Modification of the functional capacity of sarcoplasmic reticulum
membranes in patients suffering from chronic fatigue syndrome
Stefania Fullea,*, Silvia Beliab, Jacopo Vecchieta, Caterina Morabitoa,
Leonardo Vecchieta, Giorgio Fano `a
aLaboratorio Interuniversitario di Miologia, Universita ` ‘G. d’Annunzio’, Nuovo Polo Didattico, Via dei Vestini, 31, 66013 Chieti Scalo, Italy
bUniversita ` degli Studi, Perugia, Italy
Received 31 October 2002; received in revised form 22 January 2003; accepted 13 February 2003
In chronic fatigue syndrome, several reported alterations may be related to specific oxidative modifications in muscle. Since sarcoplasmic
reticulum membranes are the basic structures involved in excitation–contraction coupling and the thiol groups of Ca2þchannels of SR
terminal cisternae are specific targets for reactive oxygen species, it is possible that excitation–contraction coupling is involved in this
pathology. We investigated the possibility that abnormalities in this compartment are involved in the pathogenesis of chronic fatigue
syndrome and consequently responsible for characteristic fatigue. The data presented here support this hypothesis and indicate that the
sarcolemmal conduction system and some aspects of Ca2þtransport are negatively influenced in chronic fatigue syndrome. In fact, both
deregulation of pump activities (Naþ/Kþand Ca2þ-ATPase) and alteration in the opening status of ryanodine channels may result from
increased membrane fluidity involving sarcoplasmic reticulum membranes.
q 2003 Published by Elsevier Science B.V.
Keywords: Chronic fatigue syndrome; Membrane fluidity; Ca2þtransport; Naþ/Kþ-ATPase; Ca2þ-ATPase
Chronic fatigue syndrome (CFS) is characterized by
severe disabling fatigue and specific symptoms, such as
musculoskeletal pain, sleep disturbance, impaired concen-
tration and headaches . Initially CFS was considered to
be a disorder associated to psychiatric problems, which has
been a significant obstacle in the recognition of CFS as an
organic condition. There are two main current definitions:
one from the United States Centers for Disease and
Prevention (CDC) of 1994 and the second based on Oxford
criteria [2,3]. The most important difference between the
two criteria is that the American definition includes several
physical symptoms secondary to immunological or infective
pathologies [4,5], while the British definition focuses on
the presence of mental fatigue. CFS affects 0.2–2.6% of
the population, depending on the criteria used to define the
disease . CFS has similar prevalence in people of
different socioeconomic status, affects all ethnic groups,
and the only demographic risk factor is gender, since
females are more prone to the illness [7,8]. Several mor-
phological and biochemical alterations that may be related
to generation offree radicals have been reported . In CFS
patients various blood parameters such as malondialdehyde
(MDA), methemoglobin, 2,3-diphosphoglycerate levels,
and volume of erythrocytes are elevated . Furthermore,
the skeletal muscle tissue of CFS patients present a number
of abnormalities such as: (a) morphological alterations of
myofibrils and fatty/fibrous tissues; (b) inversion of the ratio
between cytochrome oxidase and succinate dehydrogenase;
(c) pleio/polymorphism and monstruosity of mitochondria;
(d) reduction of a number of mitochondrial enzymatic
activities and increments in common mitochondrial DNA
We previously demonstrated specific oxidative altera-
tions in the vastus lateralis muscle of CFS patients, both as
an increased value of oxidative damage markers (8OH-dG,
MDA) and membrane fluidity, as well as imbalances in
the oxidant–antioxidant system . These findings are
in agreement with the hypothesis that impairment of
0960-8966/03/$ - see front matter q 2003 Published by Elsevier Science B.V.
Neuromuscular Disorders 13 (2003) 479–484
*Corresponding author. Tel.: þ39-0871-355-4036; fax: þ39-0871-355-
E-mail address: email@example.com (S. Fulle).
mitochondrial activity underlies an increase in the pro-
duction of reactive oxygen species (ROS), leading to muscle
fatigue, similar to normal ageing . This interpretation is
supported by CFS-associated muscle magnesium defici-
ency, a possible further cause of oxidative stress . An
interesting new theory for CFS pathogenesis proposes that
excessive cytokine production due to infections is correlated
with increased nitric oxide (NO) synthesis , thus repre-
senting another potential source for oxidative damage .
All these data taken together strongly support the hypothesis
that oxidative damage plays an important role in the
pathogenesis of CFS.
The fatigue that occurs in CFS patients typically fluctu-
ates and is similar to that of multiple sclerosis or chronic
inflammatory demyelinating polyneuropathy (CIDP) .
Ion channel have been correlated to various pathological
involving fatigue symptoms. An alteration in the function-
ality of Naþand Ca2þchannels seems to play a role
respectively in CIDP and periodic hypokalemic paralysis.
Also, CFS has been associated with abnormalities of ion
channels . It is possible that excitation–contraction
(E–C) coupling is involved in this pathology since the thiol
groups of Ca2þchannels present in the terminal cisternae of
the sarcoplasmic reticulum (SR) are specific targets for ROS
. In this study, we investigate whether or not the E–C
coupling mechanism that takes place in the junctions
between sarcoplasmic reticulum and T-tubules is specifi-
cally involved in the pathogenesis of CFS.
2. Materials and methods
Our studies were carried out on samples from: (a) four
CFS patients, three of whom were males of respectively 30,
33, and 39 years of age, and the other female of 38 years of
age; and (b) three patients suffering from fibromyalgic
syndrome (FS), one male and two females of respectively
45, 40, and 60 years of age. The latter set of patients was
solely analyzed for membrane fluidity. All patients were
selected at the CFS Study Center, University of Chieti, on
the basis of CDC diagnostic criteria . The control group
consisted of six healthy age- and sex-matched subjects who
underwent elective orthopedic surgery. Biopsies from the
vastus lateralis muscle were taken following a thorough
clinical evaluation, which included detailed manual muscle
testing. The eventual risks associated with muscle biopsy,
which include discomfort during the postoperative period,
possible complications of the surgical procedure and
expected findings from the biopsy (using informed consent),
were explained to the patient.
Biopsy specimens (0.1–0.4 g) were obtained from the
vastus lateralis muscle according to the methods of Engel
and Franzini-Armstrong , and samples were immedi-
ately frozen in liquid nitrogen. The following analyses were
performed on the biopsy specimens.
2.1. Membrane fluidity
Partially purified SR membranes were prepared from
muscle biopsies, as described by Fano ` et al. . Protein
(100 mg) in 200 ml of 250 mM sucrose was incubated for
1 h in the dark in the presence of 2 mM diphenylhexatriene
(2 ml) dissolved in tetrahydrofuran, following the procedure
of Shinitzky and Barenholz . Membrane fluidity was
measured as fluorescence anisotropy, as described pre-
2.2. [3H]PN200-110 binding
Membranes derived from homogenized human skeletal
muscle biopsies were prepared according to the procedure
of Renganathan et al. . The dihydropyridine receptor
(DHPR) concentration was determined using the radio-
ligand [3H]PN200-110. Protein (100 mg) was incubated in a
final volume of 250 ml binding buffer in the presence of
1 nM [3H]PN200-110 for 1 h at room temperature, follow-
ing which samples were filtered with Whatman GF/C filters
and washed with 6 volumes of ice-cold washing buffer .
Radioactivity was determined by liquid scintillation count-
ing. Non-specific [3H]PN200-110 binding was assessed in
the presence of 10 mM unlabeled nifedipine.
2.3. Ryanodine binding
Vesicles derived from the SR were prepared as described
by Fano ` et al. , washed in binding buffer (200 mM KCl,
10 mM HEPES (pH 7.4), 100 mM CaCl2, 0.1 mM PMSF,
1 mg/ml leupeptin) and centrifuged at 100000 £ g for
90 min before resuspension at a final concentration of
1 mg/ml. Aliquots (65 mg) of protein were incubated at
25 8C in a final volume of 250 ml in the presence of 10 nM
[3H]ryanodine for 120 min. In another experiment, samples
were incubated either with or without 10 mM caffeine,
filtered with Whatman GF/C filters and washed with
6 volumes of ice-cold 200 mM KCl, 10 mM HEPES,
pH 7.4. The amount of bound [3H]ryanodine was deter-
mined by liquid scintillation counting .
2.4. Ca2þ-ATPase activity
This was measured on 30 mg aliquots of protein in a final
volume of 1 ml medium containing 5 mM ATP and 10 mM
CaCl2, similar to a previously reported procedure .
2.5. Naþ/Kþ-ATPase activity
Enzymatic activity was measured on external mem-
branes derived from skeletal muscles, using the modified
method of Rock et al. . Samples were homogenized in
20 mM HEPES, 300 mM sucrose, 1% BSA, 500 mM PMSF,
pH 7.0 ð4 £ 20 s) and centrifuged at 8000 £ g for 15 min.
The supernatant was centrifuged at 40000 £ g for 70 min,
S. Fulle et al. / Neuromuscular Disorders 13 (2003) 479–484 480
followed by 100000 £ g for 90 min. The pellet was resus-
pended in 10 mM HEPES, 150 mM KCl, pH 6.8. Protein
concentrations were determined by the Lowry method .
Protein (25 mg) was incubated in 1 ml medium (134 mM
NaCl, 21 mM KCl, 1 mM EGTA, 25 mM HEPES, 2.5 mM
ATP, pH 7.5) in the presence or absence of 1 mM ouabain.
After 10 min incubation at 37 8C, the reaction was stopped
by the addition of 12.5% TCA (1 ml) and the precipitate
removed by centrifugation at 5000 £ g for 10 min. Released
orthophosphate was determined on 1 ml clear supernatant,
following the method of Taussky .
Immunoblotting was performed on microsomal fractions
prepared using the procedure of Carmody et al. .
Proteins (50 mg) were resolved on 6% SDS–PAGE for
RyR1 detection and transferred to a nitrocellulose mem-
brane using a semi-dry blotting apparatus (500 mA, 3–4 h).
Membranes were washed with 30 mM TBS (10 mM Tris–
HCl (pH 7.5), 0.9% NaCl) and 6% BSA at room tempera-
ture for 1 h. After a further wash with 0.1% TBS/Tween-20,
PEG (1 M Tris–HCl (pH 7.5), 200 mM NaCl, 16 mM
ethylene glycol) and 8% FCS, immunoblots were incubated
overnight at 4 8C with primary antibody (anti-RyR1, Sigma)
solution (1:5000 dilution) in 8% PEG/FCS. Membranes
were washed with PEG buffer for 10 min, followed by 0.1%
TBS/Tween-20, and incubated with peroxidase-linked anti-
mouse antibody diluted in 8% PEG/FCS (1:2000 dilution).
Blots were washed with 0.1%TBS/Tween-20. Immuno-
reactive proteins were detected with enhanced chemilumi-
nescence (ECL kit, Amersham Pharmacia Biotech) and
quantified by densitometry (Imagemaster im1D, Pharmacia
2.7. Statistical analyses
Two different tests were used to analyze our data: linear
correlation (Pearson’s test) for the membrane fluidity data
while the unpaired t-test was used for the binding studies
([3H]PN200-110 and [3H]ryanodine) and Naþ/Kþ- and
Ca2þ-ATPase activity. Prism-2 from GraphPad Software
Inc. (San Diego, CA) was used to automatically perform the
3. Results and discussion
We previously reported that membrane fluidity increases
in both FS and CFS patients in comparison to control
subjects . FS patients, who generally exhibit a very
similar pathology to CFS, display higher total membrane
fluidity than CFS and control samples. Since SR membranes
are the basic structures involved in E–C coupling, we
examined the possibility that abnormalities within this
compartment are responsible for the characteristic fatigue of
CFS. Fig. 1 depicts data on membrane fluidity measured in
purified SR membranes. Evidently, CFS patients maintain
higher fluidity in comparison to controls, whereas FS
patients exhibit fluidity values comparable to controls. This
was confirmed by functional analyses on the FS muscle
Ca2þ-transport mechanism, which was similar to that of
controls (data not shown). Fluorescence polarization values
(P) of SR membranes derived from CFS and control muscles
exhibit a similar slope, but different absolute values at
temperatures between 15 and 40 8C. This finding clearly
implies different membrane fluidity in the two experimental
groups, suggesting alterations in the pathological samples at
the SR level. We hypothesize that SR modifications play a
specific role in this disease (as well as in ageing ) and
may thus be potentially used as a diagnostic marker to
distinguish CFS from FS. This modification of membrane
fluidity in the pathological samples may correlate to an
alteration of polyunsaturated fatty acid, usually target of the
lipid peroxidation . We have in fact previously demon-
strated an increase of peroxidation in CFS muscles . SR
membranes of skeletal fibers usually contain variable, but
little, amount of cholesterol [26,32]. Correlation between
variability in cholesterol amount and biological functions
have never been reported. Because of this, the alterations
of membrane properties in CFS could be ascribed to the
The main function of SR is to control cytoplasmic
concentration of Ca2þ. The release of this ion from SR
terminal cisternae is controlled by a specific interaction
between dihydropyridine receptors (DHPRs), located in the
transverse tubule membrane, and the Ca2þrelease channels
of the SR, RyR type 1 in skeletal muscle fibers. It is clear
Fig. 1. Membrane fluidity in human skeletal muscle (vastus lateralis).
Fluorescence polarization (P) values of membranes derived from CFS, FS
and healthy-matched control subjects at different temperatures (15, 20, 25,
30, 35, 40 8C) are depicted. Also, the statistical analysis of the slope and the
intercepts are reported. The values are the means ^ SD (n ¼ 3). CFS,
chronic fatigue syndrome; FS, fibromyalgic syndrome; C, control.
S. Fulle et al. / Neuromuscular Disorders 13 (2003) 479–484481
that any significant alteration of SR membrane structure
could affect this interaction and consequently the E–C
coupling mechanism. We have tested this possibility
measuring the following parameters in both CFS and
control samples: (1) the [3H]PN200-110 binding to L-type
Ca2þ-channels as a marker of the amount of DHPRs present
in T-tubules membranes; (2) the ryanodine binding to the
SR Ca2þ-release channels as a marker of the ability of SR
terminal cisternae to release Ca2þ; and (3) the activity of
Ca2þ-ATPases as an indicator of the ability of SR mem-
branes to uptake Ca2þ. As shown in Table 1 (1st row), the
small but significant decrease of DHPR binding in CFS
samples indicates that this membrane target is also modified
by the pathological state (control, 1.4 ^ 0.16 vs. CFS,
1.13 ^ 0.2 pmol/mg protein; P , 0:05). It is additionally
important to note that ageing muscle displays decreased
DHPR binding . An increased value of Ryanodine
binding suggests that SR- Ca2þchannels are in an open
state, with the consequence that Ca2þrelease from SR
vesicles increases. Table 1 (2nd row) depicts 10 nM
[3H]ryanodine binding to SR vesicles derived from the
vastus lateralis muscle of CFS patients. The data show that
the pathological samples exhibit decreased [3H]ryanodine
binding, thereby indicating a diminished capacity of Ca2þ
channels to be maintained in the open state (controls
0.130 ^ 0.016 vs. CFS 0.090 ^ 0.005 nmol/mg protein;
P , 0:01). A possible channel alteration in the muscle of
CFS subjects was indirectly confirmed by experiments in
which the ryanodine-binding assay was performed in the
presence of 10 mM caffeine (an alkaloid that stimulates the
opening of SR Ca2þchannels) . A further difference
between controls and CFS patients is that not only the Rya
binding is decreased in the latter, but also the potentiation
effect of caffeine on this binding is depressed. In fact, while
in controls 10 mM caffeine induces a drastic increase in
ryanodine binding (increase of 87 ^ 18%), the potentiation
of the alkaloid on CFS patients is much smaller (only
49 ^ 4%) indicating a possible desensitization of the
RyR/Ca2þrelease channel. The difference between the
two data is statistically significant as shown by the t-test
value: P , 0:05. This finding is not particularly surprising
and may be the consequence of an alteration of E–C
coupling or modification of excitation-Ca2þrelease mech-
anism(s). The oxidation status of RyR1 thiols is directly
correlated to the functional status of the channel. In fact,
oxidation of ca. 10 of these thiols had little effect on channel
activity, whereas more extensive oxidation reduced the
opening status . In view of the fact that ryanodine binds
its receptor in proportion to the opening status, the decrease
in binding observed in CFS muscle preparations is probably
due to a decreased SR Ca2þchannel opening status. On the
other hand, this decrease does not depend on a reduction in
specific binding sites, since RyR1 expression is not signi-
ficantly different in CFS samples compared to the controls
Table 1 (3rd row) also depicts the activity of the enzyme,
Ca2þ-ATPase type 1 (SERCA 1 ), which controls the
capacity to recover Ca2þreleased by terminal cisternae.
Vesicles prepared from CFS samples exhibited a significant
decrease in sarcoendoplasmic reticulum Ca2þATPase
activity, compared to controls (3.4 ^ 0.38 vs. 2.0 ^ 0.05
Pimg/mg/ml per min; P ¼ 0:0075). The status of Ca2þ
release described above and the correlation  between
enzyme activity and myoplasmic Ca2þconcentrations raise
the possibility that the decrease in measured activity is a
consequence of lower Ca2þavailability, secondary to
reduced SR Ca2þrelease.
Naþ/Kþ-ATPase specific activity is a marker of the
physiological capacity of plasmalemma that correlates with
both membrane excitability and muscle contractility. The
activity of this enzyme (Table 1, 4th row) is increased in the
pathological samples (0.83 ^ 0.05 vs. 1.69 ^ 0.32 Pimg/
mg/ml per min; P , 0:05) indicating that Naþ/Kþtransport
is modified in CFS muscle. Higher Naþ/Kþ-ATPase
pumping activity compared to controls may signify an
imbalance in sarcolemmal Naþ/Kþpermeability. It is
possible that this Naþ/Kþalteration results from the
functional modification of Naþand/or Kþchannels as a
consequence of alterations in membrane fluidity.
Earlier studies report that oxidative skeletal muscle fibers
of spontaneously hypertensive rats exhibit several physio-
logical defects, including a reduced ability to maintain force
secondary to increased levels of extracellular Kþ. This
finding correlates with a decrease in Naþ/Kþ-ATPase
In vitro functional parameters of E–C coupling in CFS muscles
[3H]PN200-110 binding (pmol/mg protein)
[3H]Rya binding (nmol/mg protein)
Ca2þ/Mg2þ-ATPase activity (Pimg/mg/ml/min)
Naþ/Kþ-ATPase activity (Pimg/mg/ml/min)
1.4 ^ 0.16
0.13 ^ 0.016
3.40 ^ 0.38
0.83 ^ 0.05
1.13 ^ 0.2
0.09 ^ 0.005
2.00 ^ 0.05
1.69 ^ 0.32
P , 0:05
P , 0:01
P ¼ 0:0075
P , 0:05
First row, specific [3H]PN200-110 binding; 2nd row, specific [3H]ryanodine binding; 3rd row, specific enzymatic Ca2þ/Mg2þ-ATPase activity; 4th row,
specific enzymatic Naþ/Kþ-ATPase activity. All assays were performed on homogenized tissue (DHPR binding) and membrane preparations (external for
Naþ/Kþ-ATPase and SR derived for [3H]ryanodine binding and Ca2þ/Mg2þ-ATPase) derived from skeletal muscle (vastus lateralis) of CFS patients (n ¼ 3)
and control (n ¼ 5). Experiments were performed in quintuplicate and data are presented as mean ^ SEM. [3H]Rya, [3H]ryanodine; CFS, chronic fatigue
syndrome; Pi, inorganic phosphate.
S. Fulle et al. / Neuromuscular Disorders 13 (2003) 479–484482
activity, but not with an alteration in the number of sites
or the binding affinity of the pump . Interestingly, a
decrease in pump site number, but not binding affinity, has
been demonstrated in the skeletal muscle of rats with
chronic heart failure . In our opinion, upregulation of
pump activity may reflect an attempt by the muscle to
counteract the effect of dysfunction that is established in this
CFS is a pathological state characterized by alterations of
muscle capacity in which the rapid onset of fatigue is
associated with muscle pain. Both these symptoms may be
due to a modification in membrane organization (mainly the
SR network) due to oxidative damage, with consequent
possible imbalance in E–C coupling. The data presented
here support this hypothesis and indicate that the sarco-
lemmal conduction system, as well as some aspects of Ca2þ
transport, are negatively influenced in CFS muscle samples.
In fact, both the deregulation of pump activities (Naþ/Kþ
and Ca2þ-ATPase) and alterations in the opening status of
ryanodine channels may at least partially result from
increased membrane fluidity that probably involves SR
We wish to thank Feliciano Protasi for a critical reading
of the manuscript. This research was supported by a local
grant from CUMS (Centro Universitario Medicina dello
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