Neurochem Res (2012) 37:885–898
DOI 10.1007/s11064-011-0683-z
ORIGINAL PAPER
Oxidative Damage in Muscular Dystrophy Correlates
with the Severity of the Pathology: Role of Glutathione
Metabolism
R. Renjini • N. Gayathri • A. Nalini
M. M. Srinivas Bharath
•
Received: 1 December 2011 / Accepted: 20 December 2011 / Published online: 5 January 2012
Ó Springer Science+Business Media, LLC 2012
Abstract Muscular dystrophies (MDs) such as Duchenne
muscular dystrophy (DMD), sarcoglycanopathy (Sgpy) and
dysferlinopathy (Dysfy) are recessive genetic neuromuscular diseases that display muscle degeneration. Although
these MDs have comparable endpoints of muscle pathology, the onset, severity and the course of these diseases are
diverse. Different mechanisms downstream of genetic
mutations might underlie the disparity in these pathologies.
We surmised that oxidative damage and altered antioxidant
function might contribute to these differences. The oxidant
and antioxidant markers in the muscle biopsies from
patients with DMD (n = 15), Sgpy (n = 15) and Dysfy
(n = 15) were compared to controls (n = 10). Protein
oxidation and lipid peroxidation was evident in all MDs
and correlated with the severity of pathology, with DMD,
the most severe dystrophic condition showing maximum
damage, followed by Sgpy and Dysfy. Oxidative damage in
DMD and Sgpy was attributed to the depletion of
Electronic supplementary material The online version of this
article (doi:10.1007/s11064-011-0683-z) contains supplementary
material, which is available to authorized users.
R. Renjini M. M. Srinivas Bharath (&)
Department of Neurochemistry, National Institute of Mental
Health and Neurosciences (NIMHANS), P.B # 2900,
Hosur Road, Bangalore 560029, Karnataka, India
e-mail: bharath@nimhans.kar.nic.in
N. Gayathri
Department Neuropathology, National Institute of Mental Health
and Neurosciences (NIMHANS), Bangalore 560029, Karnataka,
India
A. Nalini
Department Neurology, National Institute of Mental Health and
Neurosciences (NIMHANS), Bangalore 560029, Karnataka,
India
glutathione (GSH) and lowered antioxidant activities while
loss of GSH peroxidase and GSH-S-transferase activities
was observed in Dysfy. Lower GSH level in DMD was due
to lowered activity of gamma-glutamyl cysteine ligase, the
rate limiting enzyme in GSH synthesis. Similar analysis in
cardiotoxin (CTX) mouse model of MD showed that the
dystrophic muscle pathology correlated with GSH depletion and lipid peroxidation. Depletion of GSH prior to CTX
exposure in C2C12 myoblasts exacerbated oxidative
damage and myotoxicity. We deduce that the pro and antioxidant mechanisms could be correlated to the severity of
MD and might influence the dystrophic pathology to a
different extent in various MDs. On a therapeutic note, this
could help in evolving novel therapies that offer myoprotection in MD.
Keywords Muscular dystrophy Duchenne muscular
dystrophy Dysferlinopathy Sarcoglycanopathy Oxidative stress Glutathione Cardiotoxin
Abbreviations
NMD
Neuromuscular disease
MD
Muscular dystrophy
DMD
Duchenne muscular dystrophy
Sgpy
Sarcoglycanopathy
Dysfy Dysferlinopathy
GSH
Glutathione
SOD
Superoxide dismutase
MDA
Malondialdehyde
3-NT
3-Nitrotyrosine
GCL
Gamma glutamyl cysteine ligase
GPx
Glutathione peroxidase
GR
Glutathione reductase
GST
Glutathione-s-transferase
CTX
Cardiotoxin
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886
ROS
MTT
DNPH
PN
Neurochem Res (2012) 37:885–898
Reactive oxygen species
3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide
Dinitrophenyl hydrazine
Peroxynitrite
Introduction
Neuromuscular diseases (NMDs) are myopathic disorders
which arise due to defects in nerves, neuromuscular junctions or in the muscle fibers that they innervate [1]. Among
these, muscular dystrophies (MDs) comprise the most
frequently encountered genetic diseases that display loss of
muscle integrity. The structure and function of the human
skeletal muscle are critically dependent on the intactness of
the sarcolemma which in turn is stabilized by several
protein complexes [2]. The Dystrophin-Glycoprotein
Complex (DGC) tethered to the sarcolemma is a transmembrane complex consisting of dystrophin, dystroglycans, sarcoglycans, syntrophins and other proteins [3]. The
absence of specific proteins in the DGC compromises the
sarcolemmal integrity thus causing muscle damage and
MD. While recessive mutations in the dystrophin gene
leading to the absence of dystrophin in the DGC cause
Duchenne muscular dystrophy (DMD) (an X-linked disorder), Sarcoglycanopathies (Sgpy) are autosomal recessive limb girdle muscular dystrophies (LGMDs) resulting
from deleterious mutations in the sarcoglycan genes (a,b,c
and d). Similarly, mutations in Dysferlin, a transmembrane
protein involved in membrane trafficking and repair, cause
Dysferlinopathy (Dysfy) (LGMD-2B/Miyoshi myopathy).
Although these MDs have comparable endpoints of
muscle pathology involving cell death, adiposis, fibrosis
etc., the onset, severity and the course of the diseases vary
[4]. While Dysfy has a later onset (30–40 years), DMD and
sarcoglycanopathies have early onset. Further, DMD
patients exhibit significantly higher severity of clinical
symptoms and dystrophic pathology compared to Sgpy and
Dysfy. This indicates that distinct mechanisms exist
downstream of genetic mutations in MDs that might alter
the course and severity of dystrophic pathology but these
pathogenic mechanisms remain elusive. Oxidative stress is
one such mechanism implicated in MD but the contribution
of this phenomenon has not been completely understood. In
MD patients, altered antioxidant enzyme activities [5, 6],
lowered activity of mitochondrial complexes [7] and NFkappaB activation [8] have been demonstrated. Systemic
[9, 10] and muscle-specific [11] oxidative damage and
altered anti-oxidant function have been reported in the
muscles of DMD patients [12]. The mdx mouse model of
DMD displays impairment of redox-sensitive metabolic
enzymes [13], alteration in antioxidant enzyme activities
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[14] and increased oxidative damage [14, 15]. Previous
research on chick [16] and hamster [17] models of MD
indicated elevated oxidative damage in the muscle. However, such studies in the models and disease samples of
Sgpy and Dysfy and their comparison with DMD are
scarce. In the current study, we have analyzed the markers
of oxidative stress and antioxidant function in the muscle
biopsies of patients with DMD, Sgpy and Dysfy to investigate and compare the contribution of these markers in
muscle pathology. We have also generated mouse and
myoblast cell models of MD and analyzed the role of
oxidative damage in muscle pathology.
Materials and Methods
Bulk chemicals from Merck & Co. Inc (Whitehouse Station, NJ, USA) and Sisco Research Laboratories (Mumbai,
Maharashtra, India), fine chemicals, anti-3-NT and antidinitrophenyl polyclonal antibodies, cardiotoxin (CTX)
from Naja mossambica mossambica and protease inhibitor
cocktail from Sigma (Eugene, OR, USA), nitrocellulose
membrane from Millipore (Billerica, MA, U.S.A.), monoclonal antibodies against Dystrophin (1,2,3), Sarcoglycans
(a, b, c, d) and Dysferlin from Novocastra Laboratories
Limited (Newcastle Upon Tyne, UK) and HRP conjugated
secondary antibodies from NCL and Bangalore Genei
(Bangalore, Karnataka, India) were procured.
Tissue Samples and Extraction
Patients (n = 55) with muscle diseases were evaluated at
the neuromuscular disorders Clinic of the National Institute
of Mental health and Neurosciences (NIMHANS), Bangalore, India. Detailed clinical history of the patients was
recorded. After obtaining the informed consent, biopsies
were conducted by a neurologist, under local anaesthesia
for adults and general anaesthesia for young children. A
moderately weak muscle (biceps/vastus lateralis) free from
previous trauma was selected for uniformity and submitted
for routine diagnosis to the Department of Neuropathology,
NIMHANS. A fragment of the fresh biopsy was snap-frozen in isopentane that was pre-cooled in liquid nitrogen.
Fifty five muscle samples included immunohistochemically
confirmed cases of DMD (n = 15), Dysfy (n = 15), Sgpy
(n = 15) and healthy paraspinal muscle samples from
patients with spinal corrections (control) (n = 10)
(Table 1). The study protocol was approved by the Institutional Ethics Committee.
For preparation of muscle extracts, frozen muscle tissue
(50 mg) was minced in 10 volumes of 19 PBS containing
protease inhibitors, homogenized and sonicated on ice for
30 s. The extract was centrifuged (14,000g, 10 min) and
Neurochem Res (2012) 37:885–898
887
Table 1 List of samples utilized in the current study
Sl. No.
Disease
Age (year)
Gender
(M/F)
Sample
number (n)
1
Control
28 ± 5
7/3
10
2
DMD
08 ± 3
15/0
15
3
Sgpy
10 ± 4
7/8
15
4
Dysfy
29 ± 5
12/3
15
and Material Science, Europa, GmbH) according to the
manufacturer’s instructions.
Cell Treatment and Viability Assay
DMD Duchenne muscular dystrophy; Sgpy sarcoglycanopathy; Dysfy
dysferlinopathy
the supernatant was subjected to different assays after
protein estimation [18].
Histological and Immunohistochemical Analyses
A portion of the skeletal muscle biopsy oriented transversely was flash frozen in isopentane that was pre-cooled
in liquid nitrogen. 8 lM thick serial cryosections were
subjected to routine Haematoxylin-eosin (HE) staining and
immunostaining for Dystrophin (1,2,3), Sarcoglycans (a, b,
c, d) and Dysferlin [19].
Cell Culture
C2C12 mouse myoblast cell line was grown in DMEM
supplemented with 10% foetal bovine serum, penicillin
(100 units/ml), streptomycin (100 lg/ml) and maintained
at 37°C in a humidified atmosphere of 5% CO2/95% air
[20]. Cells were sub-cultured via trypsin treatment. Differentiation of C2C12 myoblast cell line to myotubes was
induced as follows: Upon reaching 100% confluency, cells
were allowed to spontaneously differentiate for 6 days.
Alternately, the culture medium was replaced with DMEM
supplemented with 2% horse serum and antibiotics and
allowed to differentiate for 6 days [20]. Differentiation was
confirmed by the cellular morphology (differentiated cells
were tubular compared to undifferentiated cells; data not
shown) and by assaying the creatine kinase (CK) activity of
total cell extracts as described earlier [21].
Cells (both differentiated and undifferentiated) were
harvested and centrifuged (8509g, 1 min) and the pellet
was resuspended in 19 phosphate buffered saline (PBS),
pH 7.4, containing protease inhibitors. The cell suspension
was sonicated on ice in a sonicator (Sonics and Materials
Inc., CT, USA) (5 s each for 4 cycles) and centrifuged
(15,0009g, 10 min, 4°C). The supernatant corresponding
to the soluble extract were subjected to protein estimation
by Bradford method [18]. The extract normalized for total
protein was subjected to different biochemical assays
including CK and oxidant/antioxidant assays. The CK
activity was measured by a commercial kit (Olympus Life
Cells were seeded in 96-well plates at a density of 5 9 103
or 5 9 102 for differentiated and undifferentiated cells
respectively per well. After 24 h, the cells were exposed to
buthionine sulfoximine (BSO) (inhibitor of glutathione
(GSH) synthesis) immediately or after differentiation for
84 h. After 24 h of BSO exposure, the cells were treated
with cardiotoxin (CTX) for 40 h. After the treatments,
viable cells were measured using 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) assay [22].
Briefly, 20 ll of 5 mg/ml MTT was added to cells and
incubated at 37°C for 2 h. The medium was discarded, the
dark blue formazan crystalline product was dissolved in
dimethyl sulfoxide, and the absorbance was analyzed in a
plate reader (Tecan) at 570 nm.
Measurement of Reactive Oxygen Species (ROS)
in Cells
Total ROS in C2C12 cells was measured by a modified
method of Ohashi et al. [23]. Briefly, the medium from
treated or untreated C2C12 cells was replaced with 1 ml
Locke’s solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM
NaHCO3, 5 mM HEPES, 2 mM CaCl2 and 10 mM Glucose, pH 7.4). Dihydrodichlorofluorescein diacetate
(DCFDA) (10 lM) was then added and the cells were
incubated at 37°C (10 min) in a CO2 incubator. The
Locke’s solution was then removed and the cells were
harvested. The cell pellet was washed with 19 PBS (pH
7.4) twice and reconstituted in lysis buffer (10 mM Tris–
HCl containing 0.5% Tween-20). The lysate was centrifuged at 1,000g (10 min) and the fluorescence of the
supernatant was measured (Excitation: 480 nm; Emission:
530 nm) [24].
In Vivo Model of MD
All animal experiments were carried out in accordance with
the institutional guidelines for the Care and Use of Laboratory Animals as per the internationally accepted principles for laboratory animal use and care. All the experiments
involving animals were approved by the institutional animal
ethics committee. Adult male C57BL/6 mice (10 week-old)
(weight *30g each) (n = 6 for each treatment) were
obtained from the Central Animal Research Facility,
NIMHANS, Bangalore, India. Mice were housed five per
cage with access to standard diet and water ad libitum in a
well-ventilated room and all animals were exposed to 12 h
light and dark cycle. Mice were injected either with saline
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888
or CTX (single injection in saline; 300 ll of 10 lM per
injection) across the tibialis anterior muscle on either of the
hind limbs as described previously [25]. Care was taken to
release the CTX uniformly along the muscle tissue by
injecting the myotoxin while withdrawing the syringe
needle. Twenty four hours and 14 days after the injection,
the muscle from the ipisilateral and contralateral limbs were
dissected and utilized either for histopathological or biochemical assays as described elsewhere in the methods
section. Preparation of muscle extracts was carried out as
described previously for the human samples.
Neurochem Res (2012) 37:885–898
Assay of Oxidant Markers
a nitrocellulose membrane followed by anti-3-NT western
blot. Band intensities in westerns were quantified by a
densitometric scanner using dedicated software (QuantityOne, BioRad laboratories, Hercules, CA, USA). To determine the specificity of the antibody, soluble protein extract
was treated with Peroxynitrite (PN) at different concentrations as described earlier [29]. In brief, PN solution
(0–1,000 lM) was placed on the wall of the tube containing the extract and vortex-mixed for a few seconds to
ensure proper mixing before degradation of PN. Untreated
and PN treated samples were spotted in triplicate onto a
nitrocellulose membrane followed by anti-3-NT western
blot.
Estimation of Lipid Peroxidation
SDS PAGE
The tissue supernatant (100 ll) was added to 0.75 ml of
acetic acid (pH 3.5), 0.1 ml SDS (8%) and 0.75 ml thiobarbituric acid (0.8%). The mixture was heated (45 min)
and the adducts formed were extracted into 1.5 ml of
1-butanol followed by measurement of absorbance at
532 nm. The MDA concentration was calculated using the
molar extinction coefficient (MEC) (241 mol/cm) and
normalized per mg protein [26].
Total muscle protein extracts (*50 lg/lane) from control
and MD tissues were run on 10% SDS PAGE stained with
coomassie brilliant blue as described previously [30].
Estimation of Protein Carbonyls and Protein Nitration
10 ll of the supernatant (10 lg protein) was mixed with
the buffer containing 30 mM Tris HCl buffer (pH 9.1),
0.5 mM EDTA, 50 mM TEMED and 0.05 mM quercetin.
The rate of quercetin oxidation was monitored at 406 nm
for 10 min [27, 31]. (1U SOD activity = amount of
enzyme/mg protein that inhibits quercetin oxidation by
50%).
The tissue supernatant (4 mg/ml protein) was derivatized
by dinitrophenyl hydrazine (DNPH) in 12% SDS for
20 min at room temperature. The reaction was neutralized
by 2 M Tris in 30% glycerol. Sample (5 ll) was spotted in
triplicate onto a nitrocellulose membrane and subjected to
anti-DNP western blot [27]. DNPH alone (without protein)
and non-derivatized protein samples did not give anti-DNP
signal indicating the specificity of the antibody.
Alternately, the protein carbonyl content was determined by the spectrophotometric method described by
Levine et al. [28]. Briefly, homogenate of the muscle tissue
(10%) in 20 mM Tris–HCl–0.14 M NaCl (pH 7.4) was
prepared and centrifuged at 10,0009g for 10 min at 4°C.
To 100 ll of the supernatant, 100 ll of 20% trichloroacetic
acid (TCA) was added and centrifuged at 10,0009g for
10 min at 4°C.The supernatant was discarded and the pellet
was re-suspended in 1 ml of DNPH (10 mM in 2 N HCl)
and kept in dark for 1 h with occasional mixing. To this
mixture, 500 ll of 20% TCA was added to precipitate
protein and the pellet was washed in 1 ml acetone and
dissolved in 1 ml of 2% SDS prepared in 20 mM Tris HCl.
The absorbance was read at 360 nm and the results were
expressed as g moles carbonyls/mg protein using MEC22.0 mM-1 cm-1.
To detect protein nitration (protein 3-nitrotysorine or
3-NT), 75 lg muscle protein was spotted in triplicate onto
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Assay of Antioxidant Markers and GSH Metabolic
Enzymes
Superoxide Dismutase (SOD) Assay
Catalase Assay
The sample (50 ll) was mixed with 900 ll phosphate
buffer (0.1 M, pH 7.0) and 50 ll of H2O2 (8.8 mM). The
decrease in absorbance at 240 nm was followed for 5 min
at room temperature and the enzyme activity was expressed
as lmol H2O2 consumed/min/mg protein (MEC =
43.6 mM-1 cm-1) [32].
Estimation of Total Glutathione (GSH ? GSSG)
The tissue sonicate or soluble cell extract was mixed with 5%
sulfosalycylic acid, centrifuged (18,000g, 20 min) and 10 ll
of supernatant was mixed with assay buffer (240 ll)
[Phosphate–EDTA buffer containing 0.8 mM DTNB and
0.32U/ml glutathione reductase (GR)]. The reaction was
initiated by 0.6 mM NADPH and the kinetics of 5, 50 -dithiobis (dinitrobenzoic acid) (DTNB) recycling was monitored
at 412 nm for 5 min [27]. The GSH value was calculated
based on the maximum reaction rate compared with GSSG
Neurochem Res (2012) 37:885–898
standards. All estimations were conducted in triplicate and
the values were normalized per mg protein.
Glutathione Peroxidase (GPx) Assay
The reaction mixture containing 10 ll supernatant, 0.1 M
phosphate buffer, 0.5 mM EDTA, GSH reductase (0.24 U),
GSH (2 mM) and NADPH (0.15 mM) was incubated at
37°C for 3 min and the reaction was initiated by the
addition of tert-butyl hydroperoxide (0.12 mM) [33]. The
change in absorbance at 340 nm was monitored for 5 min
and the activity was expressed as nmoles of NADPH oxidized/min/mg protein (MEC = 6.22 mM-1 cm-1).
Glutathione Reductase (GR) Assay
To the reaction mixture (240 ll) containing Tris–HCl
buffer (0.1 M, pH 8.8), 0.1 mM EDTA and NADPH
(0.2 mM), 10 ll sample was added and the decrease in
absorbance at 340 nm was monitored for 5 min. The
enzyme activity was expressed as nmoles of NADPH
oxidized/min/mg protein (MEC = 6.220 M-1 cm-1)[34].
Glutathione-S-Transferase (GST) Assay
Sample (10 ll) was added to 240 ll of the reaction mixture
containing 0.1 M phosphate buffer (pH 6.5), 0.5 mM
EDTA, 2,4-dinitro benzene (CDNB) (1.5 mM) and 50 ll
GSH (1 mM), and the absorbance at 340 nm was monitored for 5 min. The enzyme activity was expressed as
nmoles of S-2,4, DNP-GSH formed/min/mg protein
(MEC = 9.6mM-1 cm-1) [35].
Glutamate Cysteine Ligase (GCL) Assay
Tissue supernatant (40 lg) was mixed with 100 mM Tris–
HCl (pH 8.0), 150 mM KCl, 5 mM Na2ATP, 2 mM
phosphoenol pyruvate, 10 mM l-glutamate, 20 mM MgCl2,
2 mM Na2EDTA and Pyruvate kinase/Lactate dehydrogenase mix (17U each) and the reaction kinetics was monitored at 340 nm for 10 min [36]. The reaction was initiated
by the addition of 10 mM L-alpha aminobutyrate and the
activity was normalized per mg protein.
Thioredoxin Reductase (TrxR) Assay
The reaction mixture containing 0.2 M phosphate buffer
pH 7.4, 1 mM EDTA, 0.4 mM NADPH and sample (10 ll)
was initiated by the addition of 10 ll of 2 mM DTNB and
5-thio-2-nitrobenzoic acid formed was measured at
412 nm. The activity of TrxR was expressed as nM of
DTNB reduced/min/mg protein [37].
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Statistical Analysis
Quantitative data were accumulated from at least three
independent experiments and expressed as mean ± SD
followed by the analysis of variance (ANOVA). In all the
experiments, data with P \ 0.05 were considered to be
statistically significant.
Results
Oxidative stress is implicated in muscle injury and MDs
[13, 15, 38]. To investigate and compare the oxidative
damage and antioxidant function and their contribution in
different MDs, we tested the muscle biopsies from immunohistochemically confirmed cases of DMD, Sgpy, Dysfy
and normal healthy controls (Table 1, Fig. 1). First we
tested whether MD involves oxidative damage to muscle
proteins. Analysis of protein nitration (indicated by total
protein 3-nitrotyrosine/3-NT) in muscle extracts (75 lg
total protein/spot) did not indicate significant change in any
of the MDs compared to healthy controls (Fig. 2a, b).
Control experiments with lower amounts of soluble protein
extracts (15 lg/spot) treated with peroxynitrite (PN, a
reactive nitrogen species that causes robust 3-NT modification in proteins) showed dose dependent (50–1,000 lM)
increase in 3-NT signal compared to untreated control
(Fig. 2c, d) indicating that the antibody was specific to
protein 3-NT.
On the other hand, protein oxidation levels (indicated by
total protein carbonyls) were significantly elevated in all
MDs with DMD showing maximum protein oxidation
(*65% increase; P \ 0.001) followed by Sgpy (*50%
increase; P \ 0.001) and Dysfy (*25% increase;
P \ 0.05) (Fig. 2e, f). Control experiments carried out with
DNPH alone (without protein) and protein alone (without
DNPH treatment) did not give any western signal indicating that the antibody was specific to DNP-derivatized
proteins (Fig. 2h). Analysis of protein carbonyls by an
alternate approach (spectrophotometric method-see methods section) recapitulated the same trend in the data
(Fig. 2i) as obtained in the slot blot method (Fig. 2e, f)
thereby confirming the status of protein oxidation in the
muscle samples. Quantitative analysis of lipid peroxidation
indicated *3 fold increase in DMD (P \ 0.001) while it
was increased by *2.5 and *2-fold in Sgpy (P \ 0.01)
and Dysfy (P \ 0.05) respectively (Fig. 2j). Based on these
data, oxidative stress was evident in different MDs with
DMD, the most severe dystrophic condition showing
maximum oxidative damage and Dysfy exhibiting the least
damage.
To analyze whether increased oxidative stress in MDs is
linked with the antioxidant function, we assayed the
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Neurochem Res (2012) 37:885–898
Fig. 1 Histopathological analyses of the muscle biopsies used for
the study. Muscle biopsies from suspected cases of DMD (n = 15),
Sgpy (n = 15), Dysfy (n = 15) and healthy controls (n = 10) were
confirmed by histopathology. The figure shows the Haematoxylineosin (HE) staining and immunostaining for Dystrophin (DYS2),
Sarcoglycan (Gamma-sarc) and Dysferlin in representative muscle
biopsies of DMD, Gamma-Sgpy and Dysfy compared to control
(magnification in all images = X400). All the MDs showed dystrophic pathology (indicated by HE staining) and loss of respective
structural proteins (indicated by immunostaining)
activities of the constitutive antioxidant enzymes in the
muscle extracts. While the SOD activity was unaltered in
DMD and Dysfy, it was decreased by *20% in Sgpy
(P \ 0.01) (Fig. 3a). On the other hand, catalase activity
was increased by *100% in DMD (P \ 0.01) and
by *50% in Dysfy (P \ 0.001) while it was unchanged in
Sgpy (Fig. 3b). However, Thioredoxin reductase activity
was unaltered in all the three MDs compared to control
(Fig. 3c).
Since the variation in the antioxidant enzymes was not
drastic enough to explain the oxidative damage in MDs, we
tested whether the total GSH was altered. Total GSH was
depleted by 50% in DMD (P \ 0.01) and by 40% in Sgpy
(P \ 0.05), while it was unchanged in Dysfy compared to
control (Fig. 4a, supplementary Fig. 1). We observed that
the GSH depletion in DMD was due to *50% loss in the
GCL activity (P \ 0.01) leading to decreased GSH synthesis (Fig. 4b). The GCL activity was increased by *30%
in Dysfy (P \ 0.01) while it was unaltered in Sgpy. Next,
we tested whether the antioxidant enzymes and metabolic
enzymes associated with GSH were altered. The GPx and
GST activities were drastically decreased in all the MDs
(*50% decrease in Dysfy; *60% in DMD and by *70%
in Sgpy compared to control; P \ 0.001 in all the three
MDs) (Fig. 4c, d). However, GR activity was significantly
increased in DMD (*40% increase compared to control;
P \ 0.01) and Dysfy (*55% increase compared to control; P \ 0.05) while it was unaltered in Sgpy (Fig. 4e).
Our data clearly correlated oxidative damage, GSH
depletion and altered activities of GSH-metabolic enzymes
with MD pathology in human samples. To confirm the
contribution of GSH to muscle pathology under defined
conditions, we generated CTX (from the venom of snake
Naja mossambica mossambica) mouse model of MD
(n = 6) as described earlier [25] and analyzed the status of
oxidative stress and total GSH. In vivo injection with CTX
(one injection with 300 ll solution of 10 lM CTX) into the
tibialis anterior muscle caused transient but significant
muscle cell damage and infiltration of inflammatory cells
after 24 h (day 1; d1) compared to saline control (Fig. 5a),
consistent with previous reports [25]. After 14 days of CTX
injection (d14), there was significant recovery in muscle
architecture (Fig. 5a), although most of the cells displayed
centrally located nuclei. Interestingly, CTX-induced
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Neurochem Res (2012) 37:885–898
A
891
B
n1 n2 n4 n5 n7 n8 n10 f1 f3 f4
f6
f7 f9 f10
n1 n3 n4 n6 n7 n9 n10 f2 f3 f5
f6
f8 f9
n2 n3 n5 n6 n8 n9 f1 f2
f4
f5 f7
OD (AU)
Protein 3-NT
b
f8 f10 b
Control
C
0
50 100 200
D
Dysfy
DMD
Sgpy
2.5
2.0
OD (AU)
Triplicate
250 500 750 1000
1.5
1.0
0.5
Triplicate
0
50
100
200
250
500
750 1000
PN (µM)
E
Oxyblot
F
n1 n3 n4 n6 n7 n9 n10 s2 s3 s5 s6 s8
s9
OD (AU)
n1 n2 n4 n5 n7 n8 n10 s1 s3 s4 s6 s7 s9 s10
b
n2 n3 n5 n6 n8 n9 s1 s2 s4 s5 s7 s8 s10 b
Control
H
1 2
3
4 5
6
7
8
MW (kDa)
96
66
42
Triplicate
30
DMD
Control Dysfy
DMD
Sgpy
Sgpy
Lipid peroxidation
µg MDA/mg protein
J
Dysfy
I
DMD Dysfy Sgpy Control
nM protein
carbonyls/mg protein
G
Control
Dysfy
DMD
Sgpy
Fig. 2 Quantitative analysis of oxidative markers in the muscle
samples of MDs. Soluble extracts from DMD (n = 15), Sgpy
(n = 15), Dysfy (n = 15) and healthy controls (n = 10) were used
to compare the levels of different oxidant markers. a and b respectively show representative protein 3-NT blot (75 lg total protein per
spot shown in duplicate per sample) and quantitation of nitrated
proteins compared to controls; n = normal control; f = Dysfy;
b = blank (no protein). c shows a control 3-NT dot blot of soluble
protein extracts (15 lg/spot) treated with increasing PN (0–1,000 lM
as indicated above each lane). d indicates the quantitation data of the
slot blot from c. e A representative oxyblot for analysis of protein
oxidation (10 lg derivatized protein per spot shown in duplicate per
sample); n = normal control; s = Sgpy; b = blank (no protein).
f shows the quantitation data of the slot blot from e. g shows the SDS
PAGE profile of total muscle extracts from normal and MD samples
indicating comparable protein used for slot blot experiments. h shows
control oxyblot (dot blot) carried out with DNPH alone (without
protein), protein alone (without DNPH treatment) and protein ? DNPH indicating that the antibody was specific to DNPderivatized proteins. i shows the quantitation of oxidized proteins by
spectrophotometirc determination of DN-derivatized proteins. j represents estimation of lipid peroxidation (quantitated by TBARS) in
different muscle samples compared with health controls. *P \ 0.05,
**P \ 0.01 and ***P \ 0.001 compared to healthy controls in all the
experiments
123
892
A
C
Dysfy
DMD
Sgpy
Control
Dysfy
DMD
Sgpy
Thioredoxin reductase
A
Dysfy
DMD
Sgpy
B
Total GSH
µg/mg protein)
(µ
Control
C
Dysfy
DMD
D
DMD
Sgpy
nM of 2,4-DNP-GSH
/min/ mg protein
GST
Control
Dysfy
DMD
Sgpy
Dysfy
DMD
Sgpy
DMD
Sgpy
GR
nM of NADPH oxidised
/min/ mg protein
nM of NADPH oxidised
/min/ mg protein
E
Control
Sgpy
GPx
Dysfy
GCL
nM of NADPH oxidised
/min/ mg protein
GSH
Control
123
Catalase
% Catalase activity
% SOD activity
Control
Control
Fig. 4 Analysis of GSH and
related antioxidant and
metabolic activities in muscle
samples of MDs. Soluble
extracts from DMD (n = 15),
Sgpy (n = 15), Dysfy (n = 15)
and healthy controls (n = 10)
were used to compare the levels
total GSH (a), GCL activity (b),
GPx activity (c), GR activity
(d), and GST activity (e).
*P \ 0.05, **P \ 0.01 and
***P \ 0.001 compared to
healthy controls
B
SOD
nM DTNB reduced
/min/mg
Fig. 3 Assay of antioxidant
enzymes in the muscle samples
of MDs. Soluble extracts from
DMD (n = 15), Sgpy (n = 15),
Dysfy (n = 15) and healthy
controls (n = 10) were used to
compare the levels of different
antioxidant enzymes.
a Percentage differences in
SOD activity among different
MDs (100% SOD
activity = 143.49 units/min/mg
protein). b Percentage
differences in catalase activity
among different MDs (100%
catalase activity = 2.86 nM of
H2O2 converted/min/g protein).
c corresponds to the thioredoxin
activity assayed in different
MDs. *P \ 0.05, **P \ 0.01
and ***P \ 0.001 compared to
healthy controls
Neurochem Res (2012) 37:885–898
Control
Dysfy
Neurochem Res (2012) 37:885–898
dystrophic muscle pathology at d1 correlated with *35%
depletion in total GSH (P \ 0.001) and 12-fold increase in
lipid peroxidation (P \ 0.001) in the muscle samples compared to controls (Fig. 5b, c). Following recovery of the
muscle architecture at d14, the GSH and MDA content were
restored to the level comparable with the saline control
(Fig. 5b, c), thus linking GSH depletion, oxidative damage
and muscle pathology. We recapitulated the same conditions
in undifferentiated or differentiated C2C12 murine myoblast
cell line [Differentiation in C2C12 cells into myotubes was
confirmed by *3 fold increase in CK activity (P \ 0.01)
(Fig. 6a) consistent with previous reports [21]. Dosedependent exposure to CTX (0–2 lM for 40 h) caused significant cell death with differentiated cells displaying
increased vulnerability to the myotoxin compared to undifferentiated cells [LD50 = 2 lM (P \ 0.001) and 1 lM
(P \ 0.001) for undifferentiated and differentiated cells
respectively] (Fig. 6b). Next, we tested whether selective
depletion of GSH prior to CTX exposure influenced myotoxicity. Accordingly, pretreatment with BSO (specific
inhibitor of GCL that depletes GSH in a dose dependent
manner) for 24 h (10 lM) followed by CTX treatment
exacerbated cell death and this effect was more pronounced
in differentiated cells (P \ 0.001) compared to undifferentiated cells (P \ 0.001) (Fig. 6c, d). However, exposure to
BSO alone did not impinge on cell viability either in undifferentiated or differentiated cells indicating that GSH
depletion combined with other toxic insults are required for
cell death in muscle cells (Fig. 6e). Next we tested the status
of total GSH and oxidative damage in the CTX cell model.
Exposure to BSO alone (0–10 lM) caused dose dependent
depletion of GSH both in differentiated and undifferentiated
cells (Fig. 7a, b). Interestingly, treatment with CTX alone
caused significant GSH depletion both in undifferentiated
(*50% depletion at 2 lM; P \ 0.001) and differentiated
cells (*75% depletion at 1 lM; P \ 0.001) compared to
untreated cells and this was further depleted when the cells
were pre-treated with BSO (Fig. 7c, d). In these cells, GSH
depletion was associated with increased ROS (30% increase
in undifferentiated cells, P \ 0.05; *5-fold increase in
differentiated cells; P \ 0.001) compared to the respective
controls (Fig. 7e, f). CTX-mediated GSH depletion and ROS
accumulation was exacerbated by pretreatment with BSO
(10 lM) and this was more pronounced in differentiated
cells compared to undifferentiated cells. These data correlates GSH depletion, oxidative stress and muscle pathology
with implications for MD.
Discussion
DMD, Sgpy and Dysfy are recessive NMDs characterized
by a leaky sarcolemma resulting in muscle cell death [1].
893
While DMD and Sgpy are early onset, rapidly progressive
diseases, Dysfy is a late onset slowly progressive disorder
[4]. There might be specific mechanisms underlying the
clinical and pathological differences among these NMDs.
Our study revealed varied dynamics of oxidative and
antioxidant processes in different MDs which might contribute to the differences in these pathologies. We observed
widespread oxidative damage that correlated with the
severity of the pathology with DMD displaying maximum
protein oxidation and lipid peroxidation followed by Sgpy
and Dysfy (Fig. 2). The correlation between alterations in
antioxidant function and the severity of MD has been
previously suggested [39].
The mechanisms contributing to increased oxidative
damage are quite dissimilar among the three MDs. Total
GSH was considerably depleted in the rapidly progressive
diseases DMD and Sgpy, compared to Dysfy, which represents chronic dystrophy. A recent study [40] not only
suggested that low GSH hastens the onset of cardiomyopathy in mdx mice but also substantiated the role of
GSH deficiency in DMD pathogenesis. Spassov et al. [41]
demonstrated decreased GSH and GSSG content in the
masticatory muscles of the mdx mice. Redox regulation of
specific proteins contributes to the muscle function while
dysregulation of redox-sensitive processes is linked to the
loss of muscle mass and function during aging and MDs
[42]. Dorchies et al. [43] demonstrated that natural antioxidants prevented oxidative damage, restored GSH content and protected against dystrophic pathology in the
experimental models of DMD.
In DMD and Sgpy, drastic depletion of GSH and significant reduction in GPx and GST activities contributed to
oxidative damage. In addition, lowered SOD activity contributed to the pathogenesis in Sgpy. On the other hand,
oxidative damage in Dysfy was contributed by lowered
activities of GPx and GST (Figs. 3, 4). These events are
either directly responsible for the pathological and clinical
differences among the MDs or can occur in response to
muscular degeneration. In DMD, GSH depletion was due
to a significant decrease in the activity of the GSH synthesizing enzyme GCL (Fig. 4b). This is a novel observation with therapeutic implications in MDs. However, GSH
depletion in Sgpy could not be explained by the GCL
mechanism (Fig. 4b). The oxidative damage in Sgpy could
be contributed partly via slight decrease in SOD activity
and significant decrease in GPx and GST activities. Even
though the GSH levels were unaltered in the Dysfy samples, decreased GST and GPx activities could contribute to
the oxidative damage and muscle pathology. Further, lipid
peroxidation in Dysfy could be attributed to the loss of
GST activity (Fig. 2c) since soluble lipid peroxides are
detoxified by conjugation with GSH via GST. The oxidant
effects were relatively milder in Sgpy since the GSH levels
123
894
Neurochem Res (2012) 37:885–898
A
Saline
CTX
d1
Saline
CTX
d14
1.4
GSH d14
GSH d1
1.2
1.2
1
***
0.8
0.6
0.4
Total GSH
(µg/mg protein)
Total GSH
(µg/mg protein)
B
0.8
0.4
0.2
0
0
Saline
0.2
Lipid peroxidation d1
***
0.15
0.1
0.05
0
0.10
MDA (ng/mg protein)
MDA (ng/mg protein)
C
Saline
CTX
CTX
Lipid peroxidation d14
0.08
0.06
0.04
0.02
0
Saline
CTX
Saline
CTX
Fig. 5 Analysis of histopathology, antioxidant and oxidant markers
in CTX mouse model of MD. Muscle biopsies from saline injected
and CTX injected animals (n = 6 each) were isolated after 24 h
(day1; d1) or after 14 days (d14) and subjected to histopathology.
a shows the Haematoxylin-eosin (HE) staining (Magnification = 400X) which indicates dystrophic pathology in CTX injected
muscle at d1 (comparable to the human samples in Fig. 1) which was
significantly restored at d14 compared to saline control. Dystrophic
pathology in CTX muscle at d1 also correlated with depletion of total
muscle GSH (b) and increased lipid peroxidation (c) which were
restored to control levels by d14; ***P \ 0.001 compared to saline
control
were higher compared to DMD (Fig. 4a). However, GCL
activity was unaltered indicating that the GSH loss might
be contributed by other mechanisms such as formation of
protein disulphides, and/or other GSH conjugates but these
possibilities have to be supported by additional
experiments.
123
Neurochem Res (2012) 37:885–898
D
Differentiated
Cell viability
(% control)
CPK activity (% control)
A
Undiff
2 % HS
Control
10 % FBS
CTX
(1 µM)
BSO
BSO+CTX
(10 µM)
B
Differentiated
E
Undifferentiated
Differentiated
Cell viability
(% control)
Cell viability
(% control)
Undifferentiated
0
1
2
0
1
2
CTX (µ
µM)
C
0
5
10
0
5
10
BSO (µM)
Undifferentiated
Cell viability
(% control)
Fig. 6 Effects of GSH
depletion and CTX exposure in
C2C12 muscle cell line. C2C12
cells were differentiated into
myotubes and exposed either to
CTX alone for 40 h or
pretreated with BSO for 24 h
followed by CTX treatment.
Following different treatments,
undifferentiated and
differentiated cells were
analyzed for cell viability.
a differentiation of the C2C12
myoblasts into myotubes was
confirmed by *3 fold increase
in CK activity. b shows CTX
(0–2 lM) dependent loss of cell
viability in undifferentiated and
differentiated C2C12 cells.
c and d correspond to
exacerbation of CTX
myotoxicity by BSO (10 lM).
e corresponds to the effect of
BSO alone (5 and 10 lM) on
cytotoxicity in undifferentiated
and differentiated C2C12 cells.
In all experiments, *P \ 0.05,
**P \ 0.01 and ***P \ 0.001
compared to controls
895
Control
CTX
(2 µM)
Most of the studies related to antioxidant enzymes in
MDs have concentrated on DMD using human samples and
mdx mice. But the data obtained from DMD studies are
varied and are contradictory in some cases [5, 6, 9, 14].
DMD pathology was associated with chronic oxidative
stress, impairment of the redox-sensitive metabolic
enzymes [13], decreased activity of both MnSOD and Cu/
Zn SOD and increased activity of GPx and GST [5, 16]. On
the contrary, several studies have reported unaltered or
elevated activities of antioxidant enzymes and GSH in
DMD compared to controls [9, 14, 16, 44]. The decreased
activity of the antioxidant enzymes, GPx and GST shown
in our study is in accordance with a recent report [45]
which demonstrated the inhibition of these enzymes by
reactive oxygen and carbonyl species in vitro. Although
chronic oxidative stress was evident in DMD, Ragusa et al.
[14] have suggested that it may not be the chief pathogenic
mechanism. However, our data clearly showed increased
oxidative damage, GSH depletion and decreased antioxidant function in DMD and Sgpy. This study is unique in
that it has tried to correlate the oxidative damage and
BSO
BSO+CTX
(10 µM)
antioxidant function with the severity of the dystrophic
pathology.
Compared to DMD, there are limited reports linking
oxidant and antioxidant markers with LGMD. Calpain-3
deficiency in the muscle might cause mitochondrial
abnormalities leading to oxidative stress and energy deficit
ultimately leading to pathology in calpainopathy [46].
Dioszeghy et al. [47] demonstrated that lipid peroxidation
and SOD activity were not significantly altered in erythrocytes and muscle samples from patients with LGMD
compared to controls. However, lipid peroxidation in the
muscle from patients increased with the age of the patients
and the duration of the disease. Treatment of the mouse
model of Dysfy with antioxidants decreased the dystrophic
markers and enhanced tissue integrity [48].
GSH depletion and oxidative stress have been associated
with brain aging and different diseases including neurodegenerative diseases etc. [49]. In many cases, it is not
clear whether upstream processes contribute to GSH
depletion and oxidative damage or these events by themselves trigger cell damage. On the other hand, any
123
896
A
B
0
1
2
5
7.5
0
10
1
BSO (µ
µM)
C
5
7.5
10
D
GSH-differentiated Cells
Total GSH
(% control)
Total GSH
(% control)
Control
BSO
CTX
Control
BSO+CTX
BSO
CTX
BSO+CTX
(10 µM) (1 µM)
(10 µM) (2 µM)
E
2
BSO (µM)
GSH-Undifferentiated Cells
F
ROS-differentiated Cells
AFU/cell)
ROS-Undifferentiated Cells
Control
BSO
(10 µM)
contribution from oxidative damage to the dystrophic
pathology cannot be ruled out. In MDs, interaction between
the primary genetic mutation and free radical production
has been proposed to contribute to the pathology (reviewed
by Tidball and Wehling-Henricks [50]). Accordingly, free
radical dynamics in the muscle could be altered by dystrophin deficiency in DMD via the proposed three mechanisms ultimately contributing to pathology: Firstly, altered
production of free radicals can disrupt signaling pathways
thus promoting muscle pathology. Next, in response to
dystrophic pathology, muscle cells could alter the oxidant
production which can further promote muscle damage.
Thirdly, differences in individual responses and behavior
could alter the generation and dynamics of free radicals in
the muscle. However, the specific processes linking genetic
mutations with oxidative damage are not completely
delineated. In this study, we observed that the dystrophic
pathology in DMD might decrease the total GSH via
lowered GCL activity by undefined mechanisms (Fig. 4a,
123
GSH-differentiated Cells
Total GSH
(% control)
Total GSH
(% control)
GSH-Undifferentiated Cells
AFU/cell)
Fig. 7 Effects of CTX and
BSO exposure on total GSH and
ROS in C2C12 muscle cell line.
C2C12 cells (undifferentiated
and differentiated) were
exposed to BSO (24 h) and
CTX (40 h) either alone or in
combination followed by
assessment of GSH content and
ROS concentration. a and
b dose dependent GSH
depletion in undifferentiated
and differentiated C2C12 cells
by BSO (0–10 lM; 24 h); 100%
GSH = 1.5 lg/mg protein both
in undifferentiated and
differentiated cells. c and
d Total GSH level in C2C12
cells exposed to BSO alone or
CTX alone or BSO for 24 h
followed by CTX (40 h). e and
f correspond to total
intracellular ROS corresponding
to DCF fluorescence
(AFU = arbitrary fluorescence
units). In all experiments,
*P \ 0.05, **P \ 0.01 and
***P \ 0.001 compared to
controls
Neurochem Res (2012) 37:885–898
CTX
(2 µM)
BSO+CTX
Control
BSO
(10 µM)
CTX
(1 µM)
BSO+CTX
b). To substantiate our claim on the role of GSH depletion
and oxidative damage in muscle pathology, we generated
cell and animal models of CTX. The muscle pathology
observed following exposure to CTX was comparable to
human MDs which included loss of muscle cell integrity
and infiltration of neutrophils (Fig. 5a). CTX mediated
muscle pathology in vivo is an accepted model for muscle
damage/injury and dystrophy [51, 52]. CTX mediated
dystrophy correlated with GSH depletion and oxidative
stress (Fig. 5b, c). Following recovery of muscle structure
(at 14 days after CTX exposure), the total GSH and MDA
content in the CTX-injected muscles were restored to
control levels directly correlating dystrophic pathology
with GSH depletion and oxidative stress (Fig. 5). GSH
depletion might be one of the events contributing to muscle
pathology. This led us to test whether GSH depletion prior
to the introduction of the myotoxin would exacerbate its
toxicity. GSH depletion by BSO increased CTX toxicity
and this was more pronounced in the differentiated cells
Neurochem Res (2012) 37:885–898
since the GSH depletion was higher in these cells compared to undifferentiated cells (Figs. 6, 7). These data
suggest that GSH depletion could underlie the dystrophic
pathology in the human samples and drugs that boost GSH
levels could have therapeutic implications for MD. But, as
suggested earlier [50], in DMD, the interaction between
dystrophin deficiency and oxidative stress in the dystrophic
muscle is complex and may not be rescued by antioxidant
therapy alone but probably in combination with other
compounds including anti-inflammatory agents etc.
We conclude that oxidative stress is an important
mechanism among phenotypically and genotypically
comparable pathologies, and the extent of damage significantly correlates with the severity of the disease. We presume that the structural and signaling alterations due to
genetic mutations and loss of sarcolemmal proteins might
trigger the interplay of pro-oxidant and anti-oxidant
mechanisms to a variable extent. Studies on CTX cell and
animal model demonstrated that GSH depletion and oxidative damage are closely associated with dystrophic
muscle pathology. Further, GSH depletion prior to dystrophic insult might exacerbate myotoxicity with implications for MDs. On a therapeutic note, this phenomenon
could help in evolving novel therapies to offer myoprotection against oxidative damage in MD.
Acknowledgments This work was financially supported by the
Department of Science and Technology, India. RR is a senior research
fellow of the Council for Scientific and Industrial Research, India.
The authors thank all the patients and their families for the muscle
biopsies. RR and MMSB conceived the experiments. RR and NG
carried out the experiments. AN carried out the clinical analysis.
MMSB, RR and NG analyzed the data. RR and MMSB wrote the
paper.
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