REVIEW ARTICLE Oxidative stress and pathology in muscular dystrophies: focus...

REVIEW ARTICLE

Oxidative stress and pathology in muscular dystrophies:focus on protein thiol oxidation and dysferlinopathiesJessica R. Terrill1,2, Hannah G. Radley-Crabb3,1, Tomohito Iwasaki2, Frances A. Lemckert4,Peter G. Arthur2 and Miranda D. Grounds1

1 School of Anatomy, Physiology and Human Biology, University of Western Australia, Perth, Western Australia, Australia

2 School of Biomedical, Biomolecular & Chemical Science, University of Western Australia, Perth, Western Australia, Australia

3 Curtin Health Innovation Research Institute Biosciences Research Precinct, School of Biomedical Sciences, Curtin University,

Western Australia, Australia

4 Institute for Neuroscience and Muscle Research, Children’s Hospital at Westmead, New South Wales, Australia

Keywords

antioxidants; Duchenne muscular dystrophy;

Dysferlinopathies; dystropathology; muscle

necrosis; N-acetylcysteine; oxidative stress;

protein thiol oxidation; skeletal muscle;

reactive oxygen species

Correspondence

M. D. Grounds, School of Anatomy,

Physiology and Human Biology, University

of Western Australia, Perth, Western

Australia 6009, Australia

Fax: +61 8 6488 1051

Tel: +61 8 6488 3486

E–mail: [email protected]

(Received 17 December 2012, revised 14

January 2013, accepted 15 January 2013)

doi:10.1111/febs.12142

The muscular dystrophies comprise more than 30 clinical disorders that are

characterized by progressive skeletal muscle wasting and degeneration.

Although the genetic basis for many of these disorders has been identified,

the exact mechanism for pathogenesis generally remains unknown. It is

considered that disturbed levels of reactive oxygen species (ROS) contrib-

ute to the pathology of many muscular dystrophies. Reactive oxygen spe-

cies and oxidative stress may cause cellular damage by directly and

irreversibly damaging macromolecules such as proteins, membrane lipids

and DNA; another major cellular consequence of reactive oxygen species is

the reversible modification of protein thiol side chains that may affect

many aspects of molecular function. Irreversible oxidative damage of pro-

tein and lipids has been widely studied in Duchenne muscular dystrophy,

and we have recently identified increased protein thiol oxidation in dystro-

phic muscles of the mdx mouse model for Duchenne muscular dystrophy.

This review evaluates the role of elevated oxidative stress in Duchenne

muscular dystrophy and other forms of muscular dystrophies, and presents

new data that show significantly increased protein thiol oxidation and high

levels of lipofuscin (a measure of cumulative oxidative damage) in dysfer-

lin-deficient muscles of A/J mice at various ages. The significance of this

elevated oxidative stress and high levels of reversible thiol oxidation, but

minimal myofibre necrosis, is discussed in the context of the disease mecha-

nism for dysferlinopathies, and compared with the situation for dystro-

phin-deficient mdx mice.

Introduction

Overview of oxidative stress

Reactive oxygen species (ROS) are formed during a

variety of biological processes for all eukaryotes, and

although they are essential for cell signaling, excess

generation of ROS may harm biological components.

This occurs when the action of endogenous defense

mechanisms of the cell, involving various molecules

called antioxidants, is outweighed by the generation of

ROS, a state called oxidative stress [1]. Oxidative stress

Abbreviations

DMD, Duchenne muscular dystrophy; FLM, BODIPY FL–N–(2–aminoethyl) maleimide; NAC, N–acetylcysteine; ROS, reactive oxygen

species; Texas Red, Texas Red C2-maleimide.

FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 1

is implicated in the pathology of numerous conditions,

including aging, inflammatory disorders, cancer, mus-

cle wasting and muscular dystrophies [2–5]. ROS

causes cellular damage by directly and irreversibly

altering macromolecules such as proteins, membrane

lipids and DNA [6], but another (less studied) major

cellular consequence of ROS exposure is the reversible

modification of protein thiol side chains. Thiols are

organic sulfur derivatives, identified by the presence of

sulfhydryl residues (-SH) at their active site. Biological

thiols include low-molecular-weight free thiols and

protein thiols, the functional group of the amino acid

cysteine. In the presence of ROS, sulfhydryl residues

may undergo reversible modifications, whereby sulfhy-

dryl bonds are broken and disulfides are formed.

These thiol modifications are reversible via the action

of certain antioxidant molecules that reduce disulfides

via thiol/disulfide exchange, including the enzymes thi-

olredoxin and peroxiredoxin, as well as free cysteine

and glutathione. Cysteine availability is a rate-limiting

step in the synthesis of glutathione, which is a ubiqui-

tously expressed tripeptide that is considered to be the

most important cellular antioxidant molecule [7–9].Oxidation of protein thiols may be crucial to the

normal function of a specific protein, and may affect a

vast variety of functions, including protein structure,

protein–protein interactions, catalysis, electron trans-

fer, ion channel modulation, phosphorylation-depen-

dent signal transduction, post-translational protein

modification and transcriptional activation [10,11]. In

skeletal muscle, contractile function and force produc-

tion and the development of fatigue, are directly influ-

enced by the reduction/oxidation (redox) state of

protein thiols of contractile proteins. Contractile (myo-

fibrillar) proteins such as troponin, tropomyosin, myo-

sin and actin contain thiol side chains that are

sensitive to oxidation, and modifications may alter

excitation/contraction coupling and cross-bridge

cycling, and therefore modulate muscle contraction [12

–24]. Excessive oxidative stress, which occurs in condi-

tions such as chronic inflammation, during strenuous

exercise and disease states, may cause muscle weakness

[25], and is implicated in the pathology of numerous

muscular diseases such as muscular dystrophies.

Oxidative stress in muscular dystrophies

The muscular dystrophies comprise more than 30

hereditary clinical disorders that are characterized by

progressive skeletal muscle wasting and degeneration.

They generally share common histological features,

including variation in myofibre size, myofibre degener-

ation and regeneration, and the replacement of muscle

with connective tissue and fat [26]. These conditions

vary in many aspects, including prevalence, age of

onset, severity, the muscles affected and the underlying

gene defect [27]. These disorders are due to mutations

in a wide variety of molecules, including extracellular

matrix, sarcolemmal, cytoskeletal, cytosolic and

nuclear membrane proteins [28,29]. Although the

genetic basis of many of these disorders has been iden-

tified, the exact mechanism for pathogenesis remains

unclear; however, there is evidence that interactions

between the primary genetic defect and elevated levels

of ROS contribute to the pathology of several muscu-

lar dystrophies [30]. Oxidative stress has been investi-

gated extensively in Duchenne muscular dystrophy

(DMD), which is discussed in more detail below, and

is also clearly involved in other dystrophies and myop-

athies [30–35].For example, oxidative stress is strongly implicated

in the SEPN1-related myopathies that are due to defi-

ciency in the protein selenoprotein N (SEPN1): these

comprise four congenital skeletal muscle disorders that

are characterized by severe weakness and wasting of

neck and trunk muscles, leading to scoliosis and respi-

ratory insufficiency [31]. Selenoproteins contain seleno-

cysteine and include many proteins involved in the

regulation of oxidative stress, including glutathione

peroxidases and thioredoxin reductases; selenopro-

tein N has recently been identified as a key protein in

cell protection against oxidative stress and

redox-related calcium homeostasis [32]. Another disor-

der, facioscapulohumeral muscular dystrophy is an

autosomal dominant muscle disease that is character-

ized by progressive weakness and atrophy of facial,

shoulder girdle and upper-arm muscles. Its molecular

pathogenesis is due to deletions that lead to an

increase in function of the DUX4 (double

homeobox 4) protein in muscles [33]. DUX4 is a tran-

scription factor that initiates an extensive gene de-reg-

ulation cascade, and many genes that are differentially

expressed in the muscles of facioscapulohumeral

muscular dystrophy patients are involved in oxidative

stress responses [33]. Altered levels of ROS and a

higher susceptibility to oxidative stress are also a fea-

ture of laminopathies, which result from mutations in

the LMNA gene, encoding A–type lamins, proteins

that are associated with the nuclear membrane [34].

Elevated oxidative stress is also implicated in myopa-

thies due to mutations in the ryanodine receptor

RYR1, an essential component of the excitation/

contraction coupling apparatus; these include several

congenital RYR1-related myopathies that are the most

common non-dystrophic muscle diseases of childhood

[35].

2 FEBS Journal (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

Protein thiol oxidation in dysferlinopathies J. R. Terrill et al.

In our laboratory, we have applied a wide range of

quantitative measures to analyze oxidative stress in

two forms of muscular dystrophy, using the mdx

mouse model for DMD and the dysferlin-deficient A/J

mouse model for dysferlinopathies. These two diseases

are discussed in detail below.

Oxidative stress in Duchenne muscular

dystrophy

Duchenne muscular dystrophy (DMD) is a lethal

X–chromosome-linked skeletal muscle disease, mani-

fested in children, that is caused by mutations in the

dystrophin gene resulting in the absence or decreased

function of the membrane-associated protein dystro-

phin [27,36]. Other mutations in the same gene result

in a mildly defective dystrophin protein, with a less

severe disease, usually with later onset, called Becker

muscular dystrophy [27]. Skeletal and cardiac myofi-

bres lacking functional dystrophin have an increased

susceptibility to sarcolemma damage after muscle con-

traction, which leads to myofibre necrosis; this results

in inflammation, myogenesis and new muscle forma-

tion to regenerate the tissue [37,38]. However,

repeated cycles of damage and inflammation over

months and years progress to replacement of muscle

by fat and fibrous connective tissues, with severe loss

of muscle function, resulting in premature death,

often due to respiratory or cardiac failure [36,39]. It

has been proposed that growth (as well as muscle size

and mechanical loading) increases the severity of dyst-

ropathology, which is less pronounced in animal

models of DMD, such as mdx mice and Golden

Retriever dogs [40]. The pathology of DMD also

appears to be exacerbated by oxidative stress: pre-

cisely why dystrophin deficiency leads to the genera-

tion of ROS in skeletal muscle is unclear, although

probable reasons include interactions between

excessive intracellular calcium and inflammation

[5,30,41–44]. It is well documented that elevated cyto-

solic calcium concentrations increase mitochondrial

calcium, which is an effector of ATP synthesis, and

an increase in ATP synthesis increases production of

ROS by mitochondria, via higher oxygen consump-

tion and enhanced electron flow through the electron

transport chain [45–47]. Membrane damage stimulates

degranulation of resident mast cells [48] and also

releases intracellular contents that activate the

immune system of the host, further increasing the

inflammatory cell cascade [49,50]. In addition,

immune cells such as neutrophils and macrophages

generate ROS in order to promote phagocytosis [51–53].

A role for oxidative stress in DMD is supported by

a wealth of pre-clinical studies in mdx mice that report

benefits such as improved muscle pathology and

decreased necrosis for many antioxidant drugs and

interventions, such as green tea extracts, resveratrol,

coenzyme Q10 and catalase [54–61]. Many of these

drugs may have broad-based effects in vivo, and the

potential translational benefits of these for clinical

treatment of DMD remain to be substantiated [62,63].

There is a notorious lack of success of antioxidants in

clinical trials [64] that may be due in part to a lack of

understanding of the precise nature of oxidative stress

involved with the particular pathology [5].

While irreversible oxidative damage of protein and

lipids, as measured by the carbonyl and malondiade-

hyde assays, respectively, has been widely studied and

targeted by antioxidant treatment, there has been little

information related to protein thiol oxidation in mus-

cular dystrophies. However, this topic has been a focus

of recent research in our laboratory. The development

of a specific two-tag assay to measure protein thiol

oxidation in skeletal muscle tissues has revealed signifi-

cantly elevated levels of protein thiol oxidation, as well

as elevated protein carbonylation, in dystrophic muscle

of mdx mice [65–68]. In addition, treatment with the

thiol-reducing antioxidant N–acetylcysteine (NAC)

reduces the severity of dystropathology in vivo, as

measured by decreased levels of plasma creatine kinase

and reduced myonecrosis, and this study specifically

demonstrated in vivo that NAC reduced the level of

oxidized protein thiols in dystrophic muscles [67].

NAC has previously been shown to improve force pro-

duction by mdx muscles [69] and the pathology of

mdx hearts [70], and a recent in vivo study further con-

firmed the beneficial effects of NAC on creatine kinase

levels and myonecrosis (using diaphragm muscles), as

well as reduced levels of tumor necrosis factor in mdx

mice [71]. These combined studies implicate protein

thiol oxidation in the dystropathology of DMD, and

support further evaluation of specific thiol antioxidant

drugs for clinical translation.

Oxidative stress in dysferlinopathies

Another group of muscular dystrophies of interest,

that have been far less well studied, are the dysferlin-

opathies. These encompass two disorders, limb-girdle

muscular dystrophy type 2B and Miyoshi myopathy,

which are both rare adult diseases, with weakness in

either distal muscles (Miyoshi myopathy) or proximal

limb-girdle muscles (limb-girdle muscular dystrophy

type 2B) [72,73]. Limb-girdle muscular dystrophy

type 2B and Miyoshi myopathy are considered to


J. R. Terrill et al. Protein thiol oxidation in dysferlinopathies

result from allelic variations of the same dysferlin

gene; however, the reason why mutations to this gene

give rise to two different phenotypes is unknown [74].

The dysferlin gene encodes dysferlin, a membrane-

associated protein that is involved in membrane vesicle

trafficking and fusion, that is localized to transverse

tubules and the periphery of myofibres and cardiomyo-

cytes and, to a lesser extent, other tissues such as

monocytes, brain and kidney [75–77]. Muscles lacking

dysferlin may have problems in the membrane reseal-

ing that follows mechanical injury of contraction [78],

and an absence of dysferlin reportedly leads to intra-

cellular calcium dysregulation through membrane

modifications and cell signaling dysfunction [78–81].Dysferlin deficiencies are also associated with excessive

inflammation [49,82,83], and, although it has been

hypothesized that this leads to an increased presence

of ROS, very little experimental work on the subject

has been published.

A case study in a single Miyoshi patient identified

increased protein and lipid oxidation and protein thiol

content in affected muscle, suggesting increased ROS

levels in dysferlin-deficient muscle as well as protein

thiol alterations [84]; increased levels of antioxidants,

including glutathione and catalase, were also observed.

A study investigating oxidative damage in DMD, dys-

ferlinopathies and sarcoglycanopathy identified ampli-

fied lipid peroxidation and protein oxidation in all

three human muscular dystrophies [85]. The study also

showed dysregulation of glutathione-recycling antioxi-

dants, such as glutathione reductase and peroxidase,

suggesting perturbations in glutathione metabolism in

the muscle of patients with these dystrophies. Other

studies have used a combination of the antioxidants

coenzyme Q10 and resveratrol in SJL/J dysferlin-defi-

cient mice, and reported reduced inflammation and

muscle pathology, although these results were not

quantified [86,87]. Several strains of mice that lack

dysferlin are available, and include SJL/J, A/J and

Bla/J (A/J mice bred onto a C57Bl/6 background), as

well as genetically engineered null strains such as the

B6.129-Dysf tm1Kcam/J and B10.SJL–Dysf im/AwaJ mice:

all show a late onset but relatively mild pathology

compared with the human condition (http://www.jain-

foundation.org/our-dysferlin-research-institute/research-

tools/mouse-models-dysferlin-deficiency/).

Elevated protein thiol oxidation in

dysferlin-deficient muscles of A/J mice

Here we present new data for protein thiol oxidation,

and other measures of oxidative stress, along with

severity of histopathology, in a range of muscles from

dysferlin-deficient A/J (A/Jdysf�/�) mice, a naturally

occurring dysferlin-deficient strain of mice with a retro-

transposon insertion in dysferlin intron 4 [88]. These

data are compared to those for normal control A/J

mice at various ages, and then critically compared with

data for mdx mice. The A/Jdysf�/� mice exhibit dystro-

phic changes, including necrosis and inflammation, that

are histologically evident by 12 months of age in proxi-

mal muscles, whereas distal muscles appear relatively

unaffected even in late stages of the disease [88]. The

present study examined both distal and proximal mus-

cles, including the psoas and quadriceps (severely

affected) and gastrocnemius, biceps brachii muscles

(mildly affected), at four ages; 3, 8, 12 and 19 months.

The histology of muscles was assessed, and reversible

protein thiol oxidation was quantified using a dual-

labeling technique that indicates the percentage of oxi-

dized to total (reduced and oxidized protein thiols) in

muscles [66,67], and was further analyzed using a quan-

titative method that visualizes the localization of oxi-

dized protein thiols on tissue sections. Other measures

of oxidative stress included determination of irrevers-

ible oxidative damage to proteins using the carbonyl

assay [89] and of cumulative damage by quantification

of lipofuscin granules [90]. All of these measures were

correlated with histopathology. Identifying the targets

of oxidative stress that are affected in dysferlin-deficient

muscle provides insight into the molecular basis for

pathology and may also identify more specific drugs for

possible therapeutic intervention.

Results

Preliminary semi-quantification of necrosis, fat

content and protein thiol oxidation in severely

affected 19-month-old A/Jdysf�/� mice, to select

muscles most affected by pathology

A range of muscles from 19-month-old (severely

affected) A/Jdysf�/� mice were subjected to semi-quan-

titative histological analysis and measurement of total

protein thiol oxidation. The histology results indicated

that all muscles had a low–medium score for necrosis,

apart from the biceps, which scored normal–low(Fig. 1A). For fat content (Fig. 1B), both the psoas

and quadriceps muscles scored medium–high, whilst

the gastrocnemius, biceps and deltoid scored normal–low. Quantification of total protein thiol oxidation of

the muscles of 19-month-old mice (Fig. 2) showed that

both the psoas and quadriceps muscles had signifi-

cantly higher levels of protein thiol oxidation, while

the gastrocnemius, biceps and deltoid had a low level

of oxidation.



http://www.jain-foundation.org/our-dysferlin-research-institute/research-tools/mouse-models-dysferlin-deficiency/



As all three parameters were high in psoas and

quadriceps muscles, these muscles were considered the

most affected, and were selected for quantification of

histology and oxidative stress measures in further stud-

ies, together with a relatively unaffected muscle (either

gastrocnemius or deltoid) as an additional control.

Quantification of areas of necrosis and fatty

tissue in 3-, 8-, 12- and 19-month-old A/Jdysf−/−

mice

Quantification of myofibre necrosis (Figs 3A and 4)

and fat content (Figs 3B and 4) for psoas, quadriceps

and gastrocnemius muscles was performed in 3-, 8-,

12- and 19-month-old A/Jdys�/� mice. These data were

compared with age-matched control A/J wild-type

mice (except for 19 months old, as control mice of this

age were not available); data are not shown for wild-

type muscles as all values were very low compared

with A/Jdysf�/� mice, as were A/Jdysf�/� gastrocnemius

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Fig. 1. Semi-quantitative histological analysis for myofibre necrosis

(A) and fat content (B) in skeletal muscles from 3-, 8-, 12- and 19-

month-old A/Jdysf�/� (dysferlin-null) mice. Means of semi_quantitative

data, where 0 = normal (none in whole tissue area), 1 = < 5%, 2 = 5–

10%, 3 = 10–15%, 4 = > 15%. The asterisk indicates a significant

difference (P < 0.05) between gastrocnemius and quadriceps

muscles (P < 0.05). The dollar symbol ($) indicates a significant

difference (P < 0.05) between biceps and other muscles. Values are

means � SEM (n = 6).

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Fig. 2. Total protein thiol oxidation in skeletal muscles from 19-

month-old A/Jdysf�/� mice. The asterisk indicates a significant

difference (P < 0.05) from gastrocnemius, biceps and deltoid

muscles. Values are means � SEM (n = 6).

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A

B

Fig. 3. Myofibre necrosis (A) and fat (B) (percentage cross-

sectional area) in psoas and quadriceps muscle from 3-, 8-, 12- and

19-month-old A/Jdysf�/� mice. The hash symbol (#) indicates a

significant difference from quadriceps muscle of the same age

(P < 0.05). The dollar symbol ($) indicates a significant difference

from the same muscle at 3 months (P < 0.05). Values are

means � SEM (n = 6).



values. Although necrosis was significantly elevated in

the A/Jdysf�/� psoas and quadriceps muscles compared

with normal controls, the extent of necrosis was low

(<1% of the cross-sectional area), and there were

no significant differences in necrosis between these

A/Jdysf�/� muscles at any age.

In marked contrast, large areas of the muscles were

occupied by fat (20–30% of the tissue), and this was

especially prominent in the psoas muscle (Figs 3B and

4). Fat content was significantly higher in the psoas

muscle compared with the quadriceps muscle at 8 and

12 months. Fat content was also markedly higher in

both muscles at 8, 12 and 19 months compared to

3 months (Fig. 3B).

Protein thiol oxidation in tissue samples and

histological sections

The percentage of total protein thiol oxidation in mus-

cle tissue samples was quantified in the gastrocnemius

(Fig. 5A), psoas (Fig. 5B) and quadriceps (Fig. 5C)

muscles of 3-, 8-, 12- and 19-month-old A/Jdysf�/� mice

and wild-type controls. At 3 months of age, there was

no significant difference between any groups. At

8 months, protein thiol oxidation was significantly

higher in the psoas muscle of A/Jdysf�/� compared with

control mice, and in the A/Jdysf�/� psoas muscle com-

pared with the gastrocnemius muscle. At 12 months,

protein thiol oxidation was significantly higher in the

gastrocnemius and quadriceps muscles of A/Jdysf�/�

mice, compared with controls. At 19 months, protein

thiol oxidation was significantly higher in A/Jdysf�/�

psoas and quadriceps muscles compared to the gastroc-

nemius muscle. Protein thiol oxidation was also signifi-

cantly higher in the psoas muscle of 19-month-old A/

Jdysf�/� mice than in the psoas muscle at all other ages,

and the level in the quadriceps muscle of 19-month-old

A/Jdysf�/� mice was significantly higher than in the

quadriceps muscle of 3-month-old A/Jdysf�/� mice.

We also calculated the amount of reduced, oxidized

and total thiols in muscle tissue samples. For simplic-

ity, only the level of reduced, oxidized and total thiols

in the quadriceps muscle is presented (Table 1). Total

thiols were significantly reduced in 19-month-old quad-

riceps muscles of A/Jdysf�/� mice compared to all other

mice, as were reduced thiols. This was also the case

for the psoas muscle of 19-month-old A/Jdysf�/� mice

(data not shown). At 19 months of age, reduced thiols

A B

C D

Fig. 4. Histology images of muscles from

19-month-old A/Jdysf�/� mice stained with

haematoxylin and eosin, showing

replacement of muscle tissue with large

areas of fat (arrows) in quadriceps muscle

(A) and psoas muscle (B). (C, D) Higher-

magnification views showing examples of

a small area of myonecrosis (C) and fat

(arrows) between myofibres (D).



were significantly lower in the A/Jdysf�/� psoas muscle

compared to the A/Jdysf�/� gastrocnemius muscle, and

total and reduced thiols were significantly lower in

the A/Jdysf�/� quadriceps muscle compared to the

A/Jdysf�/� gastrocnemius muscle (data not shown).

Even at 8 months of age, the A/Jdysf�/� mice had sig-

nificantly lower levels of reduced thiols than mice at

3 months, while 12-month-old A/Jdysf�/� mice had sig-

nificantly more oxidized thiols than age-matched wild-

type controls.

Protein thiol oxidation was also visualized and quan-

tified on histological tissue sections of 12-month-old

A/Jdysf�/� and wild-type controls (Fig. 6). The total area

of protein thiol oxidation was significantly higher in

A/Jdysf�/� tissue compared with control tissue, and was

evident both in areas with extensive lipid presence and

without (Fig. 6A), being mainly present throughout

intact myofibres (Fig. 6B). This pattern of histological

localisation was compared with mdx muscles, where

pronounced protein thiol oxidation (Fig. 6B) occurs

most dramatically in fragmented myofibres associated

with areas of necrosis, and there are more oxidized thi-

ols in intact myofibres compared to controls.

Oxidative damage to proteins

Carbonyl content

As the muscle most affected by oxidative stress (the pso-

as muscle) was too small to measure protein carbonyl

content, the gastrocnemius (Fig. 7A) and quadriceps

(Fig. 7B) muscles were analyzed in 3-, 8-, 12- and

19-month-old A/Jdysf�/� mice and wild-type controls.

There was no significant difference in either muscle for

protein carbonylation between strains or ages, apart

from the level in the quadriceps muscle of 19-month-old

A/Jdysf�/� mice, which was significantly higher than that

in the quadriceps muscle of 12-month-old A/Jdysf�/�

mice, and also that in the quadriceps muscle of

3-month-old A/Jdysf�/� and wild-type control mice.

Lipofuscin content

The presence of lipofuscin (also known as ceroid) indi-

cates the extent to which a tissue has been exposed

over time to irreversible oxidative stress [91], although

decreased degradation through impaired autophagy

may also increase the accumulation of lipofuscin [92].

Lipofuscin was evident in the psoas and quadriceps

muscles of A/Jdysf�/� mice as autofluorescent granules

at all ages (Fig. 8A). The amount of lipofuscin was

significantly higher in the psoas muscles of A/Jdysf�/�

mice at all ages (3, 8 and 12 months) compared with

age-matched controls, and was also higher in the psoas

muscle of 12-month-old A/Jdysf�/� mice compared

with 3-month-old A/Jdysf�/� mice (Fig. 8A). Although

there was a trend for increased lipofuscin content in

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B

C

Fig. 5. Total protein thiol oxidation in muscles from 3-, 8-, 12- and

19-month-old A/Jdysf�/� mice and wild-type controls. The asterisk

indicates a significant difference from age-matched wild-type

control (P < 0.05). The hash symbol (#) indicates a significant

difference from gastrocnemius muscle of the same age (P < 0.05).

The ampersand (&) indicates a significant difference from 3-month-

old A/Jdysf�/� mice (P < 0.05). The dollar symbol ($) indicates a

significant difference from A/Jdysf�/� and wild-type at all other ages

(P < 0.05). Values are means � SEM (n = 6).



quadriceps muscle (Fig. 8B), this was not statistically

significant, possibly due to low group numbers (n = 3).

Discussion

Correlation between pathology and thiol

oxidation in affected dysferlin-null muscles

While the level of myonecrosis was very low (< 1%) in

dysferlin-deficient muscles at all ages, the replacement

of muscle with fat was a very striking feature of the

affected muscles (psoas and quadriceps) in A/Jdysf�/�

mice. In diseases such as DMD, the increased adipos-

ity of muscle is a result of progressive replacement of

myofibres in response to repeated cycles of myofibre

necrosis [39,93], but this does not appear to be the

explanation for dysferlin-deficient muscles, in which

myofibre necrosis is relatively low: instead it must be

due to another pathogenic mechanism. An increase in

adiposity in skeletal muscle is associated with modifi-

cations to adipogenic genes and cell signaling path-

ways [94], and this may occur in dysferlin-deficient

muscle as a direct cellular consequence of dysferlin

deficiency, or indirectly as a secondary consequence of

this gene defect. Further experimental work is required

to understand the reasons for increased fat content in

dysferlin-deficient muscles and why only some muscles

are so severely affected.

Oxidative stress may lead to cellular dysfunction

through permanent damage to proteins, lipids and

DNA, as well as causing reversible modifications to

thiol side chains on cysteine residues, leading to altera-

tions in protein function [6]. The protein carbonyl

assay is commonly used as an indicator of irreversible

oxidative damage to proteins, and increased carbonyl

levels have been reported in human muscle lacking

dysferlin [84,85]. The fact that protein carbonylation

was not significantly increased in the muscles of

A/Jdysf�/� mice (compared to age-matched controls)

may reflect the relatively mild phenotype of this mouse

model up to 12 months of age, as carbonyl content

was significantly increased in the severely affected

quadriceps muscles of 19-month-old A/Jdysf�/� mice

compared with younger A/Jdysf�/� and control mice

(Fig. 7).

A more striking result in A/Jdysf�/� mice was the

reversible modification of protein thiol side chains,

with significantly elevated protein thiol oxidation in

the most severely affected psoas and quadriceps

muscles as early as 8 and 12 months, respectively (com-

pared with controls), and a significant further increase

by 19 months. This pattern of protein thiol oxidation

correlates with disease manifestation in specific muscles

and progression with age. Visualization of protein thiol

oxidation in tissue sections indicated that affected

dysferlin-deficient myofibres had high levels of protein

thiol oxidation: this was not evident in the interstitial

tissue and was not particularly pronounced in areas of

severe pathology where fat was present.

Another measure of oxidative stress that was

increased in both dystrophin- and dysferlin-deficient

muscle is the accumulation of lipofuscin: lipofuscin lev-

els are already significantly elevated in the psoas muscles

of A/Jdysf�/� mice at 3 months of age and increase fur-

ther with age and disease progression (Fig. 8). This ele-

vated lipofuscin level at the young age of 3 months

(when no striking pathology is evident in A/Jdysf�/�

muscles) appears to be of particular importance as it

indicates that there are already significant disturbances

to oxidative stress levels and implies an early role for

oxidative stress in subsequent disease manifestation.

Is the pattern of oxidative stress similar in DMD

and dysferlinopathies?

There is clearly a very different pattern in the time of

onset and the nature of pathology for DMD and

dysferlinopathies, and it is of interest to compare the

Table 1. Redox state of protein thiols in quadriceps muscle from A/Jdysf�/� mice and wild-type controls. The asterisk indicates a significant

difference from age-matched wild-type controls (P < 0.05). The ampersand (&) indicates a significant difference from 3-month-old A/Jdysf�/�

and wild-type mice (P < 0.05). The dollar symbol ($) indicates a significant difference from A/Jdysf�/� and wild-type mice at all other ages


Age Genotype

Reduced thiols

(nmol�mg protein�1)

Oxidized thiols


Total thiols


Percentage

oxidized (%)

3 months Wild-type 36.4 � 1.8 4.6 � 0.4 41.0 � 2.1 8.9 � 1.1

A/Jdysf�/� 38.3 � 0.7 5.2 � 0.1 38.2 � 1.6 10.1 � 1.1

8 months Wild-type 31.7 � 1.5 5.2 � 0.5 38.1 � 1.7 11.5 � 0.3

A/Jdysf�/� 30.6 � 2.1& 4.8 � 0.4 35.4 � 2.2 14.2 � 1.4

12 months Wild-type 36.7 � 1.0 3.1 � 0.3 39.8 � 1.2 7.7 � 0.5

A/Jdysf�/� 33.6 � 1.7 4.5 � 0.3* 36.9 � 1.1 14.3 � 1.6*

19 months A/Jdysf�/� 24.1 � 0.9$ 4.9 � 0.3 29.1 � 0.8$ 17.6 � 1.2$



parameters of oxidative stress for the mouse models of

these diseases. Myofibre necrosis and oxidative stress

measures (protein thiol oxidation, carbonyl content

and lipofuscin accumulation) are compared in Table 2

for mdx and A/Jdysf�/� mice and their respective con-

trol strains (C57BL/10ScSn and A/J) at 3 months,

with additional data at 12 months for A/Jdysf�/� mice

as the disease was evident by this later age. At

3 months, active myofibre necrosis occupies approxi-

mately 6% of the muscle area for mdx mice, whereas

necrosis is very low in the muscles of A/Jdysf�/� mice.

This correlates with protein carbonyl oxidation, which

is elevated in mdx mice but not in A/Jdysf�/� mice at

this young age. Increased protein carbonyl content has

been reported in both DMD and mdx muscle [65,85,95

–97], but whether this is a cause or consequence of the

myonecrosis (and associated inflammation) is unclear.

Protein carbonylation is also evident in dysferlinopa-

thies after disease manifestation in humans [84,85] and

for very old A/Jdysf�/� mice at 19 months, with this

late onset suggesting that this is more likely to be a

consequence rather than a cause of the pathology.

* *

A

B

Fig. 6. (A) Protein thiol oxidation on muscle sections from 12-month-old A/Jdysf�/� mice and wild-type controls, in areas with and without

extensive fat. The asterisk indicates a significant difference from age-matched wild-type control (P < 0.05). Values are means � SEM (n = 3).

(B) Comparison of protein thiol oxidation on muscle tissue sections in dysferlin-null (A/Jdysf�/�) and dystrophin-null (mdx) mice, and wild-type

controls (A/J and C57BL/10ScSn respectively). Red., reduced thiols; Ox., oxidized thiols. Protein thiol oxidation is confined to intracellular

proteins of myofibres in tissue sections of skeletal muscles, and is increased in both A/Jdysf�/� and mdx muscle compared with controls.



At 3 months of age, the lipofuscin level was signifi-

cantly increased (two to threefold) in both mdx and

A/Jdysf�/� muscles, compared with controls; this was

unexpected for the A/Jdysf�/� mice as there is little mani-

festation of the disease in young adult mice. Lipofuscin

levels increase with age, and this was evident for both

wild-type and A/Jdysf�/� mice at 12 months, with much

higher levels (approximately twofold) still present in the

musclesof A/Jdysf�/� mice. Elevated lipofuscin levels and

accumulation with age have previously been reported for

skeletal muscles from DMD patients and mdx mice [98].

Both mdx and A/Jdysf�/� muscle show an increase

(approximately twofold) in protein thiol oxidation

compared with controls. This is evident in mdx mice

at 3 months (when the disease is already advanced),

but not in A/Jdysf�/� mice at this age (before the onset

*

A

B

Fig. 7. Carbonylation of proteins in gastrocnemius (A) and

quadriceps (B) muscles from 3-, 8-, 12- and 19-month-old A/Jdysf�/�

mice and wild-type controls. The asterisk indicates a value that is

significantly higher than that for quadriceps muscle from 12-month-

old A/Jdysf�/� mice and 3-month-old A/Jdysf�/� mice and wild-type

controls. Values are means � SEM (n = 6).

* *

*$A

B

Fig. 8. Area of lipofuscin content (percentage cross-sectional area)

in muscles from 3-, 8- and 12-month-old A/Jdysf�/� mice and wild-

type controls for the psoas muscle (A) and the quadriceps muscle

(B). The asterisk indicates a significant difference from age-

matched wild-type controls (P < 0.05). The dollar symbol ($)

indicates a significant difference from A/Jdysf�/� mice at 3 months


Table 2. Comparison between two forms of muscular dystrophy in terms of extent of necrosis, protein thiol oxidation, carbonyl and

lipofuscin content in dyferlin-null (A/Jdysf�/�) and dystrophin-null (mdx) mice, and wild-type controls (A/J and C57BL/10ScSn respectively).

n/a, not applicable.

Age Genotype

Percentage

necrosis

Percentage

protein thiol

oxidation

Carbonyl content

(nmol/mg protein)

Percentage

lipofuscin

3 months C57BL/10ScSn n/a 9.7 3.1 0.03

mdx 6.1 16.8 7.8 0.06

Wild-type A/J n/a 8.9 1.9 0.02

A/Jdysf�/� 0.018 10.1 2.3 0.06

12 months Wild-type A/J n/a 7.7 2.2 0.17

A/Jdysf�/� 0.1 14.3 2.1 0.33



of pathology), although it is evident by 12 months in

A/Jdysf�/� mice when the pathology has already mani-

fested. In the mdx muscles, pronounced protein thiol

oxidation (Fig. 6B) occurs most dramatically in frag-

mented myofibres associated with areas of necrosis,

and, like the muscles of A/Jdysf�/� mice, there are also

significantly more oxidized thiols inside intact myofi-

bres (also evident as lower levels of reduced protein

thiols in Fig. 6B). This suggests that skeletal muscle

proteins are undergoing protein thiol oxidation in rela-

tively ‘unaffected’ myofibres of the dystrophic muscles,

potentially causing changes in protein function and

thus contributing to the resulting pathology and

altered muscle performance. There are a wide range of

target proteins for thiol oxidation and modulation of

function that include many contractile proteins, and

we have recently identified significant changes in the

thiol oxidation state of such proteins, specifically myo-

sin and tropomyosin in the quadriceps muscle of

3-month-old mdx mice (J. Terrill, M. Grounds,

P. Arthur, unpublished data).

The A/Jdysf�/� mouse is a useful model to study dysf-

erinopathies, with a strong correlation between the onset

and severity of pathology in specific muscles and the

incidence of various measures of oxidative stress. The

early elevation of protein thiol oxidation in affected mus-

cles (compared with the late increases in carbonylation)

suggests a potentially important role for such reversible

oxidation of key muscle proteins in the manifestation of

pathology in dysferlinopathies, and presents the opportu-

nity to assess the effects of specific thiol-reducing antioxi-

dants such as NAC in the A/Jdysf�/� mice. In mdx mice,

NAC has various benefits and reduces the severity of

pathology [67,69,71], providing further evidence for the

probable important role of protein thiol oxidation in the

pathology of DMD. It is also of interest to identify the

specific proteins that are affected by thiol oxidation in

the muscles of A/Jdysf�/� mice to determine their poten-

tial role in the mechanism of the disease. The fact that

lipofuscin is already significantly elevated by 3 months

of age in the muscles of A/Jdysf�/� mice, before the

disease is obvious, indicates that this is a particularly

sensitive measure and strongly supports an early and key

role for altered oxidative stress prior to disease manifes-

tation. Further investigations into such aspects of

oxidative stress in dysferlinopathies (as have already

been initiated for the mdx mouse and DMD) appear

warranted.

Experimental procedures

All reagents were obtained from Sigma-Aldrich (St. Louis,

MO, USA) unless otherwise specified.

Animal procedures

A/J control mice were obtained from the Animal Resources

Centre (Murdoch, Western Australia). A/Jdysf�/� (dysferlin

null) mice were obtained from the Institute for Neurosci-

ence and Musclemuscular Research, Children’s Hospital at

Westmead, (Sydney, Australia). Mice were transported to

the University of Western Australia and maintained on a

12 h light/dark cycle under standard conditions, with free

access to food and drinking water. All animal experi-

ments were performed in strict accordance with the

guidelines of the National Health and Medical Research

Council of Australia code of practice for the care and

use of animals for scientific purposes (2004), and the

Animal Welfare Act of Western Australia (2002), and

were approved by the Animal Ethics Committee at the

University of Western Australia.

Tissue collection, histology and image

acquisition

All mice were killed by complete cervical dislocation while

under terminal anesthesia (2% v/v AttaneTMisoflurane,

Bomac, Hornsby, NSW, Australia). Muscles were collected

from wild-type control mice at 3, 8 and 12 months of age,

and from A/Jdysf�/� mice at 3, 8, 12 and 19 months

(19-month-old wild-type controls were omitted as these

were not available for analysis), and immediately snap-fro-

zen in liquid nitrogen for biochemical analysis. For histol-

ogy, one whole upper limb and one lower limb were

immersed in 4% paraformaldehyde and fixed for 1 week;

samples were then processed for paraffin histology. Trans-

verse muscle sections (5 lm) were cut through the mid-

region of each muscle on a Leica microtome, as previously

described [99], and sections were stained with haematoxylin

and eosin for morphological analysis.

Groups of three control and three A/Jdysf�/� mice at

three ages (3, 8 and 12 months old) were sampled, and mus-

cles were obtained for frozen histology for the analysis of

lipofuscin and protein thiol oxidation. The fresh muscles

were bisected transversely and longitudinally and mounted

on cork squares using tragacanth gum. The muscles were

frozen in a slurry of isopentane cooled in liquid nitrogen.

Cryostat sections (8 lm) were cut directly onto silinised

glass slides, and stored at �20 °C until stained or analyzed.

Histological image analysis

Myofibre necrosis was identified as areas of myofibres with

fragmented sarcoplasms and/or increased inflammatory cell

infiltration. Fat content was identified as areas of many

large circular cells unstained by haematoxylin and eosin.

Both were assessed semi-quantitatively in all skeletal muscles

(psoas, biceps brachii, quadriceps, gastrocnemius, deltoid)

from 8-, 12- and 19-month-old A/Jdysf�/� mice. In brief,



skeletal muscles were prepared for paraffin histology,

stained with haematoxylin and eosin [67,68,99], and all mus-

cles (n = 6 per group) were examined by light microscopy at

109 magnification. For the preliminary semi-quantitative

analysis, each parameter was given a score of 0–4; where

0 = normal, 1 = < 5%, 2 = 5–10%, 3 = 10–15%, 4 => 15%. These scores for six samples were averaged to pro-

vide an overall score. After the semi-quantitative analysis

had revealed that psoas and quadriceps muscles were the

most affected, these muscles were quantitatively assessed for

necrosis and fat content using non-overlapping tiled images

of transverse muscle sections that provided a picture of the

entire muscl’e cross-section. Digital images were acquired

using a Leica Microsystems (Wetzlar, Germany) DM RBE

microscope, a Hitachi (Tokyo, Japan) HVC2OM digital

camera, IMAGE PRO PLUS 4.5.1 software (Media Cybernetics,

Rockville, MD, USA) and VEXTA STAGE MOVEMENT software

(Oriental Motor Co, Tokyo, Japan). Tiled images were

taken at 109 magnification. Muscle morphology was

drawn manually by the researcher using IMAGE PRO PLUS

4.5.1 software. The area occupied by necrotic myofibres

(i.e. myofibres with fragmented sarcoplasm and/or areas of

inflammatory cells) or fat was measured as a percentage of

the whole muscle section area. Histological analysis was

completed according to the TREAT-NMD recommended

standard protocol ‘Histological Measurements of

Dystrophic Muscle – M.1.2_007’ (http://www.treat-nmd.

eu/research/preclinical/dmd-sops/).

Carbonylated protein

Oxidative damage to proteins in muscles was determined

by measuring the carbonyl content using 2,4–dini-

trophenylhydrazine as previously described [65,100,101].

Frozen muscles were crushed under liquid nitrogen, and

protein was extracted using 20% trichloroacetic acid/

acetone. The protein pellets were washed in acetone and

ethanol, precipitated, dried, re-suspended in 10 mM

2,4–dinitrophenylhydrazine in 2 M HCl, and incubated for

30 min at room temperature in the dark. Proteins were

washed with ethyl acetate/ethanol (1 : 1) for one hour at

room temperature, dissolved in 6 M guanidine, and absor-

bance was measured at 370 nm. Protein concentration

(mg�mL�1) was determined using the Bio–Rad (Hercules,

CA, USA) Bradford protein assay. Carbonyl concentra-

tions are expressed as nmol carbonyl per mg protein.

Lipofuscin quantification in muscle

Lipofuscin is composed of autofluorescent granules that

accumulate in tissue and are generated as a consequence

of irreversible oxidative stress [91]. The granules are visible

on unstained frozen tissue sections using fluorescent

microscopy. The amount of lipofuscin in frozen muscle

sections was measured by the non-subjective boot strap-

ping method [90]. Images were captured using a fluores-

cent Nikon (Tokyo, Japan) Eclipse Ti microscope

equipped with a Roper Industries (Sarasota, FL, USA)

CoolSNAP-HQ2 camera, a 450–490 nm excitation filter, a

515 nm emission barrier filter, and Nikon NIS-Elements

software. A 409 magnification was used, and sections

were scanned using an automatic stage control setting that

generated a grid structure of images in a set area. For

each section scanned by fluorescent microscopy, eight

images were selected for lipofuscin analysis: images with

tissue edges or obvious artefacts were discarded. Quantifi-

cation of lipofuscin granules was performed using IMAGEJ

version 1.44 (http://imagej.nih.gov/ij/download/), and the

green channel of an eight-bit RBG image was analyzed.

The area occupied by fluorescent granules was expressed

as a percentage of the total image area.

Protein thiol oxidation

Reduced and oxidized protein thiol levels were measured

using a dual-labeling technique [66–68]. In brief, snap-frozen

quadriceps muscle was crushed under liquid nitrogen, and

protein was extracted using 20% trichloroacetic acid/ace-

tone. Protein was solubilized in 0.5% SDS/0.5 M Tris at pH

7.3 (SDS buffer), and protein thiols were labeled with the

first tag, the fluorescent dye BODIPY FL-N–(2-aminoethyl)

maleimide (FLM) (Invitrogen, Mulgrave, VIC, Australia).

After removal of the unbound dye using ethanol, protein

was re-solubilized in SDS buffer, pH 7, and oxidized thiols

were reduced using Tris(2–carboxyethyl)phosphine, before

labeling of the resultant unlabeled reduced thiols with a sec-

ond tag, the fluorescent dye Texas Red C2-maleimide (Texas

Red) (Invitrogen). The sample was washed in ethanol and

resuspended in SDS buffer. Samples were read using a fluo-

rescent plate reader (Fluostar Optima, BMG Labtech, Oten-

berg, Germany) with wavelengths of 485 nm for excitation

and 520 nm for emission for FLM, and 595 nm for excita-

tion and 610 nm for emission for Texas Red. A standard

curve for each dye was generated using ovalbumin, and the

results are expressed per mg of protein, quantified using a

Detergent Compatible Protein Assay (Bio–Rad).

Protein thiol oxidation on tissue sections

Reduced and oxidized protein thiols on frozen tissue sec-

tions were measured using an adaptation of the dual-label-

ing technique described above (T. Iwasaki, J. Terrill,

M. Grounds and P. Arthur, unpublished). Serial muscle

sections (9 lm) used for detecting reduced thiols were trea-

ted immediately in FLM. After washing in NaCl/Pi, sec-

tions were fixed with 4% paraformaldehyde in 0.1 M

phosphate buffer, and immersed in NaCl/Pi overnight. For

detection of oxidized thiols, frozen sections from each

muscle were treated with N–ethylmaleimide to block

free thiols. After washing in NaCl/Pi to remove unreacted



http://www.treat-nmd.eu/research/preclinical/dmd-sops/

http://www.treat-nmd.eu/research/preclinical/dmd-sops/

http://imagej.nih.gov/ij/download/

N–ethylmaleimide, sections were fixed with 4% paraformal-

dehyde in 0.1 M phosphate buffer. The fixed sections were

washed again with NaCl/Pi. Oxidized thiols were reduced

with Tris(2–carboxyethyl)phosphine); this was omitted in

negative control sections. The reduced thiols were washed

twice in NaCl/Pi to remove Tris(2–carboxyethyl)phosphine

before labeling with FLM. All sections were mounted with

polyvinyl acetate mounting medium for microscopy.

Another serial section from each muscle was stained with

haematoxylin and eosin for morphometric observation.

Fluorescence images were acquired as per lipofuscin

quantification. Sections were scanned using an automatic

stage control setting that generated a grid structure of

images covering a set area. The level of oxidized thiols in

muscle sections was estimated by image analysis using

IMAGEJ version 1.44. For each section scanned by fluores-

cence microscopy, three images were selected for all

image analysis: images with tissue edges or obvious arte-

facts were discarded. The selected fluorescence images

were used for quantification of the mean fluorescence

intensity (arbitrary units) of the section.

Statistics

Significant differences between groups were determined

using one-way ANOVA with post hoc tests, and all data

are presented as means � standard error of the mean.

Significance was set at P < 0.05.

Acknowledgements

We thank Sandra Cooper (Institute for Neuroscience

and Musclemuscular Research, Children’s Hospital at

Westmead, Sydney, Australia) for generously provid-

ing the dysferlin-deficient A/J mice. This research was

supported by funding from the Jain Foundation, the

National Health and Medical Research Council of

Australia, and an Australian Postgraduate Award

Scholarship to J.R.T.

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