas, my wonderful husband, for your patience and support in
everything I do. I love you so!
56
I GABRIELLA ELIASON Skeletal muscle characteristics and physical activity patterns…
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Publications in the series
Örebro Studies in Sport Sciences
1.
Gustafsson, Henrik (2007). Burnout in competitive and elite athletes.
2.
Jouper, John (2009). Qigong in Daily Life. Motivation and Intention
to Mindful Exercise.
3.
Wåhlin Larsson, Britta (2009). Skeletal Muscle in Restless Legs
Syndrome (RLS) and Obstructive Sleep Apnoea Syndrome (OSAS).
4.
Johansson, Mattias (2009). Qigong: Acute affective responses in a
group of regular exercisers.
5.
Cardell, David (2009). Barndomens oas. En tvärvetenskaplig studie
av kulturindustriella intressen och organisering av evenemang genom
exemplet Stadium Sports Camp. Licentiat.
6.
Isberg, Jenny (2009). Viljan till fysisk aktivitet. En intervention
avsedd att stimulera ungdomar att bli fysiskt aktiva.
7.
Oskarsson, Eva (2010). Lateral epicondylitis. Intramuscular blood
flow, pressure and metabolism in the ECRB muscle.
8.
Andersson, Helena (2010). The physiological impact of soccer on
elite female players and the effects of active recovery training.
9.
Åkesson, Joakim (2010). Idrottens akademisering.
Kunskapsproduktion och kunskapsförmedling inom idrottsforskning
och högre idrottsutbildning. Licentiat.
10. Eliason, Gabriella (2010). Skeletal muscle characteristics and physical
activity patterns in COPD.