Mitochondrial changes and oxidative stress in muscle pathology

1

Mitochondrial alterations and oxidative stress in an acute transient mouse model of muscle

degeneration: Implications for muscular dystrophy and related muscle pathologies*

Renjini Ramadasan-Nair1, Narayanappa Gayathri

2, Sudha Mishra

3, Balaraju Sunitha

1,2,

Rajeswarababu Mythri1, Atchayaram Nalini

4, Yashwanth Subbannayya

5, Hindalahalli

Chandregowda Harsha5, Ullas Kolthur-Seetharam

6 and Muchukunte Mukunda Srinivas

Bharath1@

From the Departments of

1Neurochemistry,

2Neuropathology,

3Biophysics and

4Neurology,

National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore-560029, Karnataka, India

5Institute of Bioinformatics, Whitefield, Bangalore-560066, Karnataka, India

6Division of Biological Sciences, Tata Institute of Fundamental Research, Mumbai-400005, India

*Running title: Mitochondrial changes and oxidative stress in muscle pathology

@

To whom correspondence should be addressed: M.M. Srinivas Bharath, Department of Neurochemistry,

NIMHANS, No. 2900, Hosur Road, Bangalore-560029. Tel: +91-80-26995167. Fax: +91-80-26564830.

Email: bharath@nimhans.kar.nic.in

Key words: Muscle pathology; cardiotoxin; mitochondria; oxidative stress; proteomics

Background: Human muscular dystrophies and

inflammatory myopathies share common

pathological events.

Results: The cardiotoxin (CTX) model displayed

acute and transient muscle degeneration and all the

cellular events usually implicated in human muscle

pathology.

Conclusion: Mitochondrial alterations and

oxidative stress significantly contribute to muscle

pathogenesis.

Significance: The CTX model is valuable in

understanding the mechanistic and therapeutic

paradigms of muscle pathology.

ABSTRACT

Muscular dystrophies (MDs) and

inflammatory myopathies (IMs) are debilitating

skeletal muscle disorders characterized by

common pathological events including

myodegeneration and inflammation. However,

an experimental model representing both

muscle pathologies and displaying most of the

distinctive markers has not been characterized.

We investigated the cardiotoxin (CTX)-

mediated transient acute mouse model of

muscle degeneration and compared the

cardinal features with human MDs and IMs.

The CTX model displayed degeneration,

apoptosis, inflammation, loss of sarcolemmal

complexes, sarcolemmal disruption and

ultrastructural changes characteristic of human

MDs and IMs. Cell death caused by CTX

involved calcium influx and mitochondrial

damage both in murine C2C12 muscle cells and

in mice. Mitochondrial proteomic analysis at

the initial phase of degeneration in the model

detected lowered expression of 80

mitochondrial proteins including subunits of

respiratory complexes, ATP machinery, fatty

acid metabolism and Krebs cycle, which further

decreased in expression during the peak

degenerative phase. The mass spectrometry

(MS) data was supported by enzyme assays,

western blot and histochemistry. CTX model

also displayed markers of oxidative stress and

lowered glutathione reduced/oxidized ratio

(GSH/GSSG) similar to MDs, human

myopathies and neurogenic atrophies. MS

analysis identified 6 unique oxidized proteins

from Duchenne muscular dystrophy (DMD)

http://www.jbc.org/cgi/doi/10.1074/jbc.M113.493270The latest version is at JBC Papers in Press. Published on November 12, 2013 as Manuscript M113.493270

Copyright 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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samples (n=6) (vs. controls; n=6), including two

mitochondrial proteins. Interestingly, these

mitochondrial proteins were down-regulated in

the CTX model thereby linking oxidative stress

and mitochondrial dysfunction. We conclude

that mitochondrial alterations and oxidative

damage significantly contribute to CTX-

mediated muscle pathology with implications

for human muscle diseases.

Muscular dystrophies (MDs) and

inflammatory myopathies (IMs) are neuromuscular

disorders arising due to muscle-intrinsic defects.

MDs are clinically and genetically heterogeneous,

characterized by progressive degeneration of the

skeletal muscle (1). Duchenne muscular dystrophy

(DMD), Dysferlinopathy (Dysfy) and

Sarcoglycanopathy (Sgpy) are common MDs with

DMD being the most devastating condition that

culminates in premature death (2). Various

molecular processes downstream of the genetic

mutations in these MDs, lead to dystrophic

pathology (2,3). Conversely, IMs are mostly

sporadic, characterized by intense and acute

inflammation and necrosis (4). Although most

MDs have established methods of diagnosis, most

of them are not treatable since the molecular basis

of muscle degeneration is not completely

understood. On the other hand, IMs are treatable

disorders, with good clinical outcome.

Interestingly, muscle damage in MDs and IMs

involve common pathways including cycles of

myofiber necrosis/apoptosis, regeneration and

inflammation (4,5). Muscle degeneration in these

conditions in general involves cellular events

including sarcolemmal disruption, altered

cytoskeletal network, aberrant calcium dynamics,

oxidative stress and mitochondrial damage (2,5,6).

But, the interplay of these processes, their

chronology and contribution to degeneration and

the molecular mediators involved are not well

elucidated. This is partially due to the absence of

reliable models that recapitulate all the molecular,

pathological and symptomatic features common to

MDs and IMs.

MDs have been extensively studied using

patient muscle biopsies (6,7) and genetic models

(8). However, the models do not replicate the

human condition due to species barrier, altered

chronology of events and variable severity of

muscle pathology (2). Further, a single model that

displays all the cellular events common to MDs

and IMs, exhibiting transient degeneration

followed by active regeneration and displaying

secondary processes such as inflammation and

fibrosis, needs to be characterized for mechanistic

and therapeutic assessment.

Non-genetic transient animal models that

simulate myopathic pathology could be useful to

study the pathogenesis. In this regard, two models

that display muscle damage/degeneration are the

cardiotoxin (CTX) (9) and bupivacaine (10)

models. However, the implications of the CTX

model for human MD and other myopathies and

the role of oxidative stress, calcium dynamics and

mitochondrial dysfunction have not been clearly

documented. In our study, we have characterized

the muscle damage, secondary mechanisms and

necrotic pathways mediated by CTX in vivo and in

vitro compared with human MDs and IMs and

investigated the oxidative and mitochondrial

changes involved in muscle degeneration.

EXPERIMENTAL PROCEDURES

All chemicals and solvents were of

analytic grade. Bulk chemicals and solvents were

obtained from Merck and Sisco Research

Laboratories Pvt. Ltd. (Mumbai, India). Fine

chemicals, PCR primers and consumables, tissue

culture materials, CTX from Naja mossambica

mossambica, protease inhibitor cocktail, Nagarse

and -dinitrophenyl (DNP) antibodies were

obtained from Sigma. Proteomic grade trypsin was

obtained from Promega. Fura-2 AM, Rhod-2 AM

and calcein were procured from Molecular Probes.

Tissue culture grade trypsin was obtained from

Loba Chemie (Mumbai, India). Antibodies against

Dystrophin (1,2 and 3), Sarcoglycans ()

and Dysferlin were procured from Novocastra

Laboratories Limited (NCL) (Newcastle Upon

Tyne, UK). Horseradish peroxidase (HRP)

conjugated secondary antibodies were obtained

from NCL and Bangalore Genei. IPG strips,

Coomassie Brilliant Blue G-250 and SYPRO Ruby

were obtained from Bio-Rad Laboratories.

Nitrocellulose membrane was obtained from

Millipore. Fetal Bovine Serum from Gibco Life

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Technologies and horse serum from PAN Biotech

(GmbH, Germany) were procured. RT-PCR

consumables were obtained from Roche

Diagnostics and Invitrogen. Antibody cocktail

against mitochondrial proteins and ATP:ADP

quantitation kit were obtained from Abcam.

Reagent kits to assay pyruvate, lactate, aspartate

aminotransferase and mitochondrial creatine

kinase (CK) were obtained from Grenier Bio-One

GMBH (Frickenhausen, Germany). Amplex-red

assay kit, dihydroxyethidium, BODIPY® 581/591

undecanoic acid and 4-Amino-5-Methylamino-

2’,7’-Difluorofluorescein-diacetate (DAF-FM-DA)

were obtained from Invitrogen-Life technologies.

Human tissue samples

Patients (n=103; Age=1.1-65 y) with

muscle diseases evaluated at the Neuromuscular

Disorders clinic, NIMHANS, Bangalore, India

during 2006-2012 were selected following

diagnostic procedures. The muscle strength of

patients [based on the Medical Research Council

scale(11)], was recorded by the neurologist and

graded 0 to 5 (Supplementary material). After

obtaining written informed consent, skeletal

muscle biopsies from these patients were

diagnosed by histopathology. The study included

immunohistochemically confirmed cases of

Duchenne muscular dystrophy (DMD) (n=15),

Dysferlinopathy (Dysfy) (n=15),

Sarcoglycanopathy (Sgpy) (n=15) and clinically

and histologically confirmed cases of spinal

muscular atrophy (SMA 1,2 and 3) (n=15), IM

(n=15), distal myopathy (Nonaka type) (n=15) and

mitochondrial myopathy (n=13). As control,

paraspinal muscles from patients (n=12)

undergoing spinal surgeries were procured after

obtaining written informed consent. The study

protocol was approved by the Institutional Ethics

Committee. Fresh biopsy samples obtained as

explained previously (12) were snap-frozen in

isopentane pre-cooled in liquid nitrogen and stored

at -80 0C and used for histopathological and

biochemical studies.

Animal studies

Experiments were carried out according to

the Institutional guidelines for the Care and Use of

Laboratory Animals. The study protocol was

approved by the Institutional animal ethics

committee. Adult male C57BL/6 mice (10 week-

old; ~30g each; n ≥ 6 per treatment) maintained

under standard conditions were injected either with

saline or CTX (single injection; 300 l of 10M in

saline) across the tibialis anterior (TA) muscle on

one of the hind limbs as described previously (9).

CTX was uniformly released along the muscle

tissue by injecting the myotoxin while

withdrawing the syringe needle (13). Mice were

injected with Evans Blue dye (EBD) (indicator of

muscle injury) (10 mg/kg b.w. i.p., 24 h before

CTX/ saline injection). The mice were euthanized

1, 2, 3, 5, 7, 11, 14 and 31 days after the CTX

injection, and the muscle from the ipsilateral and

contralateral limbs were dissected. A fragment of

the muscle oriented transversely and snap-frozen

in isopentane pre-cooled in liquid nitrogen was

used for enzyme and immunohistochemistry

(IHC), while another portion was snap-frozen in

liquid nitrogen for RT-PCR and biochemical

assays. Tiny pieces were fixed in 3%

glutaraldehyde for electron microscopy (EM) and

the rest of the tissue was fixed in 10% formalin for

histopathology.

Grip strength test

Grip strength of the mouse limbs was

analyzed by measuring the maximum force exerted

(in Kg) on a Grip Strength Meter (Columbus

Instruments, Ohio, USA) (14) in tension mode,

using the grid assembly of the meter. The readings

from 6 trials were averaged.

Histopathology

Cryosections [8 thick transverse

sections of frozen muscle cut in a cryostat (Leica)

at -20o C] were subjected to Haematoxylin & Eosin

(H&E) staining, Modified Gomori’s Trichrome

(MGT) staining, enzyme histochemistry (EHC)

[Nicotinamide adenine dinucleotide tetrazolium

reductase (NADH-TR), Succinic dehydrogenase

(SDH), Succinic dehydrogenase-cytochrome

oxidase (SDH-COX)] and IHC (Dystrophin,

Dysferlin and -sarcoglycan). For EBD

fluorescence analysis (excitation= 470 and 540

nm; emission= 680 nm), the cryosections were

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mounted on cover-slips with buffered glycerol and

subjected to confocal microscopy (DMIRE-TCS,

Leica). Paraffin sections (tissue fixed in 10 %

formalin, grossed, sequentially dehydrated,

embedded in paraffin and sectioned in a

microtome) were subjected to H&E, Masson’s

Trichrome (MAT) and Alizarin red staining (15).

The tissues fixed in 3% glutaraldehyde were

processed for EM as described previously (15).

Cell culture and cell viability

C2C12 mouse myoblast cell line was

grown as described previously (13).

Differentiation was confirmed by the cellular

morphology and multinucleation and creatine

kinase (CK) assay (16) using a commercial kit

(Olympus Life and Material Science, Europa,

GmbH). Cells were seeded in 96-well plates (5 X

103/well) for cell viability, oxidative stress and

mitochondrial membrane potential assays and in

90 mm dishes (2X105 cells /dish) for biochemical

and Ca2+

assays (13). Cells were harvested,

centrifuged (850Xg, 1 min) and the pellet was

resuspended in 1X phosphate buffered saline

(PBS), pH 7.4, containing protease inhibitors. The

cell suspension was sonicated (5sX 4) on ice

(Sonics and Materials Inc., CT, USA) and

centrifuged (15,000Xg, 10 min, 4oC). The

supernatant was utilized for biochemical assays

after protein estimation (17). Cell viability was

measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide (MTT) assay (13).

Estimation of cellular and mitochondrial Ca2+

levels

Cells (105) seeded on 22 mm cover-slips

were loaded with 5 μM Fura-2-acetoxymethyl

ester (Fura-2AM; a cell permeant Ca2+

sensitive

fluorescent dye; excitation= 340/380nm,

emission= 510nm) and 0.08% pluronic acid for 45

min at 37 C in HEPES buffer (20mM HEPES

130mM NaCl, 5.4mM KCl, 0.8mM MgCl2,

1.8mM CaCl2 and 15mM glucose, pH 7.4). The

cells were washed with HEPES buffer and

incubated in dark for 15 min. The cover-slip was

mounted on a fluorescence microscope (Olympus

IX 70) and the ratiometric fluorescence imaging

was performed (TILL Photonics, Germany). Fields

containing at least 25-30 cells were selected and

alternately excited at 340 and 380 nm and the

emitted fluorescence was collected using band

pass filter and fluorescence images were acquired

by a 12-bit peltier cooled CCD camera.

Ratiometric quantitation of 340/380 intensity

analyzed using TILL Vision software is a measure

of intracellular Ca2+

levels (18). CTX (1or 2M)

and antagonist (nifedipine at 10M) (± 2 mM

EGTA) were added to the static bath during

experiments. All the EGTA experiments were

carried out in Ca2+

-free HEPES buffer.

The mitochondrial Ca2+

levels were

measured as described previously (19) using

Rhod-2AM, a mitochondrial Ca2+

sensitive probe.

Cells grown on cover-slips were loaded with

Rhod-2AM (2 M) for 30 min in HEPES buffer at

35oC and washed with HEPES buffer. The regions

of interest were taken from the perinuclear

mitochondrial rich areas exhibiting punctate

loading of Rhod-2. The cells were excited at

530nm and the emission collected through a band

pass filter. The fluorescence intensity change is

represented as arbitrary units (a.u). CTX was

added to static bath and the fluorescence images

were monitored for 30min at room temperature.

The dependence of Rhod-2 fluorescence on Ca2+

influx was determined by incubating the cells with

CTX ± 2 mM EGTA, followed by quantitation of

fluorescence in a fluorimeter (Tecan).

To determine the membrane damage, cells

grown in 60mm dishes to 70% confluency were

loaded with 5 M calcein AM for 45 min at 37 C

in HEPES buffer (20). The cells were washed

thrice with HEPES buffer and incubated with CTX

(2M) for 0-30 min. Later, the emission spectra of

the culture supernatant were recorded at different

time points (0.5, 2, 5, 10 and 30 min) at 500-

600nm using excitation maximum of 480nm.

Study of mitochondrial membrane potential (m)

C2C12 cells (5 X 103) in 96-well plates

were treated with 20 M of 5,5',6,6'-tetrachloro-

1,1',3,3'-tetraethyl-benzamidazolo carbocyanin

iodide (JC-1) [a lipophilic fluorescent dye that

selectively accumulates in mitochondria and

responds to changes in m by forming J

aggregates from monomers (21)] and incubated for

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30 min at 37 oC in dark. JC-1 loaded cells were

washed twice with PBS (± 2mM EGTA) and

fluorescence was measured (excitation= 490 nm;

emission= 535 &590 nm, corresponding to

monomer and aggregate, respectively) in a plate

reader (Tecan) in the kinetic mode (21). Three

readings were taken at 1 min intervals after which

CTX was added and fluorescence monitored for 30

min.

Analysis of reactive oxygen species (ROS) in cells

Total ROS in C2C12 cells was measured

using Amplex® Red assay kit (Invitrogen-life

technologies) according to the manufacturer’s

instructions. Cells in 96-well dishes were loaded

with Amplex® red reaction mixture and exposed

to CTX and the fluorescence corresponding to

ROS was recorded in a fluorimeter (Tecan)

(excitation=540nm; emission= 590nm). For

detection of cellular oxidants, cells loaded with

dihydroethidium (DHE) were exposed to CTX for

1 h, followed by detection of fluorescence in a

multi-well fluorimeter (Excitation= 518nm;

Emission= 606nm) (22). Quantitation of the higher

oxides of nitric oxide (NO) in C2C12 cells was

carried out using the compound DAF-FM-DA as

described previously (23). Cells loaded with 5M

DAF-FM-DA were exposed to CTX followed by

quantitation of fluorescence (Excitation= 495nm;

Emission= 515nm).

Preparation of whole muscle extracts

Total soluble extract from frozen tissue

(50 mg) was prepared (13) followed by protein

estimation (17).

Preparation of mitochondria

Muscle mitochondria were prepared by the

method modified from Bhattacharya et al (24).

Muscle tissue (100 mg) was minced and incubated

with 10 % Nagarse in ionic medium (100 mM

Sucrose, 10 mM EDTA, 100 mM Tris-HCl, 46

mM KCl, pH 7.4) for 5 min, washed with ionic

medium containing 0.5% BSA, homogenized and

centrifuged (500 Xg, 10 min; 4oC). The

supernatant was centrifuged (12,000 X g, 10 min;

4oC), and the partially purified mitochondrial

pellet was washed twice with ionic medium

containing 0.5% BSA and the final pellet was

stored at -80oC in suspension medium (230 mM

mannitol, 70 mM sucrose, 0.02 mM EDTA, 20

mM Tris-HCl, 5 mM K2HPO4, pH 7.4).

Assay of mitochondrial complexes I (CI), II (CII)

and III (CIII)

CI activity was assayed as described (25)

and rotenone-sensitive specific activity was

calculated. CII and CIII were assayed using

methods described previously (25).

Citrate Synthase (CS) assay

The assay was initiated by adding 10 mM

oxaloacetate (20 μl) to the reaction mixture

containing 100 mM Tris–HCl (pH 8.1), 0.2 mM

DTNB, 0.1 % Triton X-100, 0.1 mM acetyl-CoA

and 5μg protein and the reaction was monitored at

412 nm (26). CS activity was expressed as nmol

DTNB/min/mg protein (MEC=13.6 mM-1

cm-1

).

Aconitase assay

Aconitase was assayed as previously

described (27). The assay mix containing

mitochondrial protein (40 g), 100 mM Tris-HC1

(pH 7.6) and 500 M cis-aconitate was incubated

at room temperature and the enzyme activity was

monitored at 240 nm for 15 min. Enzyme activity

was calculated using extinction coefficient=3.6

mM-1

cm-1

for cis-aconitate and normalized per mg

protein.

Aspartate aminotransferase and creatine kinase

(CK) assays

These assays were carried out using

commercial kits (Grenier Bio-One GMBH). Total

mitochondrial extract (300 g) from CTX-injected

muscle at d1, d3 and d7 and control were subjected

to assays in triplicate, based on the manufacturer’s

instructions.

Estimation of pyruvate, lactate, NADH and

ADP/ATP ratio

Total pyruvate and lactate in the muscle

extract was estimated by commercial kits (Grenier

Bio-One GMBH) according the manufacturer’s

instructions. Muscle tissue (50 mg) from control

and CTX-injected animals was extracted in PBS

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and 10l of the total extract was subjected to the

assays and normalized per mg protein.

ADP/ATP ratio in the muscle extract was

determined using commercial kit (Abcam)

according to the manufacturer’s instructions.

NADH was estimated according to the

method described previously (28). Briefly, NADH

was extracted from the muscle homogenate by

adding 0.1N NaOH and neutralized by adding

HCl/ Bicine to a final concentration of 0.33M. To

350µl of neutralized extracts, 310µl of reaction

buffer (0.232M Bicine, 2mg/ml BSA, 1.16M

ethanol, 9.67mM EDTA, 3.87mM phenazine

ethosulphate and 1mM MTT) was added and

incubated at 370C for 5 min. Then cycling assay

was initiated by the addition of 60µl of 0.6mg/ml

alcohol dehydrogenase. After 30 min, the reaction

was terminated by adding 300µl of 12mM

iodoacetate and the absorbance was recorded at

570nm. Total NADH was normalized to the total

protein content.

SDS PAGE and western blot

Total muscle extract (50 g/lane) was

resolved on 12 %SDS PAGE followed by either

coomasssie staining of the gel or western blot (12)

using antibody cocktail against five specific

mitochondrial proteins (Abcam) according to the

manufacturer’s instructions.

Estimation of total glutathione (GSH+GSSG),

GSH:GSSG ratio and lipid peroxidation

Total glutathione (GSH+GSSG) assay was

carried out as described previously (13).

Alternately, GSH:GSSG ratio was determined by

the o-pthalaldehyde (OPT) method as described

earlier (29). The muscle extract (10 g) was pre-

cleared with 5% Sulfosalicylic acid and

centrifuged (10,000 Xg, 10 min @ 40C). The

supernatant was treated OPT (1mg/ml; 10 l) for

20 min and the fluorescence corresponding to

GSH (excitation= 350nm; emission= 420nm) was

measured and compared to standards. For

measuring GSSG, the cleared supernatants were

incubated with 5l of N-ethyl maleimide for 15

min, followed by addition of 0.15N NaOH

(140l). The mixture was treated with OPT

followed by fluorescence measurements.

Lipid peroxidation was quantitated by

thiobarbituric acid reactive substances (TBARS)

assay as described earlier (13). Alternately, the

extract (10 l) was incubated with the fluorescent

lipid peroxidation sensor 4,4-difluoro-5- (4-

phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-

indacene-3-undecanoic acid (BODIPY® 581/591

undecanoic acid; Invitrogen-Life technologies) and

the loss of fluorescence upon interaction with

peroxyl radicals was estimated (30) and

normalized per mg protein.

Determination of protein carbonylation (oxyblot)

Tissue supernatant (10 l at 4 mg/ml

protein) was derivatized with dinitrophenyl

hydrazine (DNPH), spotted on a nitrocellulose

membrane and subjected to anti-DNP western

(13). Anti-DNP signal captured in a gel

documentation system (BioRad, Model: 2000TM

)

was densitometrically quantified (Quantity One

software version 4.2.2, BioRad).

Two dimensional gel electrophoresis (2D-PAGE)

and western blot

Muscle tissue homogenate (10%) was

prepared by a method modified from Sultana et al

(31) in HEPES buffer (10 mM HEPES pH 7.4, 137

mM NaCl, 4.6 mM KCl, 1.1 mM KH2PO4, 0.6

mM MgSO4) containing protease inhibitors. The

extract was sonicated (10 s X 3), centrifuged

(10000 X g, 10 min) and the soluble protein (300

g) was incubated in 4 volumes of 2N HCl (±10

mM DNPH) for 20 min at room temperature. The

sample was precipitated with tricholoroacetic acid

(final concentration= 30%) and centrifuged

(10,000Xg, 5 min). The pellet was washed thrice

with ice-cold ethanol:ethyl acetate (1:1) and

resuspended in 2D lysis buffer (200 l) [8M Urea,

2M thiourea, 2% CHAPS, 0.4 % ampholytes (pH

3-10), 50 mM DTT] and incubated at room

temperature for 2 h. The lysate was centrifuged

(10,000Xg, 10 min) and the supernatant (180 l)

was loaded onto IPG strips (11 cm, pH 3-10) in the

rehydration tray, overlaid with ~800μl mineral oil

and allowed for overnight rehydration. The strips

were subjected to isoelectrifocussing in the IEF

cell (BioRad) as follows: Step 1: 25 V/ 20 min

(linear ramp); Step 2: 8000 V/ 2.5 h (linear ramp);

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Step 3: 8000 V for 25,000 V-h (rapid ramp). Strips

were incubated in equilibration buffer [50 mM

Tris-HCl, 6 M urea, 1% (m/v) SDS, 30% (v/v)

glycerol and freshly prepared DTT (0.5%)] for 10

min and then in equilibration buffer containing

iodoacetamide (4.5%) for 10 min. The equilibrated

gels were washed in SDS-PAGE running buffer

and electrophoresed on 10 % SDS gel (2nd

dimension). SDS gels were placed in fixative

solution (50 ml) [10% (v/v) methanol and 7% (v/v)

acetic acid] for 1 h at room temperature and

stained with SYPRO Ruby stain (50 ml) (2h at

room temperature), destained and the images

captured in the gel documentation system (Biorad)

and analyzed by Quantity One software (BioRad)

(31).

Proteins from SDS PAGE were transferred

on to a nitrocellulose membrane and subjected to

anti-DNP western blot (13).

In-gel tryptic digestion and peptide extraction

Spots on the SYPRO Ruby stained 2D

gels that corresponded to anti-DNP western signal

were manually excised and subjected to in-gel

tryptic digestion (32,33). Gel pieces were minced

and washed with 500 μl of wash solution (50%

acetonitrile and 40 mM Ammonium Bicarbonate)

at room temperature repeatedly with gentle

agitation (15 min/ wash) until they were

completely destained. The gel pieces were

dehydrated in 100% acetonitrile for 5 min. The gel

pieces were first rehydrated in 150 μl reduction

solution (5 mM DTT in 40 mM Ammonium

bicarbonate) for 45 min at 60C and incubated

with 100 μl of alkylation solution (20mM

Iodoacetamide in 40mM Ammonium bicarbonate)

for 10 min in the dark at room temperature. The

gel pieces were dehydrated in 100 μl 100%

acetonitrile for 5 min. Gel pieces were then

incubated in 30 μl protease digestion solution (20

μg of sequencing grade trypsin reconstituted in 2

ml of 50 mM ammonium bicarbonate) on ice for

30 min. Once the gel pieces were rehydrated with

protease digestion solution, excess trypsin was

removed and substituted with 40 mM ammonium

bicarbonate and incubated overnight at 37 C. It

was then centrifuged (12,000 g for 30 s) and the

supernatant (containing tryptic peptides) was

transferred to a sterile tube. The gel pieces were

re-extracted with 25-50 μl of extraction solution

(50 % acetonitrile/5 % formic acid). The extracted

peptides were dried in a vacuum evaporator and

reconstituted in 20 µl of 0.1 % Trifluoro acetic

acid (TFA) and subjected to tandem mass

spectrometric analysis (LC-MS/MS).

LC MS/MS and database analysis

LC-MS/MS analysis of tryptic peptides

was carried out on aLTQ orbitrap Velos Fourier

Transform mass spectrometer. Peptides from each

gel piece were resolved by reversed-phase liquid

chromatography (RP-LC) interfaced with a mass

spectrometer using an RP-LC system consisting of

a column (75 µ m × 2 cm, C18 material 5 µm, 100

Å) and an analytical column (75 µ m × 10 cm, C18

material 5 µ m, 100 Å) with a nanoflow solvent

delivery and electrospray source fitted with an

emitter tip (8µm) and maintained at 2 kV ion spray

voltage. Peptides samples (20 l) were loaded onto

the trap column in 0.1% formic acid and 5%

acetonitrile. The peptides were resolved on an

analytical column using a linear gradient of 7-30%

acetonitrile and 0.1% formic acid over 80 min with

a flow-rate of 350 nL/min. The eluted peptides

were subjected to MS analysis on LTQ orbitrap

Velos. From each precursor scan, ten most

abundant ions in the scan range of m / z 350 - 1800

were subjected to MS/MS fragmentation(34).

The MS/MS data was searched against the

NCBI Ref Seq (release 52) human protein

database containing 30,083 protein sequences and

common contaminants. The parameters selected

include (i) trypsin as specific enzyme with

maximum of 1 missed cleavage permitted (ii)

fixed post-translational modification was

carbamidomethylation of cysteine residues (iii)

oxidation of methionine was set as variable

modification (iv) precursor ion mass range @ 400-

8,000 Da (v) precursor and fragment ions mass

tolerance of 20 ppm and 0.1 Da, respectively. The

peptide and protein data were extracted using high

peptide confidence with target FDR threshold set

to 1% at the peptide level.

mRNA Isolation and real-time PCR

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All solutions and buffers were prepared in

RNase free Diethyl pyrocarbonate (DEPC) treated

water (0.01%). Muscle tissues were lysed in Trizol

reagent (1 ml), mixed with 200 l of chloroform

and vigorously shaken (15-30 sec). The lysate was

centrifuged (12,000 x g, 15 min at 4 oC) and the

supernatant was mixed with isopropanol (500 l)

and incubated for 10 min. The mixture was

centrifuged (12,000 x g, 10 min at 4 oC) and the

RNA pellet was washed with 75% Ethanol, dried

and suspended in DEPC-treated water (35).

cDNA synthesis was carried out using Superscript

III RT kit (Invitrogen) using specific primers

(Supplementary material).The RNA and primer

mix were denatured at 65oC for 10 min and snap

chilled. To this mix, 4 l of the 5X buffer, 2 l of

0.1M DTT, 1l of 10 mM dNTP mix, 0.5 l of

RNase Inhibitor (Roche) and 0.5l of SSIII

Reverse Transcriptase was added and mixed. This

reaction was incubated first at room temperature

(10 min) and then at 50oC (50 min). The reaction

was stopped by heat denaturation at 75oC (15 min).

Real time PCR was carried out in the Light Cycler

480 II (Roche) (35). The quantitation of the

expression of all the genes was normalized to

alpha-actin 1.

Mitochondrial Proteomics

Mitochondria from muscle tissue [Saline-

sal; day 1 CTX-d1;day 3 CTX-d3; day 7 CTX-d7]

were resuspended in deionized water and sonicated

(30s) and the protein concentration was estimated

(36). Equal protein (250g) from each sample was

reduced by Tris (2-carboxyethyl) phosphine

(TCEP) at 60 °C for 1 h (37). The samples were

then treated with the cysteine blocking reagent

(methyl methane thiosulfonate) for alkylation, and

incubated at room temperature for 10 min. Trypsin

digestion was carried out with sequencing grade

modified trypsin (Promega) at 370C for 16h and

the tryptic digests were evaporated to dryness.

Peptides from sal, d1,d3 and d7 were labeled with

iTRAQ reagents containing 114, 115, 116 and 117

reporter ions respectively. Since our study focused

on the degenerative phase, data for d1 and d3

compared with sal are presented in the results

section.

The labeled samples were pooled,

reconstituted using 10 mM potassium phosphate

buffer containing 30% Acetonitrile pH 2.7 (solvent

A) and subjected to strong cation exchange (SCX)

chromatography (37). SCX fractionation was

carried out on a Poly-sulfoethyl A column (Poly

LC, Columbia, MD) (300 Å, 5 µm, 100 × 2.1 mm)

with an Agilent 1200 HPLC system (Agilent

Technologies, Santa Clara, USA) with a binary

pump, UV detector and a fraction collector. A

linear gradient composed of 0-35% 10 mM

potassium phosphate buffer containing 30% ACN,

350 mM KCl, pH 2.7 (solvent B) at a flow rate of

200 µl/ min was used for peptide separation. The

fractions obtained were dried, reconstituted in

0.1% TFA and desalted using C18 stage-tips. The

desalted samples were subjected to LC-MS/MS

analysis.

For LC-MS/MS analysis, the iTRAQ

labeled samples were analyzed on LTQ-Orbitrap

Velos mass spectrometer (Thermo Electron,

Bremen, Germany) interfaced with Proxeon Easy

nLC system (Thermo Scientific, Bremen,

Germany) (37). Peptides were first loaded on to an

enrichment column (2cm x 75μm) and further

resolved on analytical columns (10 cm x 75 μm)

packed in-house with Magic C18 reverse phase

material (Michrom Biosciences Inc Magic

C18AQ, 5m, 100Å) prior to introducing the

peptides into mass spectrometer. The peptide

enrichment was carried out at a flow rate of

3l/min while the peptides were resolved in the

analytical column at a flow rate of 350 nl/min

employing a linear gradient of 7-30% ACN over

80 min. Data-dependent MS analysis was carried

out by acquiring full scans in the Orbitrap mass

analyzer between mass range of 350-1800 at a

mass resolution of 60,000 at 400 m/z. Top twenty

precursor ions from each survey scan were

selected for MS/MS fragmentation and the

fragment ions were acquired at a mass resolution

of 15,000 at 400 m/z. Fragmentation was carried

out using higher-energy collision dissociation

(HCD) mode with normalized collision energy of

41%. Isolation width was set to 2 m/z. The ions

selected for fragmentation were dynamically

excluded for 30s. The automatic gain control for

full FT MS was set to 1 million ions and for FT,

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MS/MS was set to 5 × 104 ions. The lock mass

option was enabled using poly dimethyl cyclo

siloxane (m/z, 445.1200025) ion for accurate mass

measurement.

MS/MS data was searched against mouse

RefSeq56 database (containing 26,707 sequences

with common contaminants) using Sequest

algorithm in Proteome Discoverer 1.3.0.339. The

search criteria included oxidation of methionine as

dynamic modification and iTRAQ 4-plex

modification at peptide N-terminus and Lysine (K)

and methyl thio modification of cysteine as static

modifications. A precursor mass tolerance of 20

ppm, fragment mass tolerance of 0.1 Da and 1

missed cleavage site were allowed for the

searches. The data was searched against the decoy

database and a target FDR of 1% was employed

for protein identification. The relative quantitation

was carried out using the reporter ions quantifier

node of Proteome Discoverer.

Statistical Analyses

Quantitative data represented by bar and

line graphs (excluding the MS 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

Characterization of a mouse model of muscle

degeneration

We characterized an acute transient model

of muscle degeneration and compared its cardinal

pathological features with human MDs and IMs.

We utilized snake cardiotoxin (CTX; 60 a.a) (38),

which causes cardiotoxicity when injected into the

blood, but causes tissue injury when administered

into the muscle. A single injection of 300 l CTX

(10M) was administered into the TA muscle (9)

followed by behavioral, histopathological and

inflammatory analyses at 1, 3, 5, 7, 11, 14 and 31

days (d1 to d31) after injection. Although CTX

caused immediate, but momentary paralysis in the

injected limb, the toxin did not cause any

mortality. CTX exposure did not alter the body

weight, food and water intake and other behavioral

parameters compared with controls (data not

shown).

We investigated whether CTX induced

muscle weakness is, as seen in human DMD and

IMs (Table 1; Figure1A). CTX caused complete

loss of grip strength of the injected limb from d1

up to d11. The muscle strength was restored by

~40 % at d14 and by ~90 % at d31 indicating

recovery of muscle function (Figure 1B).

The reduced muscle strength was

associated with muscle damage at d1 as indicated

by EBD fluorescence, which persisted at d3, but

decreased significantly by d7, indicating transient

muscle damage (Figure 1C). Histological analysis

(H&E staining) showed widespread damage of the

muscle fibers at d1, which persisted until d11

(Figure 1D). Muscle degeneration represented by

myofiber fragmentation and shrinkage of the

muscle cells was relatively higher at d3, consistent

with previous data (39). However, the muscle

structure significantly recovered at d14 (13), and

was comparable to the control at d31. These

histopathological features were consistent with

human DMD, Dysfy, Sgpy and IM (Figure 1E).

MAT staining of the sections indicated that the

muscle degeneration was not accompanied by

apparent fibrosis, possibly because collagen

deposition entails chronic degeneration as seen in

human MDs (Figure 1G). The recovery of muscle

architecture was concomitant with the appearance

of centrally nucleated regenerating cells (as seen in

human DMD and Sgpy; figure 1E), which was

negligible at d1 and d3, but predominant at d5 to

d14 (Figures 1E and 1F). At d7 and beyond, repair

and regeneration were evidenced by (i) centrally

nucleated cells with perinuclear glycogen granules

and (ii) fibroblasts with prominent rough ER,

engaged in active reconstruction of the

extracellular matrix. The number of fibroblasts

decreased by d11 and was not conspicuous at d14

and d31 (Data not shown). Activated satellite cells

were prominent at d7, reduced at d11 and d14 and

inconspicuous by d31 (Figure 4E-H and 6F).

Muscle degeneration in human MDs is

associated with apoptotic (40) and inflammatory

mechanisms (41,42). CTX-injected muscle

revealed infiltration of neutrophils, macrophages

and lymphocytes as seen in human DMD and

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myopathies (Figures 1D-E). Inflammatory cells

infiltrated the extracellular spaces and

phagocytosed the muscle cells at d1-d3 (Figure

4B-C), but their number decreased at d7 and

further reduced at subsequent time points (data not

shown). The degenerative changes at d1-d14 was

associated with elevated expression of apoptotic

markers (TNF-FasL and LITAF) and

inflammatory genes including cytokines

[chemokines (CCL2, CCL3 andCCL6) and

Interleukins (IL-6 and IL-1], MRC1 and

cytokine receptors (CCR1, CXCR4and CCR5)

(Figure 2). The apoptotic markers were elevated

from d1, with the highest level at d3 followed by

reduction to the control level by d14 (Figure 2A).

All cytokines showed increased expression

between d1 and d7, which decreased to the control

level at d14 (Figure 2B).The cytokine receptors,

showed a general trend with elevated expression

from d1, peaking at d3 and subsequent reduction at

d7, ultimately decreasing to the control level at

d14 (Figure 2C). However, the increase in the

relative expression varied among the different

genes (43). Up-regulation of the apoptotic and

inflammatory genes tested in the CTX model was

previously reported in human dystrophies

(Supplementary Table 1). Based on the

behavioural, histopathological and qRT-PCR data,

it could be surmised that the CTX-dependent

myotoxicity elicited all the cardinal features

observed in human pathologies and showed acute

transient degeneration at d3.

CTX-injected muscle displays sarcolemmal

disruption and ultrastructural perturbations

Muscle tissue from MD patients display

loss of sarcolemmal integrity (44), altered

cytoskeletal structure and ultrastructural

perturbations (45). The sarcolemmal integrity in

the CTX model was ascertained by the status of

the sarcolemmal complexes viz., dystrophin,

dystrophin-associated protein and dysferlin

complex (13). Loss of immunoreactivity of

dystrophin, sarcoglycan (dystrophin-associated

protein) and dysferlin (dysferlin complex) at d1-d7

indicated sarcolemmal perturbation (Figure 3A) as

observed in human DMD, Sgpy and Dysfy (Figure

3B). The restoration of immunoreactivity was

visible at d7 and was conspicuously re-established

atd14 to d31.

We investigated whether the transient loss

of immunoreactivity of the membrane proteins in

the CTX model was due to membrane disruption

(Supplementary figure 2 and Figure 4). CTX-

injected muscle displayed sarcolemmal breach

(“leakiness”) at d1 and d3 (Figure 4B-C), which

was restored at d7 (Figure 4E). CTX-mediated

sarcolemmal disruption also affected the T-system

(sarcolemmal invaginations that are continuous

with the sarcoplasmic reticulum). CTX caused

formation of tiny vesicles, predominantly at d3

(Figure 4C), probably due to the pinching-off of

the damaged sarcolemma, thus destabilizing the T-

system. CTX disrupted the cytoskeletal

architecture at d1 and d3, as indicated by complete

homogenization of the myosin-actin filaments

(Figure 4B-C). The ultrastructural alterations in

the CTX model were similar to the features

observed in the muscle biopsies of human MDs

(Figure 4D).

Altered calcium dynamics following CTX

administration in vivo and in vitro

CTX-mediated sarcolemmal disruption

consistent with human pathologies could alter the

intracellular Ca2+

dynamics. CTX injected muscle

stained positively for alizarin red at d1 and d3

indicating intracellular accumulation of Ca2+

, as

seen in human DMD (Figure 5A) (40). We

investigated the Ca2+

dynamics in the C2C12

myoblast cell line (16). CTX induced time-

dependent myotoxicity with LD50 of 2 M and 1

M at 40 h in undifferentiated and differentiated

cells respectively (Figures 5B-C) (13). CTX

treatment of C2C12 cells loaded with the Ca2+

sensitive dye Fura-2AM significantly increased the

fluorescence (340/380 nm ratio) within 60s

indicating elevated intracellular Ca2+

(Figure 5D

and 5E). While this effect was abolished by EGTA

treatment, subsequent exposure to Ca2+

(20mM)

increased the fluorescence ratio thus confirming

CTX-dependent Ca2+

influx (Figures 5F-G). This

also suggested that the elevated intracellular Ca2+

was probably due to the influx from the

extracellular milieu rather than the intracellular

stores. Pre-treatment of C2C12 cells with

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nifidepine, a blocker of L-type voltage gated

Ca2+

channel (VGCC; commonest Ca2+

channel in

skeletal muscle) did not prevent CTX-mediated

Ca2+

influx suggesting that the immediate Ca2+

rise

was VGCC-independent and is probably due to

sarcolemmal disruption to a certain extent (Figure

5H) along with other unknown mechanisms. When

cells loaded with calcein AM were exposed to

CTX, the fluorescence signal was detectable in the

extracellular medium (Figure 5I) after 30 min

confirming loss of membrane integrity. CTX

treatment also enhanced Rhod-2 (mitochondrial

Ca2+

-specific dye) fluorescence indicating

Ca2+

influx into the mitochondria (Figure 5J).

These data suggest that CTX causes significant

intracellular Ca2+

influx, which subsequently

accumulates in the mitochondria.

Evidences for mitochondrial dysfunction in the

CTX models and human tissues

The dystrophic muscle in human MDs

display mitochondrial dysfunction (46). In C2C12

cells, CTX treatment decreased the fluorescence

signal of JC-1, indicating mitochondrial

depolarization. This was abrogated by EGTA,

indicating that Ca2+

influx precedes mitochondrial

damage (Figure 6A). Mitochondrial dysfunction

was evident in CTX-injected muscle, as indicated

by loss of enzyme histochemistry (EHC) signal of

the mitochondrial activities including SDH, SDH-

COX and NADH-TR activities at d1, and more

drastically at d3 (Figures 6B-D). At d7, the EHC

signal recovered significantly indicating the

restoration of mitochondrial function. These

changes were comparable to the mitochondrial

damage evident in the human dystrophic

pathologies (Figures 6B-D).

Mitochondrial damage during muscle

degeneration could involve either mitochondrial

aggregation or altered morphology. Mitochondrial

aggregation observed in human myopathies was

not evident in the CTX model (Figure 6E).

However, the muscle mitochondria showed altered

cristae at d3, which was restored at d7 (Figure 6F).

This is consistent with the EM images of muscle

biopsies from human DMD and Dysfy (Figure 6F).

Mitochondrial dysfunction was also indicated by

significantly elevated cellular ADP/ATP ratio at

d1-d3, which recovered at d7 (Figure 6G). The

mitochondria also displayed lowered NADH

content at d1-d7 (Figure 6H). The overall

metabolic activity of the muscle was significantly

lowered, as indicated by decreased pyruvate at d1-

d7 (Figure 6I) and decreased lactate content at d1-

d3, which recovered at d7 (Figure 6J).

Analysis of the mitochondrial proteome in the

CTX-injected muscle

While the dystrophic pathology in the

CTX model at d1 and d3 was associated with

mitochondrial damage, at d7 and beyond,

regenerative processes corroborated with restored

mitochondrial function. To further characterize the

global mitochondrial changes associated with

CTX-mediated muscle degeneration, we compared

mitochondrial proteomic changes at d1 (initial

degenerative phase) andd3 (peak degenerative

phase) compared with saline control (Figure 7A)

by LC MS/MS (Supplementary figure 1). Data

analysis revealed differential expression of226

mitochondrial proteins at d1 (Figure 7B;

supplementary table 2), of which, 8 were up-

regulated (≥1.5 fold) and 80 proteins were down-

regulated (≤0.5 fold), compared to the control

(Table 2, Figure 7C-H). There were 7 under-

expressed proteins having potential

structural/functional interactions with

mitochondria, but their mitochondrial localization

was not confirmed (Table 3).

Most of the down regulated proteins at d1

were part of the mitochondrial energy metabolism

(Figure 7B). Among these, the most striking was

the down-regulation of 20 subunits of

mitochondrial complex I (CI) (Figure 7D).

Interestingly, 17 out of these 20 subunits were

further down-regulated at d3 (Figure 7D and table

2). Apart from this, 29 proteins of other complexes

of the electron transport chain and ATP

synthesizing machinery (succinate dehydrogenase-

SDH, cytochrome oxidase-COX, cytochrome b-c1

and ATP synthase) were also down-regulated at d1

(Table 2) and among these, 22 proteins were

further down-regulated at d3 (Figure 7E). Five

proteins involved in fatty acid oxidation, 4 proteins

related to Krebs cycle and 22 other mitochondrial

proteins were found to be down-regulated at d1

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(Figure 7F-H and table 2). Most of these proteins

(3 out of 5 fatty acid oxidation proteins, 2 out of 4

Krebs cycle proteins and 14 out of 22 other

mitochondrial proteins) were relatively up-

regulated at d3. Taken together, lowered

expression of the respiratory complexes might

predominantly contribute to the mitochondrial

dysfunction in the CTX model and this effect

could be reinforced by other down-regulated

proteins.

The proteomics data was corroborated by

biochemical and histochemical analysis of the

respiratory complexes and other mitochondrial

proteins. Enzyme assay of CI in the CTX model

confirmed 55 % activity at d1, 48 % at d3 and ~ 55

% at d7 compared to control (Figure 8A). Loss of

CI activity was also evident in the muscle tissues

from Dysfy patients (n=10) compared to controls

(n=6) (Figure 8B). The CTX model also displayed

loss of CII and CIII activities especially at d1-d3,

consistent with poorer activity of these complexes

in the muscle tissues from Dysfy patients (n=10)

vs. controls (n=6) (Figure 8C-F).

Western blot experiments confirmed the

down-regulation of specific subunits of different

respiratory complexes at d1-d3 compared to

control (Figure 8G-H).The down-regulation of

other mitochondrial proteins including aspartate

aminotransferase (ASAT) [or glutamic oxaloacetic

transaminase (GOT)], mitochondrial creatine

kinase (CK) and citrate synthase (CS) was

validated by lowered enzyme activity in the CTX

model (Figure 8I-K).

Lowered expression of respiratory

complexes in the CTX model was also

corroborated by the loss of EHC signal for NADH-

TR, SDH and SDH-COX (Figure 6B-D).

Role of oxidative stress in the CTX model and

human muscle diseases

In accordance with muscle degeneration-

associated oxidative stress in human MDs (13), the

dystrophic muscle in the CTX model displayed ~2

fold lower glutathione reduced/oxidized ratio

(GSH/GSSG) at d1 and d3, which significantly

improved at d7 (Figure 9A). This is consistent

with lowered GSH/GSSG ratio in the muscle

tissues from Dysfy patients (n=6) [vs. controls

(n=6)] (Figure 9B). Lowered GSH/GSSG in the

CTX model was probably not due to decreased

synthesis since the expression of gamma-glutamy

cysteine ligase (GCL-c) gene, the rate limiting

enzyme of GSH synthesis was unaltered from d1-

d14 (Figure 9C). Lowered red-ox status in the

CTX model was associated with elevated lipid

peroxidation (by 1.2-2 fold at d1-d7) (Figure 9D),

consistent with elevated lipid peroxidation in the

muscle tissues from DMD patients (n=6) [vs.

controls (n=6)] (Figure 9E) (13). C2C12 cells

incubated with CTX displayed elevated cellular

oxidants within 30-60 min of toxin exposure.

Pretreatment with EGTA decreased the CTX-

dependent oxidants, suggesting that oxidative

stress is dependent to a limited extent on Ca2+

dynamics (Figure 9F-I). CTX treatment also

elevated the DAF-FM fluorescence (indicator of

higher oxides of NO) in C2C12 cells, which was

slightly decreased by EGTA treatment (Figure 9J-

K).

These data indicate direct correlation

between muscle pathology and oxidative stress as

previously reported in human MDs (13). We

assessed whether the red/ox markers are altered in

non-dystrophic muscle disorders including distal

myopathies (DMs), mitochondrial myopathies

(MMs), IMs and SMA (Table 5). Except IMs,

other pathologies displayed elevated lipid

peroxidation (Figure 9L) and protein oxidation

(Figure 9N-O). While GSH depletion was evident

in MMs, IMs and SMA (1 and 2), it was

unchanged in DMs and SMA3 (Figure 9M).Hence,

there is credible correlation among muscle

diseases, oxidative damage and GSH depletion.

However, the intracellular targets of oxidative

damage in human muscle pathology are not

completely delineated.

Oxidative damage of proteins, which alters

their structure-function relationship and

contributes to aging and disease (47) could target

specific proteins. Among the pathologies from the

current and previous study (13), protein oxidation

was strikingly higher in DMD. We set out to

identify carbonylated proteins associated with the

dystrophic pathology in the total muscle extracts

of DMD (n=6) and control (n=6) by 2D oxyblot

followed by MS analysis. Comparison of 2D blots

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between DMD and control revealed 23 distinct

protein spots among which, spot 17 to 23 were

specific only to DMD while the others were

common between DMD and control (Figure 10A).

MS analysis of the spots 17-23 identified 6

potentially oxidized proteins (Figure 10B and

Table 4), two of which [Aconitate hydratase (spot

no.17) and dienoyl CoA isomerase (spot no. 22

and 23)] were mitochondrial, indicating that

oxidative stress in DMD can specifically damage

mitochondrial proteins thus impinging on its

function. Interestingly, these two proteins were

down-regulated in the CTX model at d1 and d3

(Supplementary Table 2).Validation of proteomics

data by enzyme assay confirmed (i) loss of

aconitase activity in DMD muscle (Figure 10C)

and (ii) ROS-dependent inactivation of the

aconitase enzyme activity (Figure 10D).

DISCUSSION

The limited therapeutic option for muscle

pathologies has necessitated the investigation of

the underlying mechanisms employing various

experimental models (8,48). However, a single

model that meticulously mimics all the features of

MD and IMs is non-existent (2). While transgenic

models involving higher mammals have breeding

and ethical issues (49), transgenic mice are limited

by stringent maintenance, restricted ability to

recapitulate the human pathology and species-

related variations. For e.g., the mdx mouse model

of DMD displays moderate pathology limited to

early stages, predominantly in the diaphragm

muscle (8). This is in contrast to the fast

progressive pathogenesis in different skeletal

muscles, worsening symptoms and reduced life-

span in DMD patients. Variants of mdx (45) and

other MD models have not provided optimal

results (7,46). Further, the MD models should, in

addition to genetic mutations, display muscle

weakness, persistent inflammation, fibrosis,

necrosis and/or apoptosis and impaired

regeneration. This is pertinent to sporadic diseases

like IMs which require non-genetic acute models

of muscle inflammation and degeneration. The

CTX model described in this study reflects the

acute dystrophic pathology and muscle weakness,

with down-stream events such as sarcolemmal

disruption, intense inflammation and

necrotic/apoptotic cell death at d1 and d3, as seen

in MDs and IMs (Figure 1 and 11B). The myofiber

death is segmental, involving homogenization of

cytoskeleton, organelle damage, breakdown of the

T-system and vesicle formation as seen in MDs

(Figure 4). While CTXs have been studied in cell

lines to understand signal transduction, toxin

internalization and Ca2+

dynamics (50-52), direct

and comprehensive comparison to human muscle

pathologies have not been explored, making it the

novel feature of the current study (Figure 11C).

Regenerative processes in the muscle

tissue (53) are inadequate in MD (54) and IM (55).

Hence, models that display regenerative processes

are important in MD therapeutics. Since most

genetic MD models do not display active

regeneration (48), researchers have utilized

alternate models including the CTX model to

study muscle regeneration (56). The CTX model

displayed a regenerative phase that commenced at

d7, was fully active from d11-d14 and nearly

complete by d31 (Figures 4 and 11A).

Muscle degeneration in different

pathologies (57,58) could be both apoptotic and

necrotic (59). The CTX model displayed increased

expression of apoptotic mediators FasL and TNF-

alpha during the degenerative phase, comparable

to the mdx model and human MDs and myopathies

(Figure 2 and supplementary table 1) (60-62). The

inflammatory pathways that contribute to the

dystrophic changes during MD (2,15,63) including

infiltration of inflammatory cells (Figure 1) and

elevated expression of the inflammatory genes

(Figure 2) were observed in the CTX model.

Muscle degeneration in MDs also involve necrotic

events including pore formation in the membranes,

mitochondrial damage (64) and elevated

intracellular Ca2+

(65). Loss of immunoreactivity

of the membrane proteins (Figure 3) in addition to

EM findings (Figure 4) and calcein data (Figure

5I) provided evidence for sarcolemmal disruption

and leakage in the CTX model. This bears

similarity to the loss of sarcolemmal proteins

leading to a leaky sarcolemma (15,59,66) (Figures

3B and 4D).

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Ca2+

is critical for muscle function and its

dynamics is tightly regulated (67). DMD muscle

displays elevated intracellular Ca2+

due to

increased activity of the sarcolemmal channels

(5,68,69) and decreased Ca2+

sequestering proteins

and pumps in the sarcolemmal reticulum (SR)

(69,70). The dystrophic phenotype in MD models

is improved by over-expression of sarcoplasmic

reticulum Ca2+

ATPase (SERCA1) (70) and down-

regulation of sarcolemmal transient receptor

potential cation (TRPC) channels (71). The CTX

mouse model displayed Ca2+

deposits at d1 and d3

(Figure 5A) due to sarcolemmal disruption (Figure

5I) and resultant influx from the external milieu

(Figure 5F and 5G). Apart from the Ca2+

release

via SR (72), free entry of Ca2+

through

sarcolemmal breaches or via other mechanisms

might add to the pathology (73), as reflected by the

Ca2+

influx independent of VGCC channels in the

CTX model (Figure 5H). Increased intracellular

Ca2+

in DMD results in increased protein

degradation, oxidative stress and necrosis

(5,13,72). In the CTX model, the Ca2+

influx

elevated mitochondrial Ca2+

(Figure 5J), caused

mitochondrial depolarization (Figure 6A) and

increased oxidative stress to a limited extent

(Figure 9), ultimately leading to myotoxicity

(Figure 11B).

Elevated mitochondrial Ca2+

alters the

mitochondrial homeostasis and turns the ETC

leaky causing increased ROS, mitochondrial

depolarization and ATP depletion, leading to

apoptosis (10,74,75). Elevated cytosolic Ca2+

,

abnormal activity of the mitochondrial

permeability transition pore (MPTP) and necrosis

is observed in -sarcoglycan-deficient hamster

cardiomyocytes (76). Analysis of Facio scapulo

humeral dystrophy revealed decreased cytochrome

c oxidase activity and reduced ATP synthesis (46),

altered MPTP and apoptosis (77). The CTX cell

model displayed mitochondrial depolarization and

concomitant production of ROS (Figures 6A and

9F-I). The mitochondria showed altered cristae

(Figure 6F), loss of NADH-TR, SDH and SDH-

COX activities (Figure 6B-D) and lowered

activities of respiratory complexes CI, CII and CIII

(Figure 8A, 8C and 8E) predominantly at d1 and

similar to Dysfy (Figures 8B, 8D and 8F) and

other MDs (6).

Mitochondrial dysfunction in MD and

other pathologies could be associated with altered

expression of mitochondrial proteins. Comparative

proteomics of the mdx TA muscle showed

significantly altered mitochondrial proteins in the

aged mice (78). Interestingly, muscle from DMD

and aged animals share common gene signatures

that coordinate mitochondrial metabolism (79).

Analysis of the mitochondrial proteome from

CTX-injected muscle at d1 and d3 showed down-

regulation of mitochondrial proteins involved in

energy metabolism and included proteins of the

respiratory complexes, ATP synthesis, fatty acid

metabolism and Krebs cycle (Table 2). The

subunits of CI formed a major class of the

significantly down regulated proteins thus

explaining the drastic reduction in CI specific

activity at d1, d3 and d7 (Figure 8A-B). Down-

regulation of the CI subunits was also confirmed

by western blot (Figure 8G-H) and lowered

NADH-TR EHC at d1 and d3 (Figure 6B). Under-

expression of different subunits of Succinic

dehydrogenase and cytochrome oxidase was also

supported by lowered CI and CII activities (Figure

8) and SDH and SDH-COX EHC staining at d1-d3

(Figure 6).

Intracellular Ca2+

dysregulation and

mitochondrial damage might contribute to

oxidative stress during MD (5,80). Altered red/ox

markers has been reported in MD patients and

animal models (7,8,13,81). ROS produced in mdx

mice and Col6a1(-/-) mice might contribute to

muscle pathology (80). Although the role of

oxidative damage in MD pathogenesis is not been

completely understood, it could directly correlate

with the severity of the dystrophic pathology (13).

Our study showed elevated lipid peroxidation and

protein carbonylation in the chronic diseases, DM,

MM and SMA (Figure 9L and 9N-O).

The CTX model displayed markers of

oxidative stress including lowered GSH/GSSG

ratio and elevated lipid peroxidation (Figure 9A

and 9D). GSH is essential for cellular

differentiation (16) and stress response in the

muscle (82). GSH depletion might contribute to

oxidative stress in MD (83), cachexia and muscle

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atrophy (82) and might increase CTX toxicity in

C2C12 cells (13). The GSH level which was low

at d1 significantly increased beyond control level

at d3 and d7. This is probably a stress response to

lipid peroxidation (78), which in turn might not be

due to increased GSH synthesis, since the

expression of the GSH synthesizing enzyme

gamma glutamyl cysteine ligase (GCL) was

unchanged (Figure 9C). Analysis of non-

dystrophic myopathies in this study showed GSH

depletion in MMs, IMs and more drastically in

SMA1 and 2 (Figure 9M). Following recovery of

muscle structure, the GSH and MDA content in

the CTX-injected muscles were restored to control

levels indicating a direct correlation of dystrophic

pathology with GSH depletion and oxidative stress

(Data not shown). Hence, GSH depletion could be

linked to the dystrophic pathology and drugs that

boost GSH levels could offer myoprotection.

Treatment of C2C12 myotubes with lipoic acid

plus coenzyme Q10 increased expression of genes

involved in GSH synthesis and recycling (84).

Unpublished data from our laboratory

demonstrated that curcumin, a dietary polyphenol

from turmeric, induced GSH synthesis and

protected against CTX toxicity both in vitro and in

vivo.

MS analysis detected six proteins that

were carbonylated only in the DMD muscle

(Figure 10A-B). One such mitochondrial protein is

aconitate hydratase (aconitase),which converts

citrate to isocitrate and is indirectly required by

GSH reductase for its redox activity (85).

Aconitase is susceptible to oxidative damage and

is deregulated in DMD (86). Carbonylation of

aconitase might inhibit its activity (87). Aconitase

activity was significantly down-regulated in DMD

muscle compared to control (Figure 10C). Kinetic

assay in mouse muscle extract demonstrated H2O2-

dependent inhibition of aconitase activity thus

confirming its susceptibility to oxidative stress

(Figure 10D). Interestingly, the CTX model

showed down-regulation of aconitate hydratase by

1.4 and 1.7 fold in d1 and d3 respectively

compared to control (Supplementary table 2). The

other carbonylated mitochondrial protein in DMD

is delta (3,5)-Delta (2,4)-dienoyl-CoA isomerase,

which is involved in the fatty acid -oxidation

pathway and is localized to the matrix of

mitochondria and peroxisomes (88). Two variants

of this protein are expressed in human striated

muscle (89). Interestingly, the CTX model

displayed down-regulation of proteins involved in

fatty acid oxidation including limited down-

regulation of enoyl-CoA delta isomerase 1 by 1.3

and 1.2 fold in d1 and d3 respectively compared to

control (Supplementary table 2).

Heat shock proteins (HSPs) (90) HSPA1A

and HSPA8 were carbonylated in DMD (Figure

10; Table 4). Carbonylated HSPA1A is associated

with lysosomal rupture during necrotic neuronal

death (91) and hippocampal neurodegeneration

(92). Carbonylated HSPs are detected in plant

mitochondria during stress (93). Interestingly,

Hspd1 (60 kDa Hsp) was down regulated by 1.7

and 3.3 fold at d1 and d3 in the CTX model (Table

2), while Hspa9 (stress protein similar to Hsp70)

was down-regulated by 2.5 fold at d3 (Table 2).

The other carbonylated protein Transferrin, which

is involved in iron metabolism (94) accumulates in

the dystrophic muscles (95). Another carbonylated

protein-enolase is a glycolytic enzyme (96)

implicated in myoblast fusion and differentiation

(97) and is modulated in mdx and muscle injury

models (97). Enolase is carbonylated in

experimental models of myopathy (87). These data

suggest that oxidative damage of specific proteins

is pertinent to DMD pathology and most of these

proteins were down-regulated in the CTX model.

In summary, the current study has

characterized the CTX model of muscle pathology

with direct implications for MDs and IMs. The

study also emphasized the role of mitochondrial

dysfunction and oxidative stress in the model and

in muscle pathology.

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REFERENCES

1. Emery, A. E. (2002) The muscular dystrophies. Lancet 359, 687-695

2. Hoffman, E. P. (2001) Dystrophinopathies. in Disorders of voluntary muscle (Karpati, G., Hilton-

Jones, D., Griggs, R. C. ed.), 7 Ed., Cambridge University Press, Cambridge. pp 385-432

3. Fairclough, R. J., Perkins, K. J., and Davies, K. E. (2012) Pharmacologically targeting the primary

defect and downstream pathology in Duchenne muscular dystrophy. Curr Gene Ther 12, 206-244

4. Dimitri, D. (2009) [Inflammatory myopathies: diagnosis and classifications]. Presse Med 38, 1141-

1163

5. Gissel, H. (2005) The role of Ca2+ in muscle cell damage. Ann N Y Acad Sci 1066, 166-180

6. Jongpiputvanich, S., Sueblinvong, T., and Norapucsunton, T. (2005) Mitochondrial respiratory chain

dysfunction in various neuromuscular diseases. J Clin Neurosci 12, 426-428

7. Burr, I. M., Asayama, K., and Fenichel, G. M. (1987) Superoxide dismutases, glutathione peroxidase,

and catalase in neuromuscular disease. Muscle Nerve 10, 150-154

8. Ng, R., Banks, G. B., Hall, J. K., Muir, L. A., Ramos, J. N., Wicki, J., Odom, G. L., Konieczny, P.,

Seto, J., Chamberlain, J. R., and Chamberlain, J. S. (2012) Animal models of muscular dystrophy.

Prog Mol Biol Transl Sci 105, 83-111

9. Kherif, S., Lafuma, C., Dehaupas, M., Lachkar, S., Fournier, J. G., Verdiere-Sahuque, M., Fardeau,

M., and Alameddine, H. S. (1999) Expression of matrix metalloproteinases 2 and 9 in regenerating

skeletal muscle: a study in experimentally injured and mdx muscles. Dev Biol 205, 158-170

10. Bernardi, P. (1999) Mitochondria in muscle cell death. Ital J Neurol Sci 20, 395-400

11. Paternostro-Sluga, T., Grim-Stieger, M., Posch, M., Schuhfried, O., Vacariu, G., Mittermaier, C.,

Bittner, C., and Fialka-Moser, V. (2008) Reliability and validity of the Medical Research Council

(MRC) scale and a modified scale for testing muscle strength in patients with radial palsy. Journal of

rehabilitation medicine : official journal of the UEMS European Board of Physical and

Rehabilitation Medicine 40, 665-671

12. Renjini, R., Gayathri, N., Nalini, A., and Srinivas Bharath, M. M. (2012) Analysis of calpain-3

protein in muscle biopsies of different muscular dystrophies from India. Indian J Med Res 135, 878-

886

13. Renjini, R., Gayathri, N., Nalini, A., and Srinivas Bharath, M. M. (2012) Oxidative damage in

muscular dystrophy correlates with the severity of the pathology: role of glutathione metabolism.

Neurochem Res 37, 885-898

14. Smith, J. P., Hicks, P. S., Ortiz, L. R., Martinez, M. J., and Mandler, R. N. (1995) Quantitative

measurement of muscle strength in the mouse. J Neurosci Methods 62, 15-19

15. Dubowitz, V., Sewry CA. (2007) Muscle biopsy – A practical approach. 3 Ed., Saunders Elsevier,

Philadelphia. pp 293-323

16. Ardite, E., Barbera, J. A., Roca, J., and Fernandez-Checa, J. C. (2004) Glutathione depletion impairs

myogenic differentiation of murine skeletal muscle C2C12 cells through sustained NF-kappaB

activation. Am J Pathol 165, 719-728

17. Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of

protein utilizing the principle of protein-dye binding. Anal Biochem 72, 248-254

18. Grynkiewicz, G., Poenie, M., and Tsien, R. Y. (1985) A new generation of Ca2+ indicators with

greatly improved fluorescence properties. J Biol Chem 260, 3440-3450

19. Peng, T. I., and Greenamyre, J. T. (1998) Privileged access to mitochondria of calcium influx through

N-methyl-D-aspartate receptors. Mol Pharmacol 53, 974-980

20. Neri, S., Mariani, E., Meneghetti, A., Cattini, L., and Facchini, A. (2001) Calcein-acetyoxymethyl

cytotoxicity assay: standardization of a method allowing additional analyses on recovered effector

cells and supernatants. Clin Diagn Lab Immunol 8, 1131-1135

by guest on February 2, 2020http://w

ww

.jbc.org/D

ownloaded from

Mitochondrial changes and oxidative stress in muscle pathology

17

21. Reers, M., Smith, T. W., and Chen, L. B. (1991) J-aggregate formation of a carbocyanine as a

quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480-4486

22. Budd, S. L., Castilho, R. F., and Nicholls, D. G. (1997) Mitochondrial membrane potential and

hydroethidine-monitored superoxide generation in cultured cerebellar granule cells. FEBS Lett 415,

21-24

23. Lopez-Figueroa, M. O., Caamano, C., Morano, M. I., Ronn, L. C., Akil, H., and Watson, S. J. (2000)

Direct evidence of nitric oxide presence within mitochondria. Biochem Biophys Res Commun 272,

129-133

24. Bhattacharya, S. K., Thakar, J. H., Johnson, P. L., and Shanklin, D. R. (1991) Isolation of skeletal

muscle mitochondria from hamsters using an ionic medium containing ethylenediaminetetraacetic

acid and nagarse. Anal Biochem 192, 344-349

25. Trounce, I. A., Kim, Y. L., Jun, A. S., and Wallace, D. C. (1996) Assessment of mitochondrial

oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines.

Methods Enzymol 264, 484-509

26. P.A, S. (1969) [1] Citrate synthase: [EC 4.1.3.7. Citrate oxaloacetate-lyase (CoA-acetylating)]. in

Methods in Enzymology (John, M. L. ed.), Academic Press. pp 3-11

27. Castro, L., Rodriguez, M., and Radi, R. (1994) Aconitase is readily inactivated by peroxynitrite, but

not by its precursor, nitric oxide. J Biol Chem 269, 29409-29415

28. Jacobson, E. L., and Jacobson, M. K. (1976) Pyridine nucleotide levels as a function of growth in

normal and transformed 3T3 cells. Arch Biochem Biophys 175, 627-634

29. Hissin, P. J., and Hilf, R. (1976) A fluorometric method for determination of oxidized and reduced

glutathione in tissues. Anal Biochem 74, 214-226

30. Choi, A. O., Cho, S. J., Desbarats, J., Lovric, J., and Maysinger, D. (2007) Quantum dot-induced cell

death involves Fas upregulation and lipid peroxidation in human neuroblastoma cells. J

Nanobiotechnology 5, 1

31. Sultana, R., and Butterfield, D. A. (2011) Identification of the oxidative stress proteome in the brain.

Free Radic Biol Med 50, 487-494

32. Owen, J. B., Opii, W. O., Ramassamy, C., Pierce, W. M., and Butterfield, D. A. (2008) Proteomic

analysis of brain protein expression levels in NF-kappabeta p50 -/- homozygous knockout mice.

Brain Res 1240, 22-30

33. Thongboonkerd, V., McLeish, K. R., Arthur, J. M., and Klein, J. B. (2002) Proteomic analysis of

normal human urinary proteins isolated by acetone precipitation or ultracentrifugation. Kidney Int 62,

1461-1469

34. Marimuthu, A., O'Meally, R. N., Chaerkady, R., Subbannayya, Y., Nanjappa, V., Kumar, P., Kelkar,

D. S., Pinto, S. M., Sharma, R., Renuse, S., Goel, R., Christopher, R., Delanghe, B., Cole, R. N.,

Harsha, H. C., and Pandey, A. (2011) A comprehensive map of the human urinary proteome. J

Proteome Res 10, 2734-2743

35. Banerjee, K. K., Ayyub, C., Sengupta, S., and Kolthur-Seetharam, U. (2012) dSir2 deficiency in the

fatbody, but not muscles, affects systemic insulin signaling, fat mobilization and starvation survival in

flies. Aging 4, 206-223

36. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) Protein measurement with the

Folin phenol reagent. The Journal of biological chemistry 193, 265-275

37. Pawar, H., Kashyap, M. K., Sahasrabuddhe, N. A., Renuse, S., Harsha, H. C., Kumar, P., Sharma, J.,

Kandasamy, K., Marimuthu, A., Nair, B., Rajagopalan, S., Maharudraiah, J., Premalatha, C. S.,

Kumar, K. V., Vijayakumar, M., Chaerkady, R., Prasad, T. S., Kumar, R. V., Kumar, R. V., and

Pandey, A. (2011) Quantitative tissue proteomics of esophageal squamous cell carcinoma for novel

biomarker discovery. Cancer biology & therapy 12, 510-522

by guest on February 2, 2020http://w

ww

.jbc.org/D

ownloaded from

Mitochondrial changes and oxidative stress in muscle pathology

18

38. Patel, H. V., Vyas, A. A., Vyas, K. A., Liu, Y. S., Chiang, C. M., Chi, L. M., and Wu, W. (1997)

Heparin and heparan sulfate bind to snake cardiotoxin. Sulfated oligosaccharides as a potential target

for cardiotoxin action. J Biol Chem 272, 1484-1492

39. Yan, Z., Choi, S., Liu, X., Zhang, M., Schageman, J. J., Lee, S. Y., Hart, R., Lin, L., Thurmond, F.

A., and Williams, R. S. (2003) Highly coordinated gene regulation in mouse skeletal muscle

regeneration. J Biol Chem 278, 8826-8836

40. Sandri, M., Minetti, C., Pedemonte, M., and Carraro, U. (1998) Apoptotic myonuclei in human

Duchenne muscular dystrophy. Lab Invest 78, 1005-1016

41. Chen, Y. W., Nagaraju, K., Bakay, M., McIntyre, O., Rawat, R., Shi, R., and Hoffman, E. P. (2005)

Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy.

Neurology 65, 826-834

42. Onofre-Oliveira, P. C., Santos, A. L., Martins, P. M., Ayub-Guerrieri, D., and Vainzof, M. (2012)

Differential expression of genes involved in the degeneration and regeneration pathways in mouse

models for muscular dystrophies. Neuromolecular Med 14, 74-83

43. Parker, M. H., Loretz, C., Tyler, A. E., Snider, L., Storb, R., and Tapscott, S. J. (2012) Inhibition of

CD26/DPP-IV enhances donor muscle cell engraftment and stimulates sustained donor cell

proliferation. Skelet Muscle 2, 4

44. Emery, A. E. (1977) Muscle histology and creatine kinase levels in the foetus in Duchenne muscular

dystrophy. Nature 266, 472-473

45. Sabatelli, P., Pellegrini, C., Faldini, C., and Merlini, L. (2012) Cytoskeletal and extracellular matrix

alterations in limb girdle muscular dystrophy 2I muscle fibers. Neurol India 60, 510-511

46. Turki, A., Hayot, M., Carnac, G., Pillard, F., Passerieux, E., Bommart, S., Raynaud de Mauverger, E.,

Hugon, G., Pincemail, J., Pietri, S., Lambert, K., Belayew, A., Vassetzky, Y., Juntas Morales, R.,

Mercier, J., and Laoudj-Chenivesse, D. (2012) Functional muscle impairment in facioscapulohumeral

muscular dystrophy is correlated with oxidative stress and mitochondrial dysfunction. Free Radic

Biol Med 53, 1068-1079

47. Berlett, B. S., and Stadtman, E. R. (1997) Protein oxidation in aging, disease, and oxidative stress. J

Biol Chem 272, 20313-20316

48. Kudryashova, E., Kramerova, I., and Spencer, M. J. (2012) Satellite cell senescence underlies

myopathy in a mouse model of limb-girdle muscular dystrophy 2H. J Clin Invest 122, 1764-1776

49. Yugeta, N., Urasawa, N., Fujii, Y., Yoshimura, M., Yuasa, K., Wada, M. R., Nakura, M., Shimatsu,

Y., Tomohiro, M., Takahashi, A., Machida, N., Wakao, Y., Nakamura, A., and Takeda, S. (2006)

Cardiac involvement in Beagle-based canine X-linked muscular dystrophy in Japan (CXMDJ):

electrocardiographic, echocardiographic, and morphologic studies. BMC Cardiovasc Disord 6, 47

50. Tsai, P. C., Hsieh, C. Y., Chiu, C. C., Wang, C. K., Chang, L. S., and Lin, S. R. (2012) Cardiotoxin

III suppresses MDA-MB-231 cell metastasis through the inhibition of EGF/EGFR-mediated signaling

pathway. Toxicon 60, 734-743

51. Chien, C. M., Chang, S. Y., Lin, K. L., Chiu, C. C., Chang, L. S., and Lin, S. R. (2010) Taiwan cobra

cardiotoxin III inhibits Src kinase leading to apoptosis and cell cycle arrest of oral squamous cell

carcinoma Ca9-22 cells. Toxicon 56, 508-520

52. Wang, C. H., Monette, R., Lee, S. C., Morley, P., and Wu, W. G. (2005) Cobra cardiotoxin-induced

cell death in fetal rat cardiomyocytes and cortical neurons: different pathway but similar cell surface

target. Toxicon 46, 430-440

53. Wang, Y. X., and Rudnicki, M. A. (2012) Satellite cells, the engines of muscle repair. Nat Rev Mol

Cell Biol 13, 127-133

54. Messina, S., Mazzeo, A., Bitto, A., Aguennouz, M., Migliorato, A., De Pasquale, M. G., Minutoli, L.,

Altavilla, D., Zentilin, L., Giacca, M., Squadrito, F., and Vita, G. (2007) VEGF overexpression via

by guest on February 2, 2020http://w

ww

.jbc.org/D

ownloaded from

Mitochondrial changes and oxidative stress in muscle pathology

19

adeno-associated virus gene transfer promotes skeletal muscle regeneration and enhances muscle

function in mdx mice. FASEB J 21, 3737-3746

55. Wanschitz, J. V., Dubourg, O., Lacene, E., Fischer, M. B., Hoftberger, R., Budka, H., Romero, N. B.,

Eymard, B., Herson, S., Butler-Browne, G. S., Voit, T., and Benveniste, O. (2013) Expression of

myogenic regulatory factors and myo-endothelial remodeling in sporadic inclusion body myositis.

Neuromuscul Disord 23, 75-83

56. Marshall, J. L., Holmberg, J., Chou, E., Ocampo, A. C., Oh, J., Lee, J., Peter, A. K., Martin, P. T., and

Crosbie-Watson, R. H. (2012) Sarcospan-dependent Akt activation is required for utrophin expression

and muscle regeneration. J Cell Biol 197, 1009-1027

57. Fadic, R. (2005) Cell surface and gene expression regulation molecules in dystrophinopathy: mdx vs.

Duchenne. Biol Res 38, 375-380

58. Paco, S., Ferrer, I., Jou, C., Cusi, V., Corbera, J., Torner, F., Gualandi, F., Sabatelli, P., Orozco, A.,

Gomez-Foix, A. M., Colomer, J., Nascimento, A., and Jimenez-Mallebrera, C. (2012) Muscle fiber

atrophy and regeneration coexist in collagen VI-deficient human muscle: role of calpain-3 and

nuclear factor-kappaB signaling. J Neuropathol Exp Neurol 71, 894-906

59. Sandri, M., and Carraro, U. (1999) Apoptosis of skeletal muscles during development and disease. Int

J Biochem Cell Biol 31, 1373-1390

60. Spencer, M. J., Walsh, C. M., Dorshkind, K. A., Rodriguez, E. M., and Tidball, J. G. (1997)

Myonuclear apoptosis in dystrophic mdx muscle occurs by perforin-mediated cytotoxicity. J Clin

Invest 99, 2745-2751

61. Warren, G. L., Hulderman, T., Jensen, N., McKinstry, M., Mishra, M., Luster, M. I., and Simeonova,

P. P. (2002) Physiological role of tumor necrosis factor alpha in traumatic muscle injury. FASEB J

16, 1630-1632

62. Kuru, S., Inukai, A., Kato, T., Liang, Y., Kimura, S., and Sobue, G. (2003) Expression of tumor

necrosis factor-alpha in regenerating muscle fibers in inflammatory and non-inflammatory

myopathies. Acta Neuropathol 105, 217-224

63. Enwere, E. K., Boudreault, L., Holbrook, J., Timusk, K., Earl, N., Lacasse, E., Renaud, J. M., and

Korneluk, R. G. (2012) Loss of cIAP1 attenuates soleus muscle pathology and improves diaphragm

function in mdx mice. Hum Mol Genet 22, 867-878

64. Wang, C. H., and Wu, W. G. (2005) Amphiphilic beta-sheet cobra cardiotoxin targets mitochondria

and disrupts its network. FEBS Lett 579, 3169-3174

65. Ownby, C. L., Fletcher, J. E., and Colberg, T. R. (1993) Cardiotoxin 1 from cobra (Naja naja atra)

venom causes necrosis of skeletal muscle in vivo. Toxicon 31, 697-709

66. Brown, S., Lucy JA. (1997) Dystrophin – Gene, protein and cell biology. Cambridge University

Press, Cambridge. pp 79-100

67. Berchtold, M. W., Brinkmeier, H., and Muntener, M. (2000) Calcium ion in skeletal muscle: its

crucial role for muscle function, plasticity, and disease. Physiol Rev 80, 1215-1265

68. Turner, P. R., Fong, P. Y., Denetclaw, W. F., and Steinhardt, R. A. (1991) Increased calcium influx in

dystrophic muscle. J Cell Biol 115, 1701-1712

69. Robin, G., Berthier, C., and Allard, B. (2012) Sarcoplasmic reticulum Ca2+ permeation explored

from the lumen side in mdx muscle fibers under voltage control. J Gen Physiol139, 209-218

70. Goonasekera, S. A., Lam, C. K., Millay, D. P., Sargent, M. A., Hajjar, R. J., Kranias, E. G., and

Molkentin, J. D. (2011) Mitigation of muscular dystrophy in mice by SERCA overexpression in

skeletal muscle. J Clin Invest 121, 1044-1052

71. Iwata, Y., Katanosaka, Y., Arai, Y., Shigekawa, M., and Wakabayashi, S. (2009) Dominant-negative

inhibition of Ca2+ influx via TRPV2 ameliorates muscular dystrophy in animal models. Hum Mol

Genet 18, 824-834

by guest on February 2, 2020http://w

ww

.jbc.org/D

ownloaded from

Mitochondrial changes and oxidative stress in muscle pathology

20

72. Hopf, F. W., Turner, P. R., and Steinhardt, R. A. (2007) Calcium misregulation and the pathogenesis

of muscular dystrophy. Subcell Biochem 45, 429-464

73. Fraysse, B., Liantonio, A., Cetrone, M., Burdi, R., Pierno, S., Frigeri, A., Pisoni, M., Camerino, C.,

and De Luca, A. (2004) The alteration of calcium homeostasis in adult dystrophic mdx muscle fibers

is worsened by a chronic exercise in vivo. Neurobiol Dis 17, 144-154

74. Dalle-Donne, I., Scaloni, A., Giustarini, D., Cavarra, E., Tell, G., Lungarella, G., Colombo, R., Rossi,

R., and Milzani, A. (2005) Proteins as biomarkers of oxidative/nitrosative stress in diseases: the

contribution of redox proteomics. Mass Spectrom Rev 24, 55-99

75. Bernardi, P., Krauskopf, A., Basso, E., Petronilli, V., Blachly-Dyson, E., Di Lisa, F., and Forte, M. A.

(2006) The mitochondrial permeability transition from in vitro artifact to disease target. FEBS J 273,

2077-2099

76. Fraysse, B., Nagi, S. M., Boher, B., Ragot, H., Laine, J., Salmon, A., Fiszman, M. Y., Toussaint, M.,

and Fromes, Y. (2010) Ca2+ overload and mitochondrial permeability transition pore activation in

living delta-sarcoglycan-deficient cardiomyocytes. Am J Physiol Cell Physiol 299, C706-713

77. Tews, D. S. (2002) Apoptosis and muscle fibre loss in neuromuscular disorders. Neuromuscul Disord

12, 613-622

78. Carberry, S., Zweyer, M., Swandulla, D., and Ohlendieck, K. (2012) Profiling of age-related changes

in the tibialis anterior muscle proteome of the mdx mouse model of dystrophinopathy. J Biomed

Biotechnol 2012, 691641

79. Baron, D., Magot, A., Ramstein, G., Steenman, M., Fayet, G., Chevalier, C., Jourdon, P., Houlgatte,

R., Savagner, F., and Pereon, Y. (2011) Immune response and mitochondrial metabolism are

commonly deregulated in DMD and aging skeletal muscle. PLoS One 6, e26952

80. Menazza, S., Blaauw, B., Tiepolo, T., Toniolo, L., Braghetta, P., Spolaore, B., Reggiani, C., Di Lisa,

F., Bonaldo, P., and Canton, M. (2010) Oxidative stress by monoamine oxidases is causally involved

in myofiber damage in muscular dystrophy. Hum Mol Genet 19, 4207-4215

81. Tews, D. S., and Goebel, H. H. (1998) Cell death and oxidative damage in inflammatory myopathies.

Clin Immunol Immunopathol 87, 240-247

82. Cotgreave, I. A., Goldschmidt, L., Tonkonogi, M., and Svensson, M. (2002) Differentiation-specific

alterations to glutathione synthesis in and hormonally stimulated release from human skeletal muscle

cells. FASEB J 16, 435-437

83. Degl'Innocenti, D., Rosati, F., Iantomasi, T., Vincenzini, M. T., and Ramponi, G. (1999) GSH system

in relation to redox state in dystrophic skin fibroblasts. Biochimie 81, 1025-1029

84. Wagner, A. E., Ernst, I. M., Birringer, M., Sancak, O., Barella, L., and Rimbach, G. (2012) A

combination of lipoic acid plus coenzyme Q10 induces PGC1alpha, a master switch of energy

metabolism, improves stress response, and increases cellular glutathione levels in cultured C2C12

skeletal muscle cells. Oxid Med Cell Longev 2012, 835970

85. Comte, B., Vincent, G., Bouchard, B., Benderdour, M., and Des Rosiers, C. (2002) Reverse flux

through cardiac NADP(+)-isocitrate dehydrogenase under normoxia and ischemia. Am J Physiol

Heart Circ Physiol 283, H1505-1514

86. Dudley, R. W., Khairallah, M., Mohammed, S., Lands, L., Des Rosiers, C., and Petrof, B. J. (2006)

Dynamic responses of the glutathione system to acute oxidative stress in dystrophic mouse (mdx)

muscles. Am J Physiol Regul Integr Comp Physiol 291, R704-710

87. Barreiro, E., and Hussain, S. N. (2010) Protein carbonylation in skeletal muscles: impact on function.

Antioxid Redox Signal12, 417-429

88. FitzPatrick, D. R., Germain-Lee, E., and Valle, D. (1995) Isolation and characterization of rat and

human cDNAs encoding a novel putative peroxisomal enoyl-CoA hydratase. Genomics27, 457-466

by guest on February 2, 2020http://w

ww

.jbc.org/D

ownloaded from

Mitochondrial changes and oxidative stress in muscle pathology

21

89. Kovalyov, L. I., Kovalyova, M. A., Kovalyov, P. L., Serebryakova, M. V., Moshkovskii, S. A., and

Shishkin, S. S. (2006) Polymorphism of delta3,5-delta2,4-dienoyl-coenzyme A isomerase (the ECH1

gene product protein) in human striated muscle tissue. Biochemistry (Mosc) 71, 448-453

90. Nahleh, Z., Tfayli, A., Najm, A., El Sayed, A., and Nahle, Z. (2012) Heat shock proteins in cancer:

targeting the 'chaperones'. Future Med Chem 4, 927-935

91. Yamashima, T. (2012) Hsp70.1 and related lysosomal factors for necrotic neuronal death. J

Neurochem 120, 477-494

92. Oikawa, S., Yamada, T., Minohata, T., Kobayashi, H., Furukawa, A., Tada-Oikawa, S., Hiraku, Y.,

Murata, M., Kikuchi, M., and Yamashima, T. (2009) Proteomic identification of carbonylated

proteins in the monkey hippocampus after ischemia-reperfusion. Free Radic Biol Med 46, 1472-1477

93. Moller, I. M., and Kristensen, B. K. (2004) Protein oxidation in plant mitochondria as a stress

indicator. Photochem Photobiol Sci 3, 730-735

94. Szoke, D., and Panteghini, M. (2012) Diagnostic value of transferrin. Clin Chim Acta 413, 1184-1189

95. Matsuda, R., Spector, D., Micou-Eastwood, J., and Strohman, R. C. (1984) There is selective

accumulation of a growth factor in chicken skeletal muscle. II. Transferrin accumulation in dystrophic

fast muscle. Dev Biol 103, 276-284

96. Ghosh, A. K., and Jacobs-Lorena, M. (2011) Surface-expressed enolases of Plasmodium and other

pathogens. Mem Inst Oswaldo Cruz 106 Suppl 1, 85-90

97. Lopez-Alemany, R., Suelves, M., Diaz-Ramos, A., Vidal, B., and Munoz-Canoves, P. (2005) Alpha-

enolase plasminogen receptor in myogenesis. Front Biosci 10, 30-36

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Acknowledgements- The authors thank all the patients and their families for the muscle biopsies. The

technical help of Mrs. Hemavathy in electron microscopy experiments is gratefully acknowledged. The

authors acknowledge the technical assistance of L. Asmitha, TIFR, Mumbai, India in RT-PCR

experiments. The assistance of Dr. Sonam Kothari in the muscle strength analysis of human patients is

gratefully acknowledged. The technical help of Dr. Phalguni Alladi in confocal microscopy is gratefully

acknowledged.

FOOTNOTES

*This work was financially supported by the Indian Council of Medical Research (ICMR) (No.

54/8/2011-HUM-BMS) to MMSB. RR is a senior research fellow of the Council for Scientific and

Industrial Research (CSIR), India. @

To whom correspondence should be addressed: M.M. Srinivas Bharath, Department of Neurochemistry,

National Institute of Mental Health and Neurosciences (NIMHANS), No. 2900, Hosur Road, Bangalore-

560029. Tel: +91-80-26995167. Fax: +91-80-26564830. Email: bharath@nimhans.kar.nic.in 5Institute of Bioinformatics, Discoverer building, International Technology Park Limited, Whitefield,

Bangalore-560066, Karnataka, India. 6Division of Biological Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Colaba,

Mumbai-400005, Maharashtra, India. 7The abbreviations used are: CTX, cardiotoxin; MD, Muscular dystrophy; DMD, Duchenne muscular

dystrophy; Sgpy, Sarcoglycanopathy; Dysfy, Dysferlinopoathy; IM, Inflammatory myopathy; MM,

Mitochondrial myopathy; DM, Distal myopathy; ROS, reactive oxygen species; GSH, glutathione

(reduced); GSSG, glutathione (GSSG); Ca2+

, calcium; EM, electron microscopy; DNP, Dinitrophenyl;

H&E, Haematoxylin & Eosin; MGT, Modified Gomori’s Trichrome; EHC, enzyme histochemistry;

NADH-TR, Nicotinamide Adenine dinucleotide tetrazolium reductase; SDH, Succinic dehydrogenase;

COX, Cytochrome oxidase; IHC, immunohistochemistry; TA, Tibialis anterior; EBD, Evan’s blue dye;

MAT, Masson’s Trichrome; ETC, Electron transport chain; CI, Mitochondrial complex I; CII,

Mitochondrial complex II; CIII, Mitochondrial complex III; CK, Creatine kinase; CS, Citrate synthase;

NO, nitric oxide.

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Table 1. Details of human samples used in the human grip strength analysis (DMD: Duchenne Muscular

Dystrophy; Dysfy: Dysferlinopathy; IM: Inflammatory myopathy).

Sl. No. Disease Age (y) Sample number (n) Gender (M/F)

1. Control 28±5 10 7/3

2. DMD 7±3 15 15/0

3. Dysfy 25±5 15 9/6

4. IM 38±10 15 5/10

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Table 2. List and classification of mitochondrial proteins significantly down-regulated in the CTX mouse

model (sal: saline control). Details include ref seq accession, gene symbol, description and fold change

(d1/sal= 115/114; d3/sal= 116/114)

(i) Complex I (NADH dehydrogenase [ubiquinone] complex) subunits

No. Ref Seq Acc no. Gene

Symbol

Description Fold change over

control

d1/Sal d3/Sal

1 NP_694704.1 Ndufs2 Iron-sulfur protein 2 0.48 0.35

2 NP_080890.1 Ndufa5 Alpha sub-complex subunit 5 0.47 0.34

3 NP_035017.2 Ndufs4 Iron-sulfur protein 4 0.47 0.35

4 NP_062308.2 Ndufb11 Beta sub-complex subunit 11 0.40 0.29

5 NP_598427.1 Ndufv1 Flavoprotein 1 0.39 0.29

6 NP_080263.1 Ndufa6 Alpha sub-complex subunit 6 0.37 0.26

7 NP_080337.1 Ndufb8 Beta sub-complex subunit 8 0.37 0.22

8 NP_080960.1 Ndufb10 Beta sub-complex subunit 10 0.36 0.39

9 NP_083548.1 Ndufs7 Iron-sulfur protein 7 0.36 0.31

10 NP_663493.2 Ndufs1 75 kDa subunit 0.35 0.21

11 NP_079873.1 Ndufb3 Beta sub-complex subunit 3 0.33 0.26

12 NP_001078962.1 Ndufaf6 Complex I assembly factor 6 0.33 0.42

13 NP_001077360.1 Ndufv3 Flavoprotein 3 0.32 0.32

14 NP_079634.2 Ndufa9 Alpha sub-complex subunit 9 0.28 0.17

15 NP_079827.2 Ndufa12 Alpha sub-complex subunit 12 0.25 0.21

16 NP_075691.1 Ndufa7 Alpha sub-complex subunit 7 0.17 0.12

17 YP_002791052.1 ND5 Subunit 5 0.15 0.14

18 NP_001025445.1 Ndufs5 Iron-sulfur protein 5 0.12 0.09

19 NP_035018.1 Ndufs6 Iron-sulfur protein 6 0.10 0.09

20 NP_080964.1 Ndufs3 Iron-sulfur protein 3 0.08 0.06

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(ii) Electron transport chain and ATP synthesis

No. Ref Seq Acc no. Gene

Symbol

Description Fold change

over control

d1/Sal d3/Sal

1 NP_034404.3 Gpd2 glycerol-3-phosphate dehydrogenase 0.47 0.32

2 NP_848808.1 Coq4 ubiquinone biosynthesis protein COQ4

homolog

0.45 0.42

3 NP_031773.2 Cox5a cytochrome c oxidase subunit 5A 0.44 0.06

4 NP_080780.1 Coq5 2-methoxy-6-polyprenyl-1,4-benzoquinol

methylase

0.44 0.33

5 NP_079597.2 Sdhc succinate dehydrogenase cytochrome b560

subunit

0.43 0.42

6 NP_031531.1 Atp5a1 ATP synthase subunit alpha 0.42 0.37

7 NP_031532.2 Atp5g1 ATP synthase lipid-binding protein 0.42 0.22

8 NP_034074.1 Cox7a1 cytochrome c oxidase subunit 7A1 0.36 0.25

9 NP_079843.1 Cyc1 cytochrome c1, heme protein 0.36 0.27

10 XP_001475467.1 LOC

100046079

PREDICTED: cytochrome c oxidase subunit

5B

0.35 0.26

11 NP_659123.2 Cox15 COX15 homolog 0.35 0.38

12 NP_082333.1 Cyb5r1 NADH-cytochrome b5 reductase 1 0.31 0.21

13 NP_083296.2 Abcb8 ATP-binding cassette sub-family B member

8

0.29 0.26

14 NP_033722.1 Abcb7 ATP-binding cassette sub-family B member

7

0.28 0.25

15 NP_034071.1 Cox4i1 cytochrome c oxidase subunit 4 isoform 1 0.27 0.20

16 NP_075770.1 Sdha SDH [ubiquinone] flavoprotein subunit 0.25 0.22

17 NP_079926.1 Uqcr11 cytochrome b-c1 complex subunit 10 0.23 0.27

18 YP_001686702.1 ATP8 ATP synthase F0 subunit 8 0.21 0.32

19 NP_031834.1 Cycs cytochrome c, somatic 0.21 0.14

20 NP_001106209.1 Atp5c1 ATP synthase subunit gamma 0.20 0.16

21 NP_080728.1 Coq9 ubiquinone biosynthesis protein COQ9 0.19 0.20

22 YP_001686701.1 COX2 cytochrome c oxidase subunit II 0.16 0.13

23 NP_075863.2 Sdhb SDH [ubiquinone] iron-sulfur subunit 0.15 0.11

24 NP_080175.1 Uqcrc2 cytochrome b-c1 complex subunit 2 0.12 0.18

25 NP_613063.1 Atp5o ATP synthase subunit O 0.10 0.11

26 NP_079628.1 Uqcrq cytochrome b-c1 complex subunit 8 0.10 0.08

27 NP_444301.1 Cox6c cytochrome c oxidase subunit 6C 0.06 0.09

28 NP_079589.2 Atp5d ATP synthase subunit delta 0.06 0.07

29 NP_079917.1 Uqcrh cytochrome b-c1 complex subunit 6 0.04 0.03

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(iii) Fatty acid oxidation

(iv) Krebs cycle related

No. Ref Seq Acc no. Gene

Symbol

Description Fold change

over control

d1/Sal d3/Sal

1 NP_032836.1 Pdha1 PDH E1 component subunit alpha,

somatic form

0.48 0.83

2 NP_080720.1 Cs citrate synthase 0.47 0.42

3 NP_061289.1 Mpc1 mitochondrial pyruvate carrier 1 0.44 0.45

4 NP_083849.1 Idh3a isocitrate dehydrogenase [NAD] subunit

alpha

0.30 0.30

(v) Other mitochondrial proteins

No.

Ref Seq Acc no. Gene

Symbol

Description Fold change

over control

d1/Sal d3/Sal

1 NP_079612.1 Chchd3 coiled-coil-helix-coiled-coil-helix

domain-containing protein 3,

mitochondrial precursor

0.49 0.31

2 NP_059100.3 Mrpl39 39S ribosomal protein L39, mitochondrial 0.46 0.41

3 NP_766024.1 Slc25a12 calcium-binding mitochondrial carrier

protein Aralar1

0.43 0.29

4 NP_001156390.1 Ccdc90b coiled-coil domain-containing protein

90B, mitochondrial isoform 2

0.43 0.48

5 NP_001171043.1 Slc25a13 calcium-binding mitochondrial carrier

protein Aralar2 isoform 2

0.43 0.32

6 NP_082549.2 Pgam5 serine/threonine-protein phosphatase

PGAM5, mitochondrial isoform 2

0.42 0.45

No. Ref Seq Acc no. Gene

Symbol

Description Fold change

over control

d1/Sal d3/Sal

1 NP_444349.1 Echs1 enoyl-CoA hydratase 0.49 0.70

2 NP_849209.1 Hadha trifunctional enzyme subunit alpha 0.41 0.27

3 NP_031407.2 Acadl

long-chain specific acyl-CoA

dehydrogenase 0.35 0.49

4 NP_663533.1 Hadhb trifunctional enzyme subunit beta 0.33 0.37

5 NP_082453.2 Ndufab1 acyl carrier protein 0.14 0.12

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7 NP_038927.2 Timm10 mitochondrial import inner membrane

translocase subunit Tim10

0.39 0.41

8 NP_109642.1 Lactb serine beta-lactamase-like protein

LACTB, mitochondrial precursor

0.37 0.46

9 NP_081226.1 Ociad2 OCIA domain-containing protein 2 0.37 0.53

10 NP_064429.2 Nfu1 NFU1 iron-sulfur cluster scaffold

homolog, mitochondrial isoform 2

precursor

0.34 0.44

11 NP_031957.1 Endog endonuclease G, mitochondrial precursor 0.29 0.24

12 NP_062726.3 Htra2 serine protease HTRA2, mitochondrial 0.25 0.41

13 NP_001078969.2 Cisd3 CDGSH iron-sulfur domain-containing

protein 3, mitochondrial precursor

0.25 0.20

14 NP_032857.1 Phb prohibitin 0.24 0.25

15 NP_079568.1 Synj2bp synaptojanin-2-binding protein 0.23 0.27

16 NP_001239325.1 Slc25a19 mitochondrial thiamine pyrophosphate

carrier isoform 4

0.22 0.23

17 NP_940807.1 Ckmt2 creatine kinase S-type, mitochondrial

precursor

0.22 0.16

18 NP_034455.1 Got2 aspartate aminotransferase, mitochondrial 0.21 0.26

19 NP_570962.2 Rtn4ip1 reticulon-4-interacting protein 1,

mitochondrial precursor

0.20 0.27

20 NP_035824.1 Vdac1 voltage-dependent anion-selective channel

protein 1

0.11 0.10

21 NP_035825.1 Vdac2 voltage-dependent anion-selective channel

protein 2

0.07 0.12

22 NP_031599.2 C1qbp complement component 1 Q

subcomponent-binding protein,

mitochondrial

0.02 0.03

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Table 3. List of under-expressed proteins in the CTX model that have structural/functional interactions

with the mitochondrion (sal: saline control). Details include ref seq accession, gene symbol, description

and fold change (d1/sal= 115/114; d3/sal= 116/114)

No. Ref Seq Acc no.

Gene

Symbol Description

Fold change

over control

d1/sal d3/sal

1 NP_076186.1 Rdh14 retinol dehydrogenase 14 0.85 0.66

2 NP_849534.2 Atad3a

ATPase family AAA domain-

containing protein 3 0.85 0.74

3 NP_080841.1 Apool apolipoprotein O-like precursor 0.84 0.60

4 NP_001013031.2 Dhrs7c

dehydrogenase/reductase SDR

family member 7C precursor 0.37 0.37

5 NP_001165583.1 Dhrs7b

dehydrogenase/reductase SDR

family member 7B isoform 1 0.28 0.35

6 NP_780421.2 Tmem65 transmembrane protein 65 0.25 0.25

7 NP_075720.1 Stoml2 stomatin-like protein 2 0.76 0.92

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Table 4. List of proteins which were carbonylated in total extracts from DMD muscle.

Spot

no.

Accession Description Score Coverage MW

[kDa]

calc.

pI

17 4501867 aconitate hydratase, mitochondrial precursor

(ACO2) [NP_001089.1]

442.95 53.85 85.4 7.61

18 4557871 serotransferrin precursor (TF) [NP_001054.1] 538.60 70.63 77.0 7.12

19 194248072 heat shock 70 kDa protein 1A/1B (HSPA1A)

[NP_005336.3]

125.91 34.95 70.0 5.66

20 5729877 heat shock cognate 71 kDa protein isoform 1

(HSPA8) [NP_006588.1]

386.34 42.41 70.9 5.52

21 4503571 alpha-enolase isoform 1 (ENO1) [NP_001419.1] 456.36 61.98 47.1 7.39

22 &

23

70995211 delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase,

mitochondrial precursor (ECH1) [NP_001389.2]

195.50 54.27 35.8 8.00

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Table 5. Details of human samples used in the biochemical analyses of myopathies.

Sl. No. Disease Age (Years) Sample number (n) Gender (M/F)

1 Control 28±5 10 7/3

2 DM 33±12 15 13/2

3 MM 29±8 13 7/6

4 IM 36±10 15 4/11

5 SMA 11±6 15 8/7

DM: Distal myopathy, MM: Mitochondrial myopathy, IM: Inflammatory myopathy, SMA: Spinal

muscular atrophy.

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FIGURE LEGENDS

FIGURE 1. Muscle weakness and degeneration in the CTX mice and human muscle pathologies. A,

Muscle strength in human Duchenne muscular dystrophy (DMD; n=15), dysferlinopathy (Dysfy; n=15)

and inflammatory myopathies (IM; n=15) compared to healthy controls (C) (n=10), expressed as grade

(0-5) (***p<0.001). B, Analysis of grip strength in CTX or saline injected (C) TA muscles of C57BL/6

mice assessed from day 1 to day 31 post injection (d1 to d31), expressed as Kg force (***p<0.001). C,

Analysis of muscle damage by confocal microscopy of EBD dye fluorescence in CTX and saline injected

mice muscle. P-sal: Phase contrast image of saline injected muscle, sal: fluorescent images of saline.d1-

d7: Fluorescent images (Magnification X400). D, H&E stained sections of saline injected (sal) TA muscle

and CTX-injected muscle at d1 to d31 (Magnification X400); Infiltration of inflammatory cells into the

interstitial spaces indicated by star; Degenerating muscle fibers indicated by filled circles; centrally

nucleated cells indicated by arrow. E, H&E staining of human control (C), DMD, Dysfy and IM biopsies

(Magnification X400). Central nucleation (arrow), inflammation (star), necrotic fibers (o) are indicated. F,

The number of centrally nucleated cells expressed as % cells/ total cells/ field in control (C) and CTX-

injected muscle at d1-d31. 6 random fields were assessed per time point; **p<0.01, ***p<0.001

compared to control. G, MAT staining of saline injected (sal) and CTX-injected mice muscle (d1-

d31).The staining shows central nucleus (arrow) (Magnification X400). Sections from DMD and

Dysferlinopathy (Dysfy) show fibrosis (blue staining around muscle fibers, black arrow) (Magnification

X250).

FIGURE 2. Elevated apoptotic markers and inflammatory genes in the CTX mouse model. Gene

expression changes (Complete name of the genes and primer sequences are given in supplementary

material) in the levels of (A) apoptotic markers TNF, FasL and LITAF (B) cytokines CCL2, CCL3,

CCL6, MRC1, IL-1b and IL6 and (C) cytokine receptors CCR1, CXCR4 and CCR5 at d1-d14 in CTX

injected TA muscle of C57BL6 mice analyzed by qRT-PCR. All values expressed as fold change over

saline injected control, carried out in triplicate of 6 experimental sets and averaged; *p<0.05, **p<0.01,

***p<0.001 compared to control.

FIGURE 3. Sarcolemmal integrity is compromised in the CTX mouse model and human pathologies. A,

cryosections of control (sal) and CTX injected muscle (d1 to d31) immunostained for dystrophin, alpha-

sarcoglycan and dysferlin are shown (Magnification X400). Loss of membrane staining at d1 and d3

shows recovery at d7 and subsequent time points. B, H&E staining and immunostaining for Dystrophin

(Dys-1), Sarcoglycan (-sarc) and Dysferlin in muscle biopsies of DMD, -Sgpy and Dysfy compared to

control (Magnification X400) indicate specific loss of immunoreactivity of membrane proteins.

FIGURE 4. Ultrastructural changes in the CTX model and human pathologies. A, EM analysis of saline

(sal) injected muscle shows LS of (i) sarcolemma (X68000) (ii) T-system (X23000) (iii) cytoskeleton

(X30000) and TS of cytoskeleton (X68000) (iv). The sarcolemmal outer and inner layers are depicted by

block arrows in (i).Triads of the T-system are indicated by block arrows (ii). The isotropic-band (I),

anisotropic-band (A), Z-line (Z), and M-line (M) of the cytoskeletal sarcomeres are depicted in (iii). In

the TS of cytoskeleton (iv), the myosin bands are seen as dark dots surrounded by light actin bands. B,

EM images from d1 shows (i) inflammatory cells (IC) seen attacking the muscle cell (X2000) (ii)

sarcolemmal breakage (arrow, X5000) (iii) disrupted sarcolemma (arrows), homogenized cytoskeleton

(star) and IC (X10000) and (iv) homogenized cytoskeleton (star) (X10000). C, EM images from d3 show

(i) IC outside the degenerating muscle cells with sarcolemmal loss (arrow) (X2300) (ii) sarcolemmal

breach (arrows) and intracellular IC in the muscle cell (X2700) and (iii) extensive cytoskeletal

homogenization (star) and tiny vesicles (filled circle) indicating the degenerating T-system (X30000). D,

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EM images from DMD muscle shows homogenized cytoskeleton (arrow) (X7200) while Dysfy muscle

shows (i) disrupted sarcolemma (arrow) (X30000) and (ii) vesicles indicating degenerating T-system

(X30000). E, Ultrastructutral analysis from d7 show (i) variation in fibre size with central nucleus,

satellite cells (S) and intact membrane (arrow) (X1400) (ii) few neutrophils (N) and macropahages (M)

Fibroblasts (F) with prominent RER, a nerve twig (NT) and blood vessel (BV) (X2900). F, Images from

d11 show (i) perinuclear glycogen granules (star) (X6800) (ii) sub-peripherally placed nuclei and normal

sarcolemma (arrow) (X2900). G, EM analysis at d14 show (i) centrally nucleated cells (X2900) (ii)

satellite cells (S) and normal sarcolemma (arrow) (X9300). H, EM analysis at d31 indicating centrally

nucleated cells (X6800) with perinuclear aggregation of mitochondria (arrow) with normal and well

developed cristae (X6800).

FIGURE 5. Calcium deregulation in the CTX model with implications for human pathologies. A, Sub-

sarcolemmal calcium deposition in the CTX mouse model. Paraffin sections stained with Alizarin red;

saline control (sal) and CTX injected muscle showing sub-sarcolemmal staining at d1 and d3 (arrows),

similar to that seen in DMD (arrow), (Magnification X400). B-C, Assessment of time dependent in vitro

toxicity induced by CTX at 2M and 1M in undifferentiated and differentiated C2C12 cells

respectively, shows ~50% cell death at 40 h. **p<0.01, ***p<0.001. Calcium dysregulation in vitro in

undifferentiated (D and F) and differentiated cells (E and G) exposed to CTX. D and E, CTX increased

the fluorescence intensity ratio (340/380) in cells loaded with Fura-2 AM. F and G, Pretreatment of cells

(undifferentiated and differentiated) with EGTA (green) prevented the increase in fluorescence intensity

ratio upon CTX (red) exposure. Upon addition of exogenous Ca2+

(blue), the fluorescence intensity ratio

increased. H, Fura-2 loaded cells pretreated with 10 M nifidepine (green) were exposed to CTX (red)

and the changes in fluorescence intensity ratio depicted. I, cells loaded with or without the fluorescent dye

calcein were exposed to CTX followed by fluorescence scan of the spent medium. The fluorescence scan

(500-525 nm) and the emission wavelength maximum of calcein (510 nm) in the spent medium is

indicated (arrow). Calcein fluorescence in the spent medium following exposure of undifferentiated and

differentiated cells to CTX for different time points (0.5 to 30 min) is also shown.***p<0.001 compared

to control (C). J, Ca2+

-influx mediated mitochondrial dysfunction induced by CTX. Cells loaded with the

fluorescent mitochondrial Ca2+

probe Rhod-2 were exposed to CTX followed by fluorescence analysis.

CTX-mediated increase of Rhod-2 fluorescence is indicated by (i) images of cells before and after CTX

addition and (ii) fluorescence spectra of Rhod-2. Ca2+

-influx dependent Rhod-2 fluorescence is abrogated

in cells (both undifferentiated and differentiated) pre-treated with EGTA. ***p<0.001 compared to

control (C).

FIGURE 6. Mitochondrial dysfunction in the CTX model and human pathologies. A, CTX treatment

induced mitochondrial dysfunction in C2C12 cells. Cells (Undifferentiated and differentiated) loaded with

the mitochondria-specific fluorescent dye JC-1 were treated with or without CTX followed by detection

of fluorescence at 590 nm. CTX caused elevation in JC-1 fluorescence at 590 nm, which was abrogated

by pretreatment with EGTA; ***p<0.001 compared to control (C). Assessment of mitochondrial function

in the CTX mouse model by enzyme histochemical (EHC) staining for SDH (B), SDH-COX (C) and

NADH-TR (D) in the cryosections of saline control (sal), CTX-injected muscle at d1-d31 and in human

biopsies of DMD, Dysfy or MD and IM. Loss of EHC signal indicated by star in the images from the

CTX model and by arrows in human mayopathies (Magnification X250). E, MGT staining in saline (sal)

injected and CTX injected (d1 and d3) muscle and in muscle from human pathologies mitochondrial

myopathy-MM, Dysfy and IM to analyze mitochondrial aggregation. The characteristic sub-sarcolemmal

aggregation (ragged red fibers [RRF]) usually seen in MM (arrows) was not observed in the CTX model

(Magnification X400) or in Dysfy and IM. F, EM images of mitochondrial alterations in saline-injected

(sal) (Magnification X18500) and CTX-injected muscle. Mitochondria with altered cristae and

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vacuolation were observed at d1 (Magnification X6700) and d3 (X18500) (arrows), while images from d7

(X6800) showed fewer mitochondria with abnormal cristae (arrow) and perinuclear glycogen granules

(star). Mitochondrial structure was normal in d14 (X 30000) (yellow arrow) and normal sarcolemma

(black arrow). Human DMD sample shows muscle degeneration, sarcolemmal damage (black arrows) and

abnormal mitochondria (yellow arrow) (X7200). Dysfy sample shows sarcolemmal damage (white arrow)

normal (black arrow) and abnormal mitochondria (yellow arrow) (X30000). Mitochondrial dysfunction at

d1-d7 was also evident from increased ADP/ATP ratio at d1 and d3 (G) and lowered NADH content at

d1-d7 (H); ***p<0.001, **p<0.01 and *p<0.05 compared to saline (sal) control. The cellular metabolic

activity was also significantly affected, as indicated by decreased pyruvate (d1-d7) (I) and decreased

lactate content (d1-d3) (J); ***p<0.001and *p<0.05 compared to saline (sal) control.

FIGURE 7. Proteomic analysis of CTX injected muscle mitochondria. A, Mitochondrial isolation of

CTX injected muscle samples for proteomic analysis. Mitochondria from control (sal) and CTX injected

muscle samples (d1, d3 and d7) were isolated and the purity assessed by electron microscopy

(Magnification X23000). B, Schematic diagram depicting the number of mitochondrial proteins

displaying altered expression in d1 and d3, compared to saline control. C, Relative quantitation of over

expressed mitochondrial proteins (≥1.5-fold compared to control) at d1 and d3 compared to control. The

description of the abbreviated protein names are provided in Table 2. Relative quantitation of under

expressed mitochondrial proteins is shown in D-H. Relative expression of mitochondrial complex I

subunits (D), Electron transport and ATP synthesis (E), fatty acid oxidation (F), Krebs cycle related

proteins (G) and other under-expressed mitochondrial proteins (H) at d1 and d3 compared to control is

shown. The descriptions for the abbreviated protein names are provided in Table 2.

FIGURE 8. Enzyme assays and western blot data of respiratory complexes (A-H) and other

mitochondrial proteins (I-K) validate the proteomic data obtained from the CTX-mouse model. A, C and

E, Lowered activities of respiratory complexes I, II and III respectively, in muscle mitochondria from

CTX injected muscle at d1, d3 and d7, compared to saline control (sal); ***p<0.001 compared to control.

B, D and F, Decreased activities of complexes I, II and III respectively, in muscle mitochondria from

Dysfy patients (n=10) compared to control (n=6); *p<0.05 and ***p<0.001 compared to control. G,

Representative western blot (using antibody cocktail) of individual subunits from respiratory complexes

(complex I= CI, complex II= CII, complex III= CIII, complex IV= CIV and complex V= CV) in

mitochondrial extract of the CTX-injected muscle showing the relative quantitation of the proteins at d1-

d7 compared to saline control (C) (The description of the abbreviated protein names are provided in Table

2). The numbers above each band indicates its relative abundance as a ratio of the corresponding

coomassie stained protein bands, compared to the control (C). H, Coomassie stained SDS gels indicating

relatively equal loading of total mitochondrial protein from d1-d7. M=Molecular weight in kDa. I , J and

K correspond to lowered activities of aspartate aminotransferase (ASAT/ GOT), creatine kinase (CK) and

citrate synthase (CS) in muscle mitochondria from the CTX model at d1-d7 compared to control (sal).

*p<0.05 and ***p<0.001 compared to sal.

FIGURE 9. Oxidative stress associated with CTX-induced muscle pathology and human myopathies. A,

Determination of the red-ox status by GSH/GSSG ratio at d1-d7 following CTX administration compared

to saline control (sal); **p<0.01, ***p<0.001 compared to control. B, Lowered GSH/GSSG in human

Dysferlinopathy (Dysfy) muscle (n=6) compared to controls (n=6); ***p<0.001 compared to control (C).

C, Relative mRNA expression of gamma glutamyl cysteine ligase-catalytic subunit (GCL-c) (primer

sequences are given in supplementary material) in the CTX injected muscle at d1-d14, analyzed by qRT-

PCR. The values are expressed as fold change over saline injected control, carried out in triplicate of 6

experimental sets and averaged; ns=not statistically significant compared to control. D, Elevated lipid

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Mitochondrial changes and oxidative stress in muscle pathology

34

peroxidation indicated by the relative fluorescence (AU = arbitrary units) of the peroxidation sensor

BODIPY C11 in the CTX-injected muscle at d1-d7compared to saline control (sal); ***p<0.001

compared to control (sal). E, Elevated lipid peroxidation (using BODIPY C11 fluorescence) in human

DMD muscle compared to control (C); ***p<0.001 compared to control. F and G, Changes in total

intracellular ROS corresponding to Amplex-red fluorescence (AU = arbitrary fluorescence units) in

C2C12 cells (undifferentiated and differentiated) following CTX exposure (± EGTA); *p<0.05

***p<0.001 compared to control. H and I, Changes in total intracellular oxidants corresponding to

Dihydroethidium (DHE) fluorescence (AU = arbitrary fluorescence units) in C2C12 cells

(undifferentiated and differentiated) following CTX exposure (± EGTA); **p<0.01 ***p<0.001

compared to control. J and K, Changes in the higher oxides of NO corresponding to DAF-FM

fluorescence (AU = arbitrary fluorescence units) in C2C12 cells (undifferentiated and differentiated)

following CTX exposure (± EGTA); ***p<0.001 compared to control. L to O, Quantitative analysis of

redox markers including MDA (L), total GSH (M) and protein oxidation (N and O) in patient muscle

biopsies of distal myopathies (DM, n=15), mitochondrial myopathies (MM, n=15), IM (n=15) and spinal

muscular atrophies (SMA 1, 2 and 3, n=15) compared to control (n=10); *p<0.05, **p<0.01, ***p<0.001

compared to healthy controls. N, Specificity and concentration-dependence of the oxyblot method of

estimating protein carbonyls by dot blot (carried out in mouse muscle extract). Lane 1, total muscle

extract+DNPH, Lane 2= Extract without DNPH, Lane 3= Sample without extract+ DNPH, Lane 4=

Muscle extract (5l) +DNPH, Lane 5= Muscle extract (10l) +DNPH, Lane 6= Muscle extract (15l)

+DNPH, Lane 7= Brain extract (positive control) +DNPH, lane 8= extract + DNPH, without primary

antibody (Negative control).

FIGURE 10. Proteomic identification of oxidized proteins in DMD. A, Total extracts of DMD (n=6) and

control (n=6) were derivatized with DNPH and subjected to 2D-PAGE followed by anti-DNP western

blot. The protein spots on the western blot were compared to the sypro-ruby stained 2D gel of total

protein extract from DMD muscle to identify oxidized proteins common between control and DMD

(outlined by complete circle)and those specific to DMD (outlined by broken circle). There were 23

distinct spots in DMD blot, of which 8 (numbered 16-23) were not represented in control. Seven spots

(No. 17 to 23) were excised out for analysis by mass spectrometry. B, list of the spot numbers and the

corresponding muscle proteins that are potentially oxidized in DMD patients compared to control. C, The

activity of Aconitase, one of the proteins carbonylated in the DMD muscle is significantly decreased.

Activity indicated in % (100 % activity=0.069 mM/min/mg protein); *p<0.05 compared to control (C). D,

Aconitase activity (in mouse muscle) is inhibited by H2O2. **p<0.01 compared to untreated control (C).

FIGURE 11. Schematic representation of the cellular changes in the CTX model. A, Time line indicating

relative changes of cellular markers at different time points (d1-d31) following CTX treatment. B,

Cellular events triggered by CTX ultimately leading to muscle degeneration. C, Comparison of the

cellular effects in the CTX model with human pathologies.

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C

D

Figure 1

E

A

5

2.9 2.8 3.2

Mu

scle

str

en

gth

(g

rad

e)

C DMD Dysfy IM

***

*** ***

P-sal sal d1 d3 d5 d7

B

C d1 d2 d3 d4 d5 d7 d11 d14 d31

sal CTX

Days post CTX injection

*** *** *** *** *** *** ***

***

***

Kg

fo

rce

0

0.01

0.04

0.05

0.06

0.07

0.02

0.03

C d1 d3 d5 d7 d11 d14 d31

*** *** *** ***

**

Cen

trall

y n

uc

leate

d c

ell

s

(% c

ell

s/t

ota

l cell

s/f

ield

)

F

0

40

60

80

100

120

20

C DMD Dysfy Sgpy

IM

sal d1 d3 d5

d7 d11 d14 d31

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sal d1 d3

d7 d11 d14

d31 DMD Dysfy

IM

G

Figure 1 (Contd.)

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Figure 2

0

5

10

15

20

mR

NA

exp

ressio

n

(fo

ld in

cre

ase/c

on

tro

l)

d1 d2 d3 d7 d14

TNFa A

***

***

***

0

2

4

6

8

10

d1 d2 d3 d7 d14

FasL

**

***

***

d1 d2 d3 d7 d14 0

2

4

6

LITAF

*** *** ***

**

CCL2

5

10

15

20

d1 d2 d3 d7 d14

mR

NA

exp

ressio

n

(fo

ld in

cre

ase/c

on

tro

l)

B

0 d1 d2 d3 d7 d14 d1 d2 d3 d7 d14

CCL3 CCL6

0

10

20

30

0

10

20

30

40

*** ***

***

** *** *** ***

***

***

*** ***

***

***

d1 d2 d3 d7 d14 d1 d2 d3 d7 d14 d1 d2 d3 d7 d14

mR

NA

exp

ressio

n

(fo

ld in

cre

ase/c

on

tro

l)

0

2

4

6

MRC1 IL1b

0

20

60

40

0

2

4

6

IL6

***

**

* *

*** *

*** ***

**

d1 d2 d3 d7 d14 d1 d2 d3 d7 d14 d1 d2 d3 d7 d14

CCR1

0

10

20

30

40

50

0

5

10

15 CXCR4 CCR5

0

5

10

15

20

mR

NA

exp

ressio

n

(fo

ld in

cre

ase/c

on

tro

l)

C

***

***

***

***

***

***

***

*** ***

***

***

*

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Dystrophin

sal d1 d3 d7 d11 d14 d31

Alpha

sarcoglycan

Dysferlin

Figure 3

A

B Normal DMD a-Sgpy Dysfy

HE

Dys-1

a-sarc

Dysferlin

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A I

Z M

X68000 X23000 X68000 X30000

X2000 X5000 X10000 X10000

IC

IC

IC

A

(sal)

B

(d1)

Figure 4

(i) (ii) (iii) (iv)

(i) (ii) (iii) (iv)

X2700 X30000 X2300

IC IC IC

DMD Dysfy i ii

X7200 X30000 X30000

(i) (ii) (iii)

C

(d3)

D

(i) (ii)

S NT F

N N

M BV X1400 X2900

E

(d7)

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G

(d14)

S

X6800 X2900

X2900 X9300 X6800

X23000

(i) (ii)

(i) (ii) F

(d11)

H

(d31)

Figure 4 (Contd.)

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A

Figure 5 sal d1 d3 DMD

8 16 24 32 40 48

Time (h)

Cell

via

bil

ity (

% c

on

tro

l)

Cell

via

bil

ity (

% c

on

tro

l)

0

50

150

100

0

50

150

100

8 16 24 32 40 48

Time (h)

Differentiated cells

(1 mM CTX)

Undifferentiated cells

(2 mM CTX) B

20 mM Ca2+

2 mM CTX

2 mM EGTA

0 200 400 600 800 1000

0.4

0.2

0.6

0.8

1.0

1.2

1.4

0

Time (s)

Flu

ore

scen

ce i

nte

ns

ity

rati

o (

340/3

80)

0 200 400 600 800 1000

Time (s)

0

0.4

0.2

0.6

0.8

1.0

1.2

1.4

Flu

ore

scen

ce i

nte

ns

ity

rati

o (

340/3

80)

2 mM CTX

0 200 400 600 800 1000

0.4

0.2

0.6

0.8

1.0

0

Time (s)

Flu

ore

scen

ce i

nte

ns

ity

rati

o (

340/3

80)

Undifferentiated cells Differentiated cells

20 mM Ca2+

1 mM CTX

2mM EGTA

0 100 200 300 400 500 600

Time (s)

10 mM Nifidepine

1.0

0.5

1.5

0

Flu

ore

scen

ce i

nte

ns

ity

rati

o (

340/3

80)

Undifferentiated cells

2 mM CTX

C

E D

F

H

1 mM CTX

Differentiated cells

0 200 400 600 800 1000

Time (s)

0

0.4

0.2

0.6

0.8

1.0

1.2

1.4

Flu

ore

scen

ce i

nte

ns

ity

rati

o (

340/3

80)

G

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CTX

0 100 200 300 400 500 600 700 800

1.0

0.5

1.5

0 Rh

od

-2 f

luo

rescen

ce (

A.U

.)

Time (s)

Before CTX After CTX

J i

ii

Figure 5 (contd.)

Differentiated cells Undifferentiated cells

0

50

150

100

250

200

0

50

150

100

250

200

Rh

od

-2 f

luo

rescen

ce

(%

co

ntr

ol)

C EGTA CTX CTX

+EGTA

C EGTA CTX CTX

+EGTA

Rh

od

-2 f

luo

rescen

ce

(%

co

ntr

ol)

CTX

Calcein

Calcein+CTX

500 525 550 575 600

Flu

ore

scen

ce i

nte

ns

ity (

A.U

)

0.4

0.2

0.5

0

0.1

0.3

wavelength (nm)

I

0

500

1500

1000

15000

10000

Calc

ein

flu

ore

scen

ce (

AU

)

***

C 0.5 2 5 10 30

Duration of CTX

treatment (min)

Differentiated cells Undifferentiated cells

C 0.5 2 5 10 30

Duration of CTX

treatment (min)

***

5000

0

Calc

ein

flu

ore

scen

ce (

AU

)

*** ***

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Figure 6

***

C EGTA CTX EGTA+CTX 0

1000

3000

2000

4000

JC

-1 f

luo

rescen

ce

(red

)

***

C EGTA CTX EGTA+CTX 0

1000

3000

2000

4000

5000 Differentiated cells Undifferentiated cells

sal d1 d3 d7 d11

d14 d31 DMD Dysfy IM

d14 d31 DMD MD IM

sal d1 d3 d7 d11

sal d1 d3 d7 d11

d14 d31 DMD Dysfy IM

JC

-1 f

luo

rescen

ce

(red

)

A

B

C

D

SDH

SDH -COX

NADH -TR

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E

MM Dysfy IM

sal d1 d3

MGT

Figure 6 (Contd.)

F

X7200 X30000

X18500 X6700 X18500 X6800

sal d1 d3 d7

d14 DMD Dysfy

X30000

G H

***

0.6

0.3

1.2

0

0.9

***

sal d1 d3 d7

AD

P/A

TP

rati

o

sal d1 d3 d7

0.05

0.15

0.10

0.20

0

0.25

NA

DH

(mM

/ m

g p

rote

in)

**

** **

Lacta

te

(mg

/ m

g p

rote

in)

sal d1 d3 d7

5

15

10

20

0

25

30

Pyru

vate

(mg

/ m

g p

rote

in)

sal d1 d3 d7

0.5

1.5

1

2

0

2.5

3

3.5

*** ***

* *

***

I J

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226 differentially expressed

mitochondrial proteins at d1

& d3

39 up-regulated:

8 proteins ≥1.5 fold

187 down regulated:

80 proteins ≤0.5 fold

20 subunits of mitochondrial complex I

29 proteins of ETC and ATP synthesis

5 proteins of b-oxidation of fatty acids

4 proteins of Krebs cycle

22 other mitochondrial proteins

A Figure 7

E

B

D Complex I subunits

Fo

ld c

han

ge o

ver

co

ntr

ol

Electron transport chain and ATP synthesis

Fo

ld c

han

ge o

ver

co

ntr

ol

Up-regulated proteins

d1 d3

d1 d3 d1 d3

Fo

ld c

han

ge o

ver

co

ntr

ol

Down-regulated proteins

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50 Ndufs2

Ndufa5

Ndufs4

Ndufb11

Ndufv1

Ndufa6

Ndufb8

Ndufb10

Ndufs7

Ndufs1

Ndufb3

Ndufaf6

Ndufv3

Ndufa9

Ndufa12

Ndufa7

ND5

Ndufs5

Ndufs6

Ndufs3 0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Gpd2

Coq4

Cox5a

Coq5

Sdhc

Atp5a1

Atp5g1

Cox7a1

Cyc1

LOC100046079

Cox15

Cyb5r1

Abcb8

Abcb7

Cox4i1

Sdha

Uqcr11

ATP8

Cycs

Atp5c1

Coq9

COX2

Sdhb

Uqcrc2

Atp5o

Uqcrq

Cox6c

Atp5d

Uqcrh

0.50

1.00

1.50

2.00

2.50

Shmt2

Hadh

Acot9

Mrpl38

Mrps24

Cox7a2l

Aldh2

Ndufaf2

X23000

C

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G

F

Figure 7 (Contd.)

H

Krebs cycle related

Fatty acid oxidation Other mitochondrial proteins

Fo

ld c

han

ge o

ver

co

ntr

ol

Fo

ld c

han

ge o

ver

co

ntr

ol

Fo

ld c

han

ge o

ver

co

ntr

ol

0.00

0.25

0.50

0.75

1.00

d1 d3

Echs1

Hadha

Acadl

Hadhb

Ndufab1

0.00

0.25

0.50

0.75

1.00

d1 d3

Pdha1

Cs

Mpc1

Idh3a 0

0.1

0.2

0.3

0.4

0.5

0.6

d 1 d 3

Chchd3

Mrpl39

Slc25a12

Ccdc90b

Slc25a13

Pgam5

Timm10

Lactb

Ociad2

Nfu1

Endog

Htra2

Cisd3

Phb

Synj2bp

Slc25a19

Ckmt2

Got2

Rtn4ip1

Vdac1

Vdac2

C1qbp

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AS

AT

/GO

T a

cti

vit

y

(U/ m

g p

rote

in)

sal d1 d3 d7 0

0.05

0.10

0.15

***

*** ***

sal d1 d3 d7

CK

(m

ito

) acti

vit

y

(U/ m

g p

rote

in)

0

5

10

***

***

*

sal d1 d3 d7 0

20

40

60

*** *** ***

Co

mp

lex III

acti

vit

y

(nM

C

yt-

c

red

uc

ed

/

min

/mg

pro

tein

)

C Dysfy 0

0.2

0.4

0.6 C

om

ple

x III

acti

vit

y

(nm

ol/

mg

pro

tein

)

***

0.8

0

0.05

0.10

0.15

sal d1 d3 d7

Co

mp

lex I a

cti

vit

y

(mM

D

CIP

red

uc

ed

/ m

in/

mg

pro

tein

)

C Dysfy

*** ***

***

***

Co

mp

lex I a

cti

vit

y

(n

mo

l D

CP

IP /m

g p

rote

in)

0

0.02

0.04

0.06

0.08 C

om

ple

x II acti

vit

y

(mM

D

CIP

red

uced

/

min

/mg

pro

tein

)

0

2

4

6

8

***

***

sal d1 d3 d7

A B

C Dysfy

*

0

0.01

0.02

0.03

0.04

Co

mp

lex II acti

vit

y

(nm

ole

/m

g p

rote

in) 0.05

Validation: Respiratory complexes

Validation: Other mitochondrial proteins

C d1 d3 d7

CV-ATP5A (55kDa)

C d1 d3 d7 M

CIII-UQCRC2 (48 kDa)

CIV-MTCO1 (40 kDa)

CII-SDHB (30 kDa)

CI-NDUFB8 (20 kDa)

C D

E F

G H

I J

Figure 8

1.0 0.18 0.18 0.77

1.0 0.18 0.10 0.94

1.0

1.0 0.62 0.42 0.92

1.0 0.86 0.83 0.86

0.31 0.38 1.06

94

66

42

30

sal d1 d3 d7

*** ***

***

CS

acti

vit

y (

nM

DT

NB

/

min

/mg

pro

tein

)

0

25

50

75

100

K

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Figure 9

C2C12 cells

A G

SH

/GS

SG

rati

o

sal d1 d3 d7

1

3

2

4

0

5

*** ***

**

C Dysfy

GS

H/G

SS

G r

ati

o

0.5

1.5

1.0

2.0

0

***

Am

ple

x r

ed

Flu

ore

scen

ce (

AU

)

sal d1 d3 d7 C DMD

2

6

4

8

0

Flu

ore

scen

ce (

AU

) X

10

3/m

g p

rote

in

Flu

ore

scen

ce (

AU

) X

10

3 /m

g p

rote

in

1

3

2

4

0

***

*** *** ***

C EGTA CTX EGTA +CTX

50

150

100

0

***

*

C EGTA CTX EGTA +CTX

*** ***

Am

ple

x r

ed

Flu

ore

scen

ce (

AU

)

50

150

100

0

250

200

C EGTA CTX EGTA +CTX

C EGTA CTX EGTA +CTX

DA

F-F

M

Flu

ore

scen

ce (

AU

) X

10

3

DA

F-F

M

Flu

ore

scen

ce (

AU

) X

10

3

10

30

20

0

40

10

30

20

0

Undifferentiated cells

Undifferentiated cells

Differentiated cells

Differentiated cells

***

***

*** ***

B C

D E

F G

H I

d1 d2 d3 d7 d11 d14

mR

NA

exp

ressio

n

(fo

ld in

cre

ase/c

on

tro

l)

1

3

2

0

GCL-c

ns

4

CTX mouse model

C EGTA CTX EGTA +CTX

C EGTA CTX EGTA +CTX

DH

E F

luo

rescen

ce (

AU

)

1

3

2

0

5

4

DH

E F

luo

rescen

ce (

AU

)

5

15

10

0

20 Undifferentiated cells Differentiated cells

J K

***

***

**

**

Lipid peroxidation Lipid peroxidation

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Human myopathies

1 2 3 4 5 6 7 8

C DM MM IM SMA1,2 SMA3

C DM MM IM SMA1,2 SMA3

OD

(A

U)

To

tal G

SH

(m

g)/

mg

pro

tein

mM

M

DA

/ m

g p

rote

in

0.5

1.5

1.0

2.0

2.5

0

20

60

40

80

100

0

1

3

2

4

5

0

Protein oxidation

C DM MM IM SMA

L M

N

O

Figure 9 (Contd.)

Triplicate

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A

98

68

44

29

16

1

2 3 8

9 10 11

Oxyblot: Control (n=6)

pH 10.0

98

68

44

29

16

pH 3.0

MW (kDa)

12

13 14

15

4 6 7

5

16 17 18 19

20

21

22

23

Oxyblot: DMD (n=6)

98

68

44

29

16

MW (kDa)

12

13 14

15

8

9 10 11

4

6

1

2 3

7

5

pH 10.0 pH 3.0

2D gel-sypro ruby: DMD (n=6) MW (kDa)

12

13 14

15

22

23

8

9 10 11

1

2 3 4

6 7

5

21

18 19

20 16

17

Spot # Protein Identity

17 Aconitate hydratase, mitochondrial precursor

18 Serotransferrin precursor

19 Heat shock 70 kDa protein 1A/1B

20 Heat shock cognate 71 kDa protein isoform 1

21 Alpha-enolase isoform 1

22 & 23 Delta(3,5)-Delta(2,4)-dienoyl-CoA isomerase,

mitochondrial precursor

B

pH 10.0 pH 3.0

C DMD

Aco

nit

ase a

cti

vit

y (

% c

on

tro

l)

0 100 300

H2O2 (mM)

20

60

40

80

100

0

** **

120

140

Aco

nit

ase a

cti

vit

y (

% c

on

tro

l)

20

60

40

80

100

0

120

140

C D

Figure 10

*

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d1 d3 d7 d11 d14 d31

Rela

tive c

han

ge

(rela

tive u

nit

s)

20

60

40

80

100

0

Regeneration

Oxidative stress

Inflammation &

Mitochondrial damage

Repair

Cell death A

B

Human pathologies

(MDs and IMs)

CTX model

Muscle weakness Yes Yes

Pathology: Onset Acute: IMs

Chronic: MDs

Acute

Pathology: Transient/ Permanent Transient: IMs

Permanent: MDs

Transient

Myofiber

Degeneration

Yes Yes

Regeneration (centrally nucleated

regenerating cells)

Yes (DMD, Sgpy) Yes

Infiltration of immune cells Yes Yes

Elevated expression of inflammatory

genes

Yes Yes

Elevated apoptotic markers Yes Yes

Necrotic markers Yes Yes

Sarcolemmal disruption Yes Yes

Disruption of cytoskeletal architecture Yes Yes

Altered T-system Yes Yes

Altered calcium dynamics Yes Yes

Mitochondrial dysfunction Yes Yes

Lowered complex activities Yes Yes

Elevated ROS Yes Yes

C

CTX Sarcolemmal

disruption

Inflammation

Oxidative stress

Ca2+ influx

Mitochondrial

dysfunction

•Elevated Ca2+

•Lowered protein expression

•Decreased enzyme activities

•Decreased membrane potential

•Abnormal structure

•Elevated ROS

•Increased lipid peroxidation

•Decreased glutathione

Muscle

degeneration

Ultrastructural

changes

?

Figure 11

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BharathChandregowda Harsha, Ullas Kolthur-Seetharam and Muchukunte Mukunda SrinivasRajeswarababu Mythri, Atchayaram Nalini, Yashwanth Subbannayya, Hindalahalli Renjini Ramadasan-Nair, Narayanappa Gayathri, Sudha Mishra, Balaraju Sunitha,

pathologiesmuscle degeneration: Implications for muscular dystrophy and related muscle

Mitochondrial alterations and oxidative stress in an acute transient mouse model of

published online November 12, 2013J. Biol. Chem. 

  10.1074/jbc.M113.493270Access the most updated version of this article at doi:

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Supplemental material:

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