ABSENCE OF DYSTROPHIN DISRUPTS SKELETAL MUSCLE SIGNALING: ROLES OF Ca , REACTIVE

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Physiol Rev 96: 253–305, 2016
Published December 16, 2015; doi:10.1152/physrev.00007.2015
ABSENCE OF DYSTROPHIN DISRUPTS SKELETAL
MUSCLE SIGNALING: ROLES OF Ca2ⴙ, REACTIVE
OXYGEN SPECIES, AND NITRIC OXIDE IN THE
DEVELOPMENT OF MUSCULAR DYSTROPHY
David G. Allen, Nicholas P. Whitehead, and Stanley C. Froehner
Sydney Medical School & Bosch Institute, University of Sydney, New South Wales, Australia; and Department of
Physiology & Biophysics, University of Washington, Seattle, Washington
Allen DG, Whitehead NP, Froehner SC. Absence of Dystrophin Disrupts Skeletal
Muscle Signaling: Roles of Ca2⫹, Reactive Oxygen Species, and Nitric Oxide in the
Development of Muscular Dystrophy. Physiol Rev 96: 253–305, 2016. Published
December 16, 2015; doi:10.1152/physrev.00007.2015.—Dystrophin is a long rodshaped protein that connects the subsarcolemmal cytoskeleton to a complex of proteins in the surface membrane (dystrophin protein complex, DPC), with further connections via
laminin to other extracellular matrix proteins. Initially considered a structural complex that protected the sarcolemma from mechanical damage, the DPC is now known to serve as a scaffold for
numerous signaling proteins. Absence or reduced expression of dystrophin or many of the DPC
components cause the muscular dystrophies, a group of inherited diseases in which repeated
bouts of muscle damage lead to atrophy and fibrosis, and eventually muscle degeneration. The
normal function of dystrophin is poorly defined. In its absence a complex series of changes occur
with multiple muscle proteins showing reduced or increased expression or being modified in various
ways. In this review, we will consider the various proteins whose expression and function is changed
in muscular dystrophies, focusing on Ca2⫹-permeable channels, nitric oxide synthase, NADPH
oxidase, and caveolins. Excessive Ca2⫹ entry, increased membrane permeability, disordered
caveolar function, and increased levels of reactive oxygen species are early changes in the disease,
and the hypotheses for these phenomena will be critically considered. The aim of the review is to
define the early damage pathways in muscular dystrophy which might be appropriate targets for
therapy designed to minimize the muscle degeneration and slow the progression of the disease.
L
I.
II.
III.
IV.
V.
VI.
VII.
INTRODUCTION
THE DYSTROPHIN-ASSOCIATED COMPLEX
EARLY FUNCTIONAL CHANGES IN...
PROTEIN CHANGES IN THE ABSENCE...
IMPAIRED MEMBRANE REPAIR AND...
THERAPEUTIC DIRECTIONS
CONCLUDING REMARKS
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291
I. INTRODUCTION
Dystrophin is a long (110 nm), rod-shaped protein expressed primarily in muscle that connects ␥-actin of the
subsarcolemmal cytoskeletal system to a group of proteins
in the surface membrane, the dystrophin protein complex
(DPC). There are further connections via laminin from the
DPC to the extracellular basal lamina (FIGURE 1). Based on
this structure, it is generally thought that one function of
dystrophin is the transmission of force laterally across the
muscle and/or helping to maintain the registration between
the membrane and intracellular cytoskeleton matrix and
extracellular matrix. However, growing evidence suggests
that dystrophin, through its multiple protein connections,
also has a major role in regulating signaling pathways, particularly those pathways that activate nitric oxide (NO)
production, Ca2⫹ entry, and the production of reactive oxygen species (ROS).
Most of what we know about the function of dystrophin
arises from studying the diseases that result from the absence of dystrophin. The best known of these diseases,
Duchenne muscular dystrophy (DMD), occurs through mutations or other genetic rearrangments in the dystrophin
gene which lies on the X chromosome. The disease has
classical X-linked recessive genetics in which males, who
carry the mutation, express the disease while females, who
have a single copy of the mutation, do not express the
disease but are carriers (for a fuller account of the carrier
state, see Ref. 188). As a consequence of these genetic abnormalities, boys with DMD have muscles which largely or
completely lack dystrophin. In Becker muscular dystrophy
(BMD), which is a later onset and milder disease, the muscles contain reduced quantities of a truncated, partially
functional dystrophin protein. Different types of dystrophin mutations occur in DMD/BMD, including large dele-
0031-9333/16 Copyright © 2016 the American Physiological Society
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ALLEN ET AL.
Biglycan
Agrin
Laminin
Collagen VI
Laminin
Basal lamina
α-Dystroglycan
α7β1 integrin
β-Dystroglycan
Sarcoglycans
α β γ δ
Sarcolemma
H2
N
ABD
H1
4
8
12
16
H3
20
Dystrophin
24
DBD
α-Dystrobrevin N
Syn
SBS
SBS
Syn
Syn
SBS
SBS
γ-actin
SSPN
Syn
nNOSμ
γ-actin
CC CC
CC
CC
C
C
FIGURE 1. The dystrophin protein complex (DPC). Shown are the interactions between core components of
the DPC, the extracellular matrix, and nNOS. Numbers in dystrophin indicate hinge regions (H1, H2, etc.) and
spectrin-like repeat domains (4, 8, 12, etc). nNOS, neuronal nitric oxide synthase; Syn, syntrophin; SSPN,
sarcospan; ABD, actin binding domain; DBD, dystroglycan binding domain; SBS, syntrophin binding site; CC,
coiled-coil domain; N, amino terminus; C, carboxy terminus.
tions (60-65% of cases) and large duplications (5–10% of
cases), with the remaining cases due to point mutations and
small deletions or duplications (for a review of dystrophin
mutations in DMD and BMD, see Ref. 363).
The purpose of this review is to try to understand the normal function(s) of dystrophin in skeletal muscle and to consider the early stages of the disease process and how these
might result from the absence of dystrophin. Despite a large
literature on the disease, surprisingly little is known about
the earliest stages of the disease, and a coherent set of mechanisms that link the absence of dystrophin to the disease
pathogenesis is still unavailable.
A. History of DMD and the Discovery of
Dystrophin
DMD is a rare but distinctive disease so there are occasional
brief descriptions in the literature before the definitive account by Duchenne in 1868 (103). In this report Duchenne
described 13 cases, including 2 girls, and noted the progressive weakness affecting initially the lower limbs, the early
hypertrophy followed by gradual atrophy, and the characteristic histology. One of Duchenne’s innovations was the
use of muscle biopsy to obtain samples during life, and he
was able to follow the histological features during the
course of the disease and to ascribe the disease to muscle as
opposed to the nervous system. Gowers (151) also studied
many cases of the disease and described the technique for
rising from the ground utilized by patients now known as
254
“Gowers’ manoeuvre.” He also noted the low rate of disease in females and the fact that the disease was inherited
from the mother rather than the father. This pattern of
inheritance, in which females are carriers but do not generally express the disease whereas males are affected, is now
recognized as characteristic of recessive mutations on the X
chromosome.
For the next 100 years or so, remarkably little was known
about the pathogenesis of the disease apart from its Xlinked genetics and that skeletal muscle was primarily affected. Gradually other forms of muscular dystrophy were
described and characterized clinically and, in particular,
Becker (28) described a more benign version of the disease
with the same X-linked genetics.
The situation changed dramatically in the 1980s when,
within a short period, the location and sequence of the
dystrophin gene were discovered and the protein product
dystrophin was characterized (for a personal account of
these discoveries, see Ref. 225). Prior to the 1980s, it was
known only that the mutated gene lay on the X chromosome. Through studies of a series of linked genetic diseases
which changed the banding pattern on chromosome X, it
gradually became clear that the mutated gene lay in the
short arm of the X chromosome (Xp21) and was very large
(for review, see Ref. 114). Davies et al. (88) produced a
genomic library from the human X chromosome and identified two sequences which genetic studies showed to be
linked to DMD. They were able to show that these two
sequences lay on either side of the DMD locus identifying
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
the site with new precision. Worton’s group (516) focused
on a female patient with what appeared to be DMD who
had a translocation of part of the X chromosome to an
autosome. They assumed that the breakpoint in the X chromosome was at the site of the DMD gene. A nearby group of
ribosomal genes was available as a marker, and eventually a
clone was identified in close linkage to the DMD site which
was capable of detecting deletions in some DMD boys.
Kunkel et al. (226) used a subtractive hybridization technique approach which Kunkel had previously used to study
the Y chromosome. Their starting clinical material was the
DNA from a patient with a large deletion that included the
DMD gene. They then used a hybridization technique with
normal X chromosome DNA sequences that eventually
yielded clones of the sequences of the deleted DNA material
in the patient (227). Subsequently they used restriction fragment linked polymorphisms known to be linked to DMD to
identify the position and some of the deletions and mutations that can cause DMD (226, 299). The gene was sequenced and proved to be the largest in the human genome
spanning 2.5 megabases and containing 79 exons (385).
Once the gene sequence was known, antibodies could be
produced to epitopes, and the protein product dystrophin
was characterized and located on the intracellular side of
the cell membrane (184, 492). The discovery of the dystrophin gene and protein has revolutionized our understanding
of the disease and led to advances in prenatal diagnosis and
carrier detection.
provides symptomatic relief. The use of ventilators, initially
at night and eventually during the day, has had a substantial
impact on survival. Mean survival in the 1960s was 15 yr,
but this has gradually increased with improved therapy of
various sorts and is currently in the mid 20s (110, 369). As
patients are surviving longer, the cardiac consequences are
becoming of greater importance, and currently ⬃20% of
patients die of cardiac consequences of the disease (428).
B. Natural History of DMD and BMD
Just as DMD occurs in humans through mutations in the
dystrophin gene, it is also recognized in a number of animals. The golden retriever dystrophy model has many similarities to the human model (219). For instance, it is Xlinked and of comparable severity to the human disease.
Serum CK is high and the histological picture of degeneration and regeneration is similar. For these reasons, it is
thought to be a good model of the human disease, and
treatments that are successful in this model may have a
higher probability of success in humans. Unfortunately, the
mortality of pups with the disease is high and the cost of
maintaining the animals is considerable so that there are
relatively few colonies, and consequently, opportunities to
use this model for preclinical studies are limited (24, 322).
DMD is commonly diagnosed between the ages of 2 and 6
yr because of delay in walking and/or unsteady gait (for
details of clinical features, see Refs. 114, 176). Other common features are some delay in intellectual milestones and
speech development. Frequently there is muscle hypertrophy, commonly in the calf muscles, but the dominant feature is muscle weakness, starting in the legs and gradually
affecting upper limbs and trunk muscles. As the disease
proceeds, affected boys have difficulty in rising from the
ground, and Gower’s sign is the use of the arms to support
the legs as the boys slowly rise from the ground to standing.
The weakness progresses slowly, but between 7 and 12 yr,
most boys are confined to a wheel chair. In the teenage years
contractures of many muscles can occur, and atrophy becomes prominent. Curvature of the spine associated with
weakness of the postural muscles can also occur. Because of
weakness of the thoracic muscles and the diaphragm, respiratory function declines and ventilatory complications are a
common cause of death.
While there is currently no specific treatment, many forms
of therapy prolong survival. Steroids delay the time to wheel
chair use by several years (279) and slow the decline of
many aspects of the disease (176) but are associated with
many side effects. Surgery for contractures and scoliosis
Diagnosis rests on the clinical features, the presence of a
grossly elevated serum creatine kinase (CK), the histological
picture including the absence of dystrophin by antibody
staining and, usually, the identification of the mutation in
the dystrophin gene. The latter, while not strictly necessary
for diagnosis, is of value for carrier studies and prenatal
diagnosis and, increasingly in the future, for some types of
therapy (see sect. VI).
BMD is also caused by mutations to the dystrophin gene,
but while DMD nearly always leads to complete absence
of dystrophin expression in muscle, BMD is associated
with reduced levels or shortened dystrophin proteins. As
a consequence, while the clinical picture is similar, the
onset and development are slower and more variable. For
instance, the mean age of loss of walking for BMD patients is 37 yr (58).
C. Animal Models: mdx Mouse
The mdx mouse was identified in 1984 as a spontaneous,
X-linked mutation with elevated serum CK and histological
features of muscle damage and regeneration (54). Subsequently, it was shown that the muscles lacked dystrophin,
and there was a premature stop codon in exon 23 of the
dystrophin gene (for review see Ref. 322). In-breeding has
resulted in the mdx line in which both males and females
exhibit the disease and all their offspring are affected. Because of the identical genotype and easy availability, mdx
mice have become the most widely used model of DMD.
However, despite the identical genotype, the disease is
much milder than in humans. Affected animals live a near-
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ALLEN ET AL.
normal life span and reproduce normally. Muscles appear
normal at birth, but a wave of damage occurs around 4 – 8
wk, which results in substantial regeneration with most
fibers showing central nuclei (indicative of regeneration).
Thereafter, damage and regeneration remain in balance and
the animals remain mobile though voluntary performance
on a running wheel is substantially reduced compared with
wild-type (WT) animals (167). Respiratory muscles are
more affected than limb muscles, possibly reflecting the
continuous use of these muscles (432) (see sect. ID). Many
studies have shown that eccentric contractions (in which the
muscle is stretched during contraction) cause substantially
greater damage in mdx mice than in WT (77, 295, 354), and
the damage caused by eccentric contractions in the mdx
mouse is widely used as a model of the muscle damage in
DMD. Chemical mutagenesis programs have produced mutants, such as the mdx3Cv, which is deficient in both fulllength dystrophin and the short forms of dystrophin which
express in brain and other tissues and may give more insights into the role of dystrophin in other tissues (84).
An important, and only partially resolved, issue is why the
phenotype of the mdx mouse is milder than DMD. Perhaps
the best established explanation is the degree of utrophin
expression. Utrophin is a structurally similar protein to dystrophin which is expressed along the sarcolemma in developing muscle but restricted to the neuromuscular junction
and myotendinous junction in normal adults (for review,
see Ref. 37). The structural similarities between utrophin
and dystrophin have led to the suggestion that utrophin can
partially replace dystrophin. In the mouse this idea is supported by a number of findings. One is that in the mdx
mouse, sarcolemmal utrophin levels are increased (271).
Another is that the double knockout of both dystrophin and
utrophin in mice produces a severe phenotype that more
closely mimics DMD (96, 154) and may therefore be more
suitable for testing possible therapies. In addition, transgenic overexpression of utrophin in the mdx mouse further
ameliorates the phenotype (456). These ideas have also led
to the search for techniques to upregulate utrophin in humans in the hope that this will be of therapeutic value (see
sect. VIA).
A second reason for the milder phenotype of the mdx mouse
relates to the ability of the satellite cells to divide repeatedly
to allow repair of damaged fibers. Sacco et al. (395) have
proposed that the longer telomeres and shorter life span in
mouse muscle provide additional capacity for repair and
can contribute to the greater resistance to damage. This
topic is further explored in section VA2.
A third reason for the milder phenotype is the presence of
the cytidine monophosphate sialic acid hydroxylase gene
(CMAH) in mice. This gene has been inactivated in humans
and appears to exacerbate the consequences of dystrophin
absence in humans. The evidence for this is that the inser-
256
tion of a CMAH inactivating mutation into the mdx mouse
produced a much more severe phenotype (65).
A fourth possible reason is that the small size of the mdx
mouse is of itself protective. This could arise because forces
on muscle fibers are reduced in small animals so that muscle
damage would tend to be less (40, 336). In support of this
idea, small golden retrievers have a milder phenotype than
large (469), and humans with short stature, due to growth
hormone deficiency or natural variability, are partially protected (530, 532).
D. Preliminary Ideas About Mechanisms
Even before the site and structure of dystrophin were
known, structural evidence suggested that membrane damage and subsequent damage secondary to Ca2⫹ entry were
important elements in the pathology. Mokri and Engel
(298) noted focal disruptions of the cell membrane in EM
studies of DMD muscle and described contractures of the
myofibrils in the vicinity, suggestive of Ca2⫹ entry through
the membrane breach. Bodensteiner and Engel (39) subsequently showed that total Ca2⫹ was greatly increased in
DMD muscle and suggested that raised free intracellular
Ca2⫹ concentration ([Ca2⫹]i) had a role in the developing
pathology. As the site and structural connections of dystrophin became clearer (184), it seemed likely that dystrophin
exerted a structural role though the details of that remain
unclear and are explored in section IIB. These ideas still have
wide support, but the pathway of Ca2⫹ entry remains under
debate.
Evidence has shown that muscle damage in mdx and DMD
is dependent on contractile activity. In mdx mice, extensive
damage and regeneration of the hindlimb muscles begins
within a week after the mice are weaned (3– 4 wk of age),
and by ⬃8 wk of age more than 50% of fibers have undergone a damage-regeneration cycle as quantified by central
nuclei. If, however, contractile activity is prevented in 3- to
5-wk-old mdx mice, by immobilizing the EDL and soleus at
a fixed length, either short or long, these muscles do not
accumulate centrally nucleated fibers (297). These data suggest that the onset of chronic contractile activity in mdx
mice is necessary for the initial muscle damage to occur. The
elevated resting intracellular Ca2⫹ concentration in spontaneously contracting mdx myotubes is also dependent on
chronic activity, as it was reduced by long-term application
of tetrodotoxin (189). In both of these cases, mdx mice
running in a cage and myotubes in culture, the activity is
chronic over several days. Therefore, dystrophic muscles
are susceptible to damage from both acute eccentric contractions as well as chronic, submaximal noneccentric activity. Perhaps this is another important difference between
the time courses of muscle damage in mdx and DMD in that
mice are very active immediately after weaning (⬃21 days
old), whereas activity levels in humans are minimal after
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
birth and during the first year or two before the onset of
walking.
cular dystrophies (LGMDs) are caused by loss of the
sarcoglycan complex, while dystrophin remains unperturbed at the sarcolemma (106), prompted reassessment of
the hypothesis that loss of the basal lamina-cytoskeletal
linkage was the major causative defect in muscular dystrophies. The very mild dystrophic phenotype in mice lacking
␥-actin also suggests that the membrane stabilization theory
is too simplistic to fully account for the severity of the
disease (421).
Thus a key question is how the combination of the loss of
dystrophin and contractile activity lead to muscle and membrane damage.
II. THE DYSTROPHIN-ASSOCIATED
COMPLEX
The DPC can be roughly divided into two subcomplexes:
the structural complex and the signaling complex. The delineation is not intended to be absolute as some elements of
the DPC have roles in both functions.
A. The Dystrophin Complex: Structural
Versus Signaling
In the 25 years since the discovery of the DMD gene, the
function of dystrophin and the proteins associated with it
(the DPC) has been the subject of intense study. Although
skeletal, cardiac, and smooth muscles are the organs most
severely affected by abnormalities in the DPC, growing evidence suggests that many cell types, including several in the
brain, have some form of the dystrophin complex (161). A
full understanding of the DPC in different cells and tissues
will likely inform us about its critical role in muscle cells and
the mechanistic deficiencies that lead to muscle degeneration in the muscular dystrophies. In this review, we focus
primarily on the DPC in skeletal muscle.
B. The Structural Complex
For many years (essentially since the identification of dystrophin as the DMD gene product), the DPC has been considered a structural complex that protects the sarcolemma
from damage during repeated cycles of muscle fiber contraction. In its simplest form, the structural complex provides a linkage between the submembrane cytoskeleton and
components of the extracellular matrix, with dystrophin
binding to ␥-actin and ␤-dystroglycan, which is in turn
linked to laminin via ␣-dystroglycan.
Based largely on dystrophin’s key role in linking the extracellular matrix to the subsarcolemmal actin cytoskeleton
(FIGURES 1 and 2), and functional studies that revealed
increased damage to the muscle membrane following eccentric contractions, a structural role for dystrophin became a
widely accepted hypothesis. More recently, the signaling
component of the DPC has received growing attention. The
discovery that signaling molecules of major importance in
muscle function, such as nitric oxide synthase (NOS), require an intact DPC for sarcolemmal association has stimulated reconsideration of the function of dystrophin. Furthermore, the demonstration that certain limb girdle mus-
Actin
Actin
Par-1b
12
Synemin
Keratin 19
2
16
8 9
15 17 20
23 26
30
34
20
24
Plectin
nNOS
38 41 44
DBD
48
51
55
59
63
SBS
SBS
CC
8
Microtubules
CC
4
The most prominent feature of dystrophin is the large central section of 24 spectrin-like repeats (FIGURE 1), each
comprised of a triple-helical coiled-coil structure (for recent
review, see Ref. 238). This presumably flexible repeat region has properties suggestive of a shock absorber that
could transmit force laterally during muscle contraction. In
fact, atomic force microscopy analyses have demonstrated
that the force generated by only a few myosin molecules is
sufficient to unfold and extend the molecule (34). However,
Cholesterol
Anionic lipids
ABD
1. Dystrophin
AnkB
68
73
74
75
79
FIGURE 2. The dystrophin molecule. Alignment of dystrophin protein structure with gene exons. Top:
dystrophin protein domains with binding sites for structural and signaling proteins and lipids. ABD, actin binding
domain; DBD, ␤-dystroglycan binding domain; SBS, syntrophin binding domain; CC, coiled-coiled region that
binds ␣-dystrobrevin. Numbers identify spectrin-like repeats (for clarity, only 4, 8, 12, 16, 20, and 24 are
indicated). Par-1b, polarity regulating kinase; AnkB, ankyrin B binding site. Bottom: corresponding exons in the
human dystrophin gene. Note that the exons are arranged to correspond with the dystrophin protein structure.
Therefore, the widths of the boxes do not correspond to the relative sizes of the exons. Data used to construct
this figure are from the Leiden Muscular Dystrophy Pages (http://www.dmd.nl/).
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ALLEN ET AL.
the spectrin repeats are not merely structural components.
Several repeats, or groups of tandem repeats, bind actin,
anionic lipids (241), and signaling proteins such as neuronal
NOS (nNOS) (6, 49) and polarity regulating kinase,
PAR-1b (FIGURE 2). PAR-1b binds to spectrin repeats 8 –9
and phosphorylates sites in this region (520). These results
suggest that the spectrin repeats of dystrophin are much
more than shock absorbers and likely serve as a scaffold for
signaling proteins (for review, see Ref. 239).
Proline-rich hinge regions (labeled H1-H3 in FIGURE 1) interrupt or border on the repeat region at four sites and are
necessary for full dystrophin function (25, 60, 218). The
amino- and carboxy-terminal sections of dystrophin contain defined protein motifs that bind other proteins, including actin, ␤-dystroglycan, syntrophins, and ␣-dystrobrevin.
2. ␣/␤-Dystroglycan
Dystroglycan has important functions in many different cell
types (for review, see Ref. 46). In skeletal muscle, the extracellular glycosylated protein ␣-dystroglycan and its transmembrane partner, ␤-dystroglycan, are central to the linkage function of the DPC. Current evidence supports the idea
that dystroglycan is both a cell adhesion protein and a signaling receptor (for review, see Ref. 300). ␣-Dystroglycan
interacts directly with extracellular matrix proteins, including laminin, agrin, and perlecan, via their LG domains.
Phosphorylated intracellular tyrosine residues on ␤-dystroglycan recruit signaling proteins that bind to phosphotyrosine. The GTPase dynamin 1 associates with ␤-dystroglycan, an interaction that appears to regulate endocytosis
(533). Cells lacking dystroglycan exhibited greater transferrin uptake, a process that requires dynamin. A large number
of congenital muscular dystrophies are linked to dystroglycan abnormalities (285; reviewed in Refs. 405, 501). Many
of these abnormalities stem from deficiencies in glycosylation and the subsequent failure to bind extracellular proteins.
Recently, Johnson et al. (204) reported that only a small
fraction of ␣-dystroglycan in normal skeletal muscle is associated with dystrophin. In fact, the dystroglycan to dystrophin molar ratio may exceed 40:1. Dystrophin-free dystroglycan is largely in skeletal muscle fibers (not in vasculature or nerve) and is not associated with utrophin.
Dystroglycan remains on the sarcolemma in mdx and dystrophin/utrophin double knockout (DKO) mouse muscle
and in DMD muscle, although in lower amounts than in
normal muscle. Some sarcoglycans, syntrophins, and ␣-dystrobrevin remain associated with the core dystroglycan
complex in mdx muscle extracts, presumably by association
with utrophin.
What then is the function of the dystrophin-independent
dystroglycans? Using a proteomics approach, Johnson et al.
(204) identified two interesting possibilities. In one case,
258
dystroglycan is associated with cavin-1, a central organizer
of caveolae (179). In a separate complex, dystroglycan is
associated with the calcium channel regulatory subunit
Cav␤2 (55, 56). Although the calcium channel itself was not
found, this is may be due to technical limitations in detecting transmembrane proteins by mass spectrometry. However, regulation of Cav 1.1 by cAMP-dependent protein
kinase is altered in mdx mouse muscle (203). Based on these
findings, Johnson et al. (204) propose a novel model of
three distinct dystroglycan complexes in skeletal muscle: 1)
the well-known DPC, 2) a dystroglycan complex containing
Cavin-1, and 3) a dystroglycan complex with calcium channels. Only the latter is unperturbed by the loss of dystrophin. An intriguing possibility is that the remaining dystroglycan-calcium channel complex is involved in the
mechanisms that lead to higher intracellular calcium concentrations in dystrophic muscle.
3. ␥-Actin
The three major filamentous structures common to many
cell types, F-actin, microtubules, and intermediate filaments, are directly associated with the DPC.
The association of ␥-actin with dystrophin has been thoroughly examined and documented (118). The canonical
actin-binding domain is necessary but not sufficient for
high-affinity binding of dystrophin to filamentous ␥-actin.
Spectrin repeats in the amino terminus of the repeat region
are essential contributors to a strong interaction (15, 392).
These results are consistent with a model in which ␥-actin
lies laterally along the dystrophin rod for approximately
half of its length. In view of its predicted critical role in the
DPC structural hypothesis, the finding that genetic ablation
of ␥-actin resulted in a slowly progressive myopathy, similar to the phenotype of centronuclear myopathy (421), was
unexpected. Key features of muscular dystrophy in the mdx
mouse, including fibrosis, inflammation, and sarcolemmal
damage, were not observed. Nevertheless, transgenic overexpression of ␥-actin in mdx skeletal muscle, albeit at very
high amounts (⬃200-fold), attenuated contraction-induced
force loss (23). The mild phenotype of ␥-actin null muscle
might be explained by compensatory effects of other filamentous structures, although further studies are required to
test this hypothesis.
4. Microtubules
In skeletal muscle, microtubules are uniquely arrayed in a
nontraditional stationary lattice (328). The strands of the
lattice are sometimes formed by only a few microtubules,
some of which are dynamic and grow along existing static
microtubules. Nuclei and Golgi elements serve as nucleation sites for the microtubule lattices, a process that involves proteins that are common in microtubule organizing
centers in other types of cells. One model suggests that
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dystrophin may be an important guide or may restrict the
arrangement of the microtubule lattice. At the same time,
microtubule lattices appear to be important for localization
of intracellular organelles, particularly Golgi elements
(113, 344). The microtubule lattice pattern is severely disrupted in dystrophic muscle, but is largely restored by AAVmediated rescue with a microdystrophin construct (345).
Restoration of the lattice is accompanied by partial normalization of Golgi element distribution. These changes in microtubule structure in dystrophic muscle may be explained
by the observation that microtubules interact directly with
dystrophin (361).
Microtubules likely play a dual structural and signaling
role. As discussed in section VD, alterations in microtubules
are intimately involved in ROS production in dystrophic
muscle (213). Furthermore, microtubule-associate protein
1B (MAP1B) binds ␣-syntrophin (133) and nNOS (440).
5. Intermediate filaments
Desmin is the most abundant intermediate filament protein
in skeletal muscle. Together with keratin 8 and keratin 19,
these intermediate filaments link contractile elements to
each other and to the sarcolemma. Intermediate filaments
that contain keratin 19 associate with the actin-binding
domain of dystrophin (437). Genetic ablation of desmin
resulted in the loss of costameres, but otherwise had surprisingly mild effects (326). Tibialis anterior muscles in keratin 19 null mice displayed a mild myopathy, characterized
by modest decreases in mean fiber diameter and specific
tetanic force and mislocalized mitochondria (438).
Several other members of the intermediate filament family
of proteins may interact with the DPC. Syncoilin is expressed at highest levels in skeletal and cardiac muscle and
binds directly to ␣-dystrobrevin (321). It is most highly
expressed at the neuromuscular junction. Syncoilin does
not appear to form intermediate filaments, even when overexpressed. However, it may link desmin filaments to the
DPC (359). In contrast to most DPC proteins, syncoilin
expression is increased in mdx muscle, and also in muscle
from DMD, BMD, and congenital muscular dystrophies,
often concentrated in intracellular core structures (52). The
structure, interactions and possible functions of syncoilin
are discussed in a recent review (301).
Desmuslin (synonymous with ␤-synemin) is an intermediate filament related protein that was identified as a potential
DPC protein based on its interaction with ␣-dystrobrevin
(293). ␤-Synemin/desmuslin is an A-kinase associated protein (AKAP) in heart (391), but its function in skeletal muscle, both normal and dystrophic, remains unknown.
The plectin cytolinker stabilizes and regulates intermediate
filament networks in many cell types (513). As a consequence, mutations in plectin are involved in multiple dis-
eases, including muscular dystrophy, myofibrillar myopathies, skin blistering, and neurological syndromes. Of the
eight isoforms generated by alternative splicing, four are
found in skeletal muscle. Plectin 1 and 1f are present on
the sarcolemma where they interact with dystrophin (the
EF-ZZ domains) and multiple sites on ␤-dystroglycan (for a
model of a complex interaction, see Ref. 381). Recent studies of mdx mice lacking plectin 1f implicated this isoform in
metabolism. Mice lacking both dystrophin and plectin 1f
exhibited a more severe dystrophic phenotype, despite the
fact that sarcolemmal integrity (determined by serum CK
levels and Evans Blue dye uptake) and glucose uptake were
restored to normal levels (368). Increased amounts of glucose transporter 4 at the sarcolemma in the double knockout may result from alterations in the microtubule network
in the absence of plectin.
C. The Core Signaling Complex
Six protein groups make up the core DPC signaling complex: sarcoglycans, ␣-dystrobrevin, syntrophins, sarcospan,
integrin, and biglycan (see FIGURE 1). These core proteins
bind numerous other signaling proteins and, in some cases,
regulate their interaction with the DPC.
1. Sarcoglycans
The sarcoglycans are transmembrane glycoproteins that
form a tetrameric complex. Of the six genes encoding sarcoglycans, four are expressed in skeletal muscle and mutations in each of the genes results in a specific LGMD (for
reviews, see Refs. 126, 270). The absence of one of the
sarcoglycan subunits usually results in the loss of the entire
sarcoglycan complex. In these cases, sarcolemmal dystrophin remains largely unaffected (216). The sarcoglycan
complex interacts directly with some of the other core signaling proteins, including biglycan (367), ␣-dystrobrevin
(524), and sarcospan (292), and with other proteins such as
filamin 2 (454). Despite its central role in DPC-mediated
signaling and its critical importance in muscle health, the
molecular function of the sarcoglycan complex remains
largely unknown (177; for reviews, see Refs. 333, 402).
2. Dystrobrevins
The two forms of dystrobrevin, a dystrophin-related protein (36, 153, 483), are encoded by distinct genes. ␣-Dystrobrevin, but not ␤-dystrobrevin (351), is expressed in
skeletal muscle as a component of the sarcolemmal DPC.
Five primary splice forms of ␣-dystrobrevin have been identified (36). ␣-Dystrobrevin-1 and -2 are the major forms in
skeletal muscle and have different carboxy-terminal tail sequences. The longer tail of ␣-dystrobrevin-1 contains numerous serine, threonine, and tyrosine phosphorylation
sites, suggesting that kinases may regulate ␣-dystrobrevinmediated interactions of signaling proteins with the DPC
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ALLEN ET AL.
(22, 152, 484). Furthermore, these two isoforms are differentially localized in skeletal muscle and preferentially interact with different members of the dystrophin complex and
utrophin (352). Mice lacking ␣-dystrobrevin exhibit a moderate muscular dystrophy (153). Human skeletal muscle
diseases due to mutations in the ␣-dystrobrevin (DTNA)
gene have not been reported.
3. Syntrophins
The syntrophins are a family of adapter proteins that contain a PDZ domain and 2 PH domains. Five closely related
isoforms are encoded by separate genes (5, 8, 357). Four of
the isoforms, ␣, ␤1, ␤2, and ␥2, are expressed in skeletal
muscle. ␣-, ␤1-, and ␤2-syntrophins are part of the DPC on
the sarcolemma (350), whereas ␥2-syntrophin is located on
the sarcoplasmic/endoplasmic reticulum (12). Each member of the dystrophin protein family has two syntrophin
binding domains (9, 320, 444). Thus the potential number
and isoform combination of syntrophins associated with
the DPC are very large and, in turn, may result in assembly
of different functionally interdependent signaling proteins.
The list of signaling proteins known to bind syntrophins is
large and includes nNOS, ion and water channels, protein
and lipid kinases, transporters, G protein receptors, and
others (TABLE 1). Thus the dystrophin-dystrobrevin-syntrophin signaling scaffold has enormous potential for assembling signaling pathways at the muscle sarcolemma. Mutations in the syntrophin genes that result in skeletal muscle
disease have not been identified, but changes in syntrophin
sarcolemmal expression are associated with numerous myopathies and muscular dystrophies (80). For example, mutations in contactin-1 alter the syntrophin and ␣-dystrobrevin isoform composition on the sarcolemma and cause a
lethal congenital myopathy (79). Mutations that cause single amino acid changes in the PH domains are the basis for
one form of human cardiac long QT syndrome (467, 517).
4. Sarcospan
A member of the tetraspanin family of proteins involved in
signaling, the role of sarcospan (SSPN) in muscular dystrophy has been elusive (reviewed in Ref. 265). Recently, mice
lacking SSPN were shown to have impaired diaphragm contractile function, perhaps resulting from a decreased level of
dystrophin and utrophin (265). Furthermore, moderate
overexpression of SSPN in skeletal muscle ameliorates dystrophic pathology in the mdx mouse (349). Like other members of the tetraspanin family of proteins, sarcospan forms
higher ordered oligomers in the sarcolemma (292). Sarcospan is tightly associated with the sarcoglycan complex (86)
and may serve as a chaperone in the assembly of the DGC
(266). An alternatively spliced isoform, ␮SPN, lacking two
of the transmembrane domains is localized in the sarcoplasmic reticulum (SR) (291) and may be involved in excitationcontraction coupling.
260
5. Integrin
The ␣7␤1 isoform of integrin is intimately involved in the
pathological development of muscular dystrophy. Although this integrin was initially thought to be a sarcolemmal stabilization structure separate from the DGC, recent
results revealed that ␣7␤1 integrin is linked to the DGC by
interaction with sarcospan (264). Transgenic and AAV-mediated expression of ␣7␤1 integrin in the mdx mouse ameliorates the dystrophic pathology, increases muscle regenerative capacity, and reduces contraction induced injury
(43, 57, 175). Mice that lack both ␣7 integrin and dystrophin exhibit a severe dystrophy (389). These results suggested that the ␣7␤1 integrin complex could be a therapeutic target for treatment of DMD and other dystrophies and
myopathies. Injection of laminin-111, an extracellular ligand for ␣7 integrin, markedly improves the dystrophic
phenotype in the mdx and the merosin-deficient mouse
model (388). Laminin-111 and other basement membrane
proteins mediate cell adhesion and are linked to signaling
platforms (reviewed in Ref. 528). Thus the role of integrin
in DMD likely involves perturbations in both structural and
signaling functions.
6. Biglycan
Originally defined as a structural protein of the extracellular matrix, biglycan has emerged as a ubiquitously expressed signal modulating protein (for review, see Ref.
316). In part, the evidence for a signaling role is based on
biglycan’s interaction with cytokines and growth factors
implicated in muscular dystrophy pathology, including
transforming growth factor (TGF)-␤, tumor necrosis factor
(TNF)-␣, and several forms of bone morphogenetic proteins. Biglycan also interacts with components of the DPC:
␣-dystroglycan and the ␣- and ␥-sarcoglycans (45, 367).
The expression of ␣- and ␥-sarcoglycans are also regulated
by biglycan during muscle development (367). Finally, and
perhaps most importantly, biglycan regulates nNOS␮ association with the DPC (284). This process is mediated by
association with the sarcoglycan complex and involves
␣-dystrobrevin and the syntrophins. The therapeutic potential of biglycan for DMD has been demonstrated in the mdx
mouse model (525). Injection of recombinant biglycan improves several key pathological features in skeletal muscle,
perhaps by upregulation of utrophin and increased levels of
␥-sarcoglycan, ␤2-syntrophin, and nNOS␮ at the sarcolemma (16).
Although not part of the core signaling complex defined
above, several other signaling/scaffolding proteins are associated with the DPC. These include Grb2 (327), Ca2⫹/calmodulin (319), and G␤␥ (518). Some of these will be discussed below in their functional context. Also, the physiological importance and therapeutic potential of nNOS␮ will
be discussed in detail.
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Table 1. Signaling proteins associated with the dystrophin complex
Protein
Function
DPC Binding Site
Signaling enzymes/proteins
Ca2⫹/calmodulin-dependent NO synthesis ␣-Syntrophin,
dystrophin
Tyrosine phosphatase N/ Catalytically inactive receptor tyrosine
␤2-Syntrophin
ICA 512
phosphatase
TAPP1, TAPP2
Tandem pleckstrin homology domain
Syntrophins
adapters
(multiple)
ARMS
EphA4 receptor-associated protein and a ␣-Syntrophin
substrate for ephrin receptors
Grb2/P66shc
Involved in many signaling pathways
␣-Syntrophin
Tks5
Adapter protein involved in many signaling ␤-Dystroglycan
pathways
␣-Catulin
Multipurpose scaffolding protein for G
␣-Dystrobrevin-1
protein signaling pathways
Tiam1
Rac signaling in cell adhesion
␤2-Syntrophin (with
utrophin and
␤-dystrobrevin)
Mixed-lineage leukemia 5, Regulator of myogenin expression
␣-Syntrophin
MLL5
PTEN
Phosphatase and tensin homolog;
␣-Syntrophin
dephosphorylates PIP3 to PIP2
Channels, receptors, and transporters
Nav 1.4, Nav 1.5
Skeletal and cardiac muscle sodium
␣-Syntrophin
channels
Kir2.1, Kir4.1
Inward rectifier potassium channels
Syntrophins
(multiple)
nNOS/NOS1
TRPC1, TRPC4
Aquaporin-4
Non-voltage-gated cation channels
Water channel
ABCA1
Cholesterol/lipid transporter
␣1D-Adrenergic receptor
Cell/Tissue
Reference Nos.
Skeletal muscle
6, 49
Pancreatic ␤-cells
408
B-cells
83
Skeletal muscle
253
Human breast cancer cells 35
Myoblasts
453
HEK cells, skeletal muscle 258, 329
Epithelial cells
259
Skeletal myoblasts
215
Skeletal myoblasts
215
Skeletal muscle
142
Skeletal and cardiac
muscle, brain,
astrocytes
␣-Syntrophin
Skeletal muscle
␣-Syntrophin
Skeletal muscle, astrocytic
endfeet
Syntrophins
Macrophages, Schwann
(multiple)
cells, liver
␣- and ␤2-syntrophin Vascular smooth muscle
G protein receptor regulator of blood
pressure
Adiponectin receptor
Metabolic regulation
␣- and ␤1-syntrophin Liver
Kinases
Diacylglycerol kinase zeta Metabolizes diacylglycerol to phosphatidic ␥1- and ␣-syntrophin Skeletal muscle
acid
Stress-activated kinase 3, Mitogen-activated protein kinase
␣-Syntrophin
Skeletal muscle
ERK6, p38␥
MAST205
Microtubule-associated serine/threonine ␤2-Syntrophin
Heart, brain, skeletal
kinase
muscle
SAST
Syntrophin-associated S/T kinase related ␤2-Syntrophin
Brain, testes
to MAST
Par-1b
Cell polarity-regulating kinase; maintains Dystrophin, utrophin MDCK, HEK293 cells
dynamic state of microtubules
Src kinase
Non-receptor tyrosine kinase
␤-Dystroglycan
Skeletal muscle
Other
Myocilin
Modulator of muscle hypertrophy pathway ␣-Syntrophin
Skeletal muscle
MAP1A/MAP1B LC1
Microtubule-associated proteins (light
␣-Syntrophin
Schwann cells, brain
chain 1)
Cavin-1
Central organizer of caveolae
Unknown
Cardiac muscle
Ahnak1
Large scaffold protein associated with the Unknown
Cardiac muscle
calcium channel ␤-subunit
Cypher/ZASP
Sarcomeric Z-line AKAP associated with Unknown
Cardiac muscle
L-type calcium channel
CryAB
Heat shock protein
Unknown
Cardiac muscle
81, 242
394, 472
6, 17, 317
10, 53, 306,
330
256, 257
318
2, 3
169
251
251, 522
269, 520
424, 453
202
133
205, 511
89, 205
205, 247
205
See text for definitions.
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D. DPC Diversity and Dynamic Regulation of
Protein Composition
The function of the DPC in other tissues and cell types is
likely to yield important information for DMD. Cardiac
and smooth muscle are especially relevant to DMD. Some
form of the DPC, often containing utrophin or shorter
forms of dystrophin, is present in many cell types. These
include endothelial and epithelial cells, neurons, Schwann
cells, astroglial cells, and pancreatic ␤-cells (10, 69, 161,
252, 332, 449). In these cases, membrane stabilization is
not likely to be relevant. Results from studies of numerous
tissues cells types have demonstrated that many other signaling proteins can associate with the DPC. These results
are summarized in TABLE 1. Further studies of the dystrophin complex in nonmuscle cells types and in nonmammalian models (32, 62, 146, 209) will be important in understanding the full range of signaling pathways mediated by
the DPC and the therapeutic potential of drugs that target
these pathways.
Even in muscle cells, the molecular composition of the DPC
differs depending on the muscle and fiber type. Within specialized regions of the sarcolemma of a single muscle fiber,
individual DPCs may have different syntrophin and ␣-dystrobrevin isoform compositions. Consequently, the associated signaling proteins may differ from one complex to
another. Identification of signaling proteins associated with
the DPC in different regions of the sarcolemma (postsynapse, myotendious junction, costameres, extra-costameric
membrane, caveolae, etc.) will be very informative in understanding the signaling role of the DPC and related complexes, and how disruption of these signaling pathways
contribute to the dystrophic pathology. Finally, dystrophin
is also located in the transverse tubules (invaginations of the
surface membrane; abbreviated t-tubules) (492). In contrast
to the t-tubular network in cardiac myocytes, the composition of the DPC in skeletal muscle t-tubules is relatively
unknown. It is possible, perhaps likely, that the signaling
function of the t-tubular DPC is very different from the
sarcolemmal form. Because of its proximity to the sarcoplasmic reticulum, the t-tubular DPC could have a major
impact on calcium homeostasis.
In the context of DMD, differences in the signaling function
of the DPC are especially important, since any treatment
will need to target both skeletal and cardiac muscle. In fact,
improvement in skeletal muscle by targeted expression of
microdystrophin heightens cardiac injury in the mdx mouse
(463). Proteomic studies conducted on skeletal and cardiac
muscle revealed major differences in signaling proteins associated with the DPC. In addition to differences in the
syntrophin and dystrobrevin isoform composition, nNOS is
not associated with the DPC in cardiac muscle (205). Furthermore, four novel DPC proteins, specific to cardiac
muscle, were identified: Cypher, Ahnak1, Cavin-1, and
CRYAB.
262
The protein composition of the DPC is likely to be dynamic,
but the regulation of its association with signaling proteins
is poorly understood. The phosphorylation state of DPC
proteins appears to be important. In the insulin secretory
granules in pancreatic ␤-cells, binding of the granule protein ICA512 to the PDZ domain of ␤2-syntrophin is
thought to link the granules to actin filaments, thereby restricting their mobility (205, 331). This interaction is regulated by phosphorylation of ␤2-syntrophin (332). Cdk5
phosphorylates a serine residue near the PDZ domain of
␤2-syntrophin and decreases binding to ICA512. Phosphorylation of a serine residue only 15 residues away (by a
yet to be identified kinase) has the opposite effect (408).
Similar regulatory mechanisms may apply in skeletal muscle to nNOS␮ association with ␣-syntrophin, for example.
Phosphorylation of ␤-dystroglycan is important in regulating its interaction with dystrophin, modulating the degradation of dystroglycan and perhaps initiating a signal transduction pathway. The interaction with dystrophin is disrupted by phosphorylation of a tyrosine residue near the
carboxy-terminal region of ␤-dystroglycan (196, 424). Tyrosine 980 (Y980) in the WW binding motif of mouse
␤-dystroglycan is especially critical in the interaction between dystroglycan and dystrophin. Also, Y980 phosphorylation recruits several SH2 domain proteins, including several tyrosine kinases (424). In addition to disrupting the
dystroglycan-dystrophin interaction, Y980 phosphorylation causes translocation of ␤-dystroglycan from the sarcolemma to an intracellular vesicle population, perhaps a subset of recycling endosomes. Tyrosine phosphorylation of
␤-dystroglycan is stimulated by ligands for ␣-dystroglycan,
including laminin and agrin.
Using gene knock-in technology, Miller et al. (290) generated a mouse line with a phenylalanine substitution at
Y980, a key tyrosine phosphorylation site in dystroglycan.
Introduction of this modification into mdx mice improved
several dystrophic features, including fewer central nuclei,
reduced Evans blue dye uptake, lower serum CK activity,
resistance to muscle damage, and force loss due to eccentric
contractions. ␣-Sarcoglycan and sarcospan (but not utrophin) were restored to the sarcolemma and the levels of
plectin were also increased.
Recently, global analysis has identified 18 phosphorylated
residues in dystrophin. Many of these sites are located in the
cysteine-rich region downstream of the spectrin repeats
(446). Phosphorylation of serine3059 enhances the interaction with dystroglycan. Clearly, phosphorylation is important in regulation of dystroglycan localization and interaction with dystrophin. Phosphorylation of other DPC proteins, including syntrophins (169, 260, 332, 408, 484, 537)
and dystrobrevins (22, 152, 407, 483), is likely to modulate
the composition of the DPC in skeletal muscle and the signaling proteins bound to it. Further studies on the regula-
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
tion of DPC assembly and function by phosphorylation are
clearly warranted. Other modes of posttranslational modification, especially nitrosylation, should also be examined.
In summary, we have learned much about the DPC since the
discovery of dystrophin, but the full function of the complex and its regulation are not yet known. The shift from the
structural hypothesis to greater emphasis on dystrophin as a
signaling complex has greatly enhanced the potential for
identification of therapeutic targets that slow the progression of muscle degeneration. Additional studies are needed
so that informed design of genetic treatments restore as
much of the signaling capability of the DPC as possible.
Combinatorial approaches involving genetic correction and
drug enhancement of signaling pathways will most likely be
needed for the most effective treatments.
III. EARLY FUNCTIONAL CHANGES IN THE
ABSENCE OF DYSTROPHIN
A. Muscle Weakness
Muscle weakness is an early and progressive feature of
DMD. Its most obvious manifestations are the delayed
walking, unsteady gait, and difficulty in rising from the
ground (Gowers sign) which commonly lead to the diagnosis at the age of 2– 6 yr. The magnitude of the weakness is
not commonly quantified in humans, partly because it is
very variable in different patients and different muscles, but
a variety of clinical stagings are used (114). As an example,
the maximum voluntary force produced by hand flexors in
normal 10-yr-old boys was 15.1 ⫾ 1.7 (SE) kg (n ⫽ 10),
whereas in DMD boys of the same age it was 4.5 ⫾ 0.5 kg
(401). As the disease progresses there is gradual onset of
severe atrophy which compounds the muscle weakness.
The 6-min walk test is a useful test of global muscle and
cardiovascular/respiratory activity (274) and is becoming
the gold standard for assessing therapies. While simple to
perform, it has some practical difficulties. In normal boys,
the distance increases with age as the boys become bigger
and better coordinated. In DMD patients, the distance initially increases with age and then falls off as the disease
becomes more debilitating. Thus an increase in distance in a
therapeutic trial is only unequivocally the result of the drug
if the stage of disease is one of decline. Another important
practical issue is that the enthusiasm of the assistant during
the test can influence the patient and the outcome so that
adequate placebo controls and double blinding of all concerned is critical.
Muscle fatigue during and after exercise is also a prominent
symptom in muscular dystrophies (for review, see Ref.
481). Again there are very few quantitative studies, and it is
unclear to what extent the fatigue arises simply because the
weakened muscles are being used at nearer their maximum
capacity and hence fatigue faster. Inactivity will also contribute to fatigue, and the preferential damage to fast rather
than slow fibers (494) may partially offset this factor since
slow fibers fatigue more slowly than fast. It is possible that
some of the muscle weakness derives from a very slow component of recovery from fatigue (217). In normal muscles,
fatigue is closely related to the metabolic and ionic changes
that accompany intense muscle activity (for review, see Ref.
13). Whether these and/or additional mechanism contribute to fatigue in dystrophic muscle requires further research.
Muscle damage is another likely cause of weakness in
DMD. Operationally, muscle damage is defined as weakness which recovers very slowly after activity, typically with
a time course similar to repair or regeneration of damaged
muscle (4 – 8 days). Activities in which the muscle is
stretched during contraction (eccentric contractions) are
particularly prone to cause damage, which is characterized
by reduced force, membrane damage leading to loss of soluble muscle proteins, overstretched and understretched sarcomeres, and swelling and tenderness of muscles (for review, see Ref. 129). If a muscle fiber is damaged to the point
at which it is unexcitable, then it will not contribute to the
overall force until regeneration is complete, and it is likely
that such damaged fibers contribute to the muscle weakness
that characterizes DMD.
There is good agreement in the literature that the absolute
force from isolated mdx muscles is similar to WT but that
mdx muscles are somewhat bigger and that specific force
(force per unit area) is moderately reduced (for review, see
Ref. 489). Most mammalian muscles produce a maximum
specific isometric force of ⬃200 – 400 mN/mm2, whereas
values for mdx muscles are typically 150 –200 mN/mm2
(255), except for the diaphragm where values range from
50 –150 mN/mm2 (120).
In considering the many possible causes of muscle weakness, one approach is to consider those factors within the
intact fiber, specifically 1) Ca2⫹ release and 2) Ca2⫹ sensitivity and maximum force production of the myofibrillar
proteins. There are also a range of possible mechanisms
associated with 3) muscle damage, 4) impaired lateral transmission of force by cytoskeletal proteins and extracellular
collagen, and 5) the possibility that muscle ischemia impairs
recovery from fatigue.
1. Ca2⫹ release
Ca2⫹ release in skeletal muscle occurs when an action potential, having conducted along the surface membrane and
into the t-tubules, excites the voltage sensor in the t-tubules
(a modified L-type Ca2⫹ channel) which through an interaction with the ryanodine receptor (RyR1, the SR Ca2⫹
release channel in skeletal muscle) releases a quantity of
Ca2⫹ from the store within the SR. In mdx mice, the action
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ALLEN ET AL.
potential is little changed and conduction of the AP through
the t-tubule network appears to be normal (514). However,
a number of studies have shown that the amount of Ca2⫹
released, judged by the amplitude of the [Ca2⫹]i increase
caused by a single action potential or a train of action potentials, is moderately reduced (174, 187, 515, 523). This
could come about either because the amount of Ca2⫹ stored
in the SR is reduced or because the openings of the RyR1s
are reduced. There is evidence that calsequestrin, the main
buffer for Ca2⫹ in the SR, is reduced in mdx muscles (100),
and this would reduce buffering of Ca2⫹ in the SR and the
amount of Ca2⫹ released by an action potential.
A long-standing observation in studies of human muscle
fatigue is that long and demanding periods of activity lead
to a prolonged (2– 4 days) reduction in force at low frequencies of stimulation while the maximum force observed at
high frequencies is normal (for review, see Refs. 13, 112).
This phenomenon can be caused by reduced SR Ca2⫹ release (502) and can be triggered by a prolonged preceding
period of elevated resting [Ca2⫹]i (73, 232). The mechanism
by which elevated [Ca2⫹]i causes reduced SR Ca2⫹ release
has long been uncertain; calpain inhibitors have only limited effect, and proteolysis products of RyR and the voltage
sensor are not detectable (232). Recently, Corona et al. (82)
showed that junctophilin, a protein required to stabilize the
SR membrane connection to the t-tubule, was disrupted
after eccentric contractions and suggested that this might
cause the failure of Ca2⫹ release. Subsequently Murphy et
al. (308) showed that ␮-calpain, when activated by moderate elevations of [Ca2⫹]i, was capable of cleaving junctophilin which could then cause a failure of Ca2⫹ release. Given
that the resting [Ca2⫹]i is elevated in mdx muscle (see sect.
IVC), and Murphy et al. (308) showed disruption of junctophilin in mdx muscle, it seems likely that this mechanism
contributes to reduced Ca2⫹ release and the muscle weakness observed in dystrophic muscles.
There is also substantial evidence that the properties of
RyR1 are modified in mdx muscle and may contribute to
reduced Ca2⫹ release. A recently proposed mechanism is
that nitrosylation of the RyR specifically in the mdx mouse
may change the properties of the RyR1 in a way that elevates resting [Ca2⫹]i and reduces tetanic Ca2⫹ release (30).
In earlier studies, the Marks group established that a variety
of posttranslational modifications of the RyR1 lead to loss
of an ancillary protein, calstabin1 (also known as FKBP12),
which causes increased open probability of isolated RyR1s.
This, in turn, might be expected to raise the resting [Ca2⫹]i,
deplete the SR of Ca2⫹, and reduce tetanic Ca2⫹ release in
intact muscle (31). In their later study (30), a reduction in
nNOS was confirmed but there was a substantial increase
in expression of iNOS, which was proposed to be the source
of the NO leading to nitrosylation. In addition, they
showed that a calstabin1 stabilizing agent, rycal or S107,
reversed the depletion of calstabin1 and the RyR1 “leaki-
264
ness” attenuated the eccentric muscle damage in isolated
muscles and improved exercise performance in intact animals. Most previous work has suggested that the elevated
resting [Ca2⫹]i in mdx muscle following eccentric contractions arises from extracellular Ca2⫹ entry (see sect. IIIC);
these studies (31, 31) raise the possibility that changes in
RyR1 performance contribute to elevated resting [Ca2⫹]i.
Thus these experiments offer a novel and therapeutically
reversible pathway of muscle damage, which can help to
explain the damage and reduced muscle performance in the
mdx mouse.
2. Ca2⫹ sensitivity and maximum force of the
contractile proteins
The Ca2⫹ sensitivity of the contractile proteins is normally
determined after removal of the surface membrane (skinning) and measuring the force at an appropriate range of
[Ca2⫹]. The resulting curve relating force and Ca2⫹ is characterized by the steepness, or Hill coefficient, the Ca2⫹ sensitivity and the maximum Ca2⫹-activated force. Many
physiological and pathological factors that might change in
muscular dystrophies are known to affect Ca2⫹ sensitivity,
especially ROS and NO (for review, see Ref. 233). However, studies on muscle biopsies from boys with DMD (123)
and from mdx mice (174, 509) have not found changes in
Ca2⫹ sensitivity, provided comparisons are made between
comparable fiber types. It remains possible that acute production of ROS or NO associated with intense activity or
eccentric contractions produce changes in sensitivity that
may be persistent (108).
Maximum force was very substantially reduced in the
skinned fibers obtained from muscle biopsies of 5- to 8-yrold DMD boys (123). In fast (IIA) fibers, force from normal
boys was 360 mN/mm2, whereas in the DMD boys it was
only 67 mN/mm2. Slow fibers showed a lesser reduction,
and some gave almost normal force. The reasons for this
drastic reduction in force in fast fibers are not clear. It is
unlikely to be directly related to the presence or absence of
dystrophin since in the mechanical skinning procedure used
here, the entire surface membrane and underlying dystrophin complex are removed. The weakness more likely relates to poor alignment (131) or reduced numbers of myofibrils associated with the disease or the regeneration process. Alternatively some other change associated with the
disease, such as ROS/RNS or Ca2⫹-activated proteolysis,
may have reduced the force-generating capacity. In contrast, studies on mdx fibers showed reductions of ⬃20%
mainly in slow fibers while the fast fibers showed no significant reduction (509).
3. Muscle damage
Muscle damage can contribute to weakness in many ways
depending on the components of the cell that are affected.
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
In this section we focus on some new studies of the earliest
manifestations of damage. An intriguing study of damage
mechanisms in mdx muscle (5–13 mo of age) utilized direct
observation of isolated lumbrical muscles (76). Muscles
were stimulated to give maximal 1-s tetani at 1-min intervals and, in these very small muscles, it was possible to
observe the behavior of individual fibers. As expected, the
wild-type muscles maintained tetanic force well under these
nonfatiguing conditions. However, the mdx muscles
showed a progressive reduction in force to ⬃50% after 10
tetani. Observation showed localized contractures progressing to “hypercontracted clots” occurring during the
interval between tetani. This localized damage was associated with increases in resting intracellular Ca2⫹ and prevented by removal of extracellular calcium. These experiments suggest that normal tetani in the mdx cause Ca2⫹
entry that triggers local contracture and then individual cell
death. The pathway of Ca2⫹ entry was not defined but,
because the damage occurred between the tetani rather than
within a tetanus, it does not seem to be caused by “membrane tears” associated with the stress/strain of contraction.
Even extending the tetani to 5 s in the mdx muscle did not
cause failure during the tetani. A surprising aspect of these
experiments is that single fibers dissected from mdx muscles
do not show failure of this type, even after eccentric contractions (523), suggesting that the lateral connections between fibers are needed for this particular pattern of failure.
Furthermore, intact muscles of the mdx mouse do not show
such rapid decline of force for tetani at this low frequency
(295), suggesting that some aspect of isolation worsens the
situation. One hypothesis is that the Ca2⫹ entry that triggers this damage is through a channel whose opening is
triggered with a delay after tetani. If ROS are involved in the
opening of this channel (145, 507), perhaps the lack of
blood perfusion in this preparation exacerbates the accumulation of ROS and drives this very rapid damage pathway.
Head (174) has investigated the effect of branching on muscle damage, using single mdx fibers that frequently exhibit
two or more side arms (branches), thought to be a consequence of incomplete or abnormal regeneration. Single fibers were isolated by collagenase treatment from 18- to
25-mo-old mdx mice, and resting [Ca2⫹]i was found to be
the same in both WT and branched or unbranched mdx
fibers. However, when stimulated, mdx fibers had reduced
tetanic [Ca2⫹]i compared with WT, and branched fibers
showed a further reduction. When stimulated repeatedly,
branched mdx fibers frequently failed, becoming inexcitable with raised resting [Ca2⫹]i. When fibers were skinned,
the properties of WT and mdx were similar except that
branched fibers frequently ruptured at the branch site. Head
(174) suggests a two-stage process leading to damage in the
mdx. In young muscles, the absence of dystrophin leads to
channel activation that elevates Ca2⫹ followed by Ca2⫹activated damage. This damage then causes splitting of fi-
bers that subsequently leads to further damage and impaired muscle force, as a consequence of the imbalance of
forces around the split site (see FIGURE 6). Head (174) suggests that therapy needs to be implemented in the early
phase, before mechanical impairment adds an additional
component of instability (see also sect. VC4).
4. Lateral transmission of force
Muscles consist of sarcomeres in series and myofibrils and
fibers in parallel. During activation, the force produced is
developed by crossbridges between the thick and thin filaments and is transmitted by the thick and thin filaments and
the Z line proteins between thin filaments; thus the primary
route of force transmission is longitudinal along the myofibrils to the tendon via the myotendinous junction. During
normal activity, sarcomeres remain in register across a muscle fiber and muscle fibers remain in register across a whole
muscle, implying that there is some lateral connection that
retains this registration. It is also important that the sarcomeres remain in register with the sarcolemma; otherwise,
t-tubules would be subjected to shear forces. The lateral
connections between myofibrils occur between the Z lines
and are composed principally of desmin (338). At the edge
of the cell where myofibrils are close to the surface membrane, these lateral connections continue and insert into a
circumferential group of proteins associated with the membrane known as the costamere (360). Many proteins are
associated with the costamere including vinculin, a-actinin,
␥-actin, dystrophin, integrins, and spectrin (for review, see
Ref. 117). Costameres include the DPC and make further
connection via laminin to the extracellular matrix and
hence to neighboring fibers.
While the principal mode of force transmission is longitudinal, there are a variety of situations in which lateral force
transmission is liable to be important. For instance, when a
muscle is partially activated, fibers scattered randomly
throughout a muscle are activated, and because in large
muscles fibers do not run the whole length of a muscle, there
would be no force production unless force were capable of
lateral transmission as well as longitudinal transmission.
Another example would be if a small group of myofibrils is
damaged, then longitudinal force transmission through that
region may fail, but if lateral force transmission occurs, a
damaged region can be bypassed. The first clear demonstration of such lateral transmission in frog skeletal muscle was
made by Street (439). She used a preparation of a single
fiber with a cuff of damaged fibers surrounding it. Force
production was first measured conventionally from tendon
to tendon of the single fiber. Then one tendon was detached
and connection made to the cuff of damaged fibers surrounding the single fiber. When the fiber was stimulated, it
was still possible to measure 85% of the original force between one tendon and the cuff of surrounding fibers demonstrating that lateral force transmission between fibers
must occur. This work has recently been extended to mam-
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ALLEN ET AL.
malian muscles by the use of a “yoke” which was sewed to
the fibrous connective tissue (epimysium) surrounding a
small muscle (371). Force was first measured from tendon
to tendon and then from tendon to yoke with the other
tendon detached. This study confirmed that lateral force
transmission occurs between fibers and the surrounding
connective tissue, but they also extended the study to mdx
mice and old WT rats and mice. In both the mdx and the
aged groups, lateral transmission was substantially reduced
compared with appropriate controls. This important study
establishes that in mdx mice, presumably because of loss of
dystrophin, lateral force transmission is impaired, and this
is likely to contribute to the loss of force production when
there are localized regions of damage in an mdx fiber. Another possible cause of impaired lateral force transmission
in DMD is the excessive deposition of fibrotic tissue in the
endomysium between muscle fibers (97).
5. Blood flow regulation in mdx muscles
Blood flow to active muscles increases by mechanisms that
are still only partly understood (399). Sympathetic activity
increases in exercise and, while this decreases blood flow in
resting muscles, it is relatively ineffective in contracting
muscle, an effect know as “functional sympatholysis”
(452). The mechanism of this inhibition is not known in
detail, but Thomas and Victor (1998) showed that the NOS
inhibitor NG-nitro-L-arginine (L-NAME) attenuated this effect. In a later study they showed that functional sympatholysis was also attenuated in mdx muscle, in which nNOS␮ is
absent from the surface membrane, and in a nNOS␮ KO
muscle (450). Despite the absence of nNOS␮, the contraction-induced increase in blood flow was similar in
L-NAME, mdx muscle, and the nNOS␮ KO, suggesting that
other mechanisms are capable of maintaining the contraction-induced stimulation of blood flow. Functional sympatholysis is also attenuated in boys with DMD (401), raising
the possibility that impaired blood flow to muscles is an
element in the disease. Early clinical studies on DMD boys
failed to detect differences in blood flow between normal
and dystrophic muscles (47). However, a recent study in
BMD showed that tadalafil, a phosphodiesterase inhibitor,
increased cGMP and NO production and thereby restored
functional sympatholysis and increased exercise-induced
muscle blood flow (267). Whether tadalafil will affect the
pathogenesis and progression of BDM remains to be determined. For further discussion of the role of nNOS, see section IVA.
B. Membrane Properties
As noted in the introduction and FIGURE 1, the distribution
of dystrophin and other cytoskeletal proteins suggests they
might have a role in lateral transmission of force (considered in sect. IIIA4) or in strengthening the membrane or
maintaining the registration of sarcomeres with the surface
266
membrane. Dystrophin molecules are rod shaped with a
length of ⬃110 nm (403, 485). They form a network on the
intracellular side of the sarcolemma with increased concentration at the level of the Z line contributing to the
costamere (117, 512). Because t-tubules run laterally across
the fiber, it is critical that the registration of the membrane
to the sarcomeres is maintained; otherwise, there is a risk
that the t-tubules will be sheared off from the surface membrane during contraction or stretch. In the present section
we consider mechanical measurements of the membrane
designed to establish which, if any, properties are affected
by the absence of dystrophin.
An influential early paper by Menke and Jockusch (282)
studied the sensitivity of collagenase-isolated single muscle
fibers to hyposmotic solutions. Such solutions cause muscle
cells to swell, to produce numerous membrane blebs, and
eventually lead to hypercontraction and the leakage of soluble intracellular proteins from the muscle. Menke and
Jockusch (282) found that mdx fibers were more susceptible
to this type of damage and suggested that dystrophin helped
to stabilize the membrane and protect against this type of
damage. However, they also pointed out that the mechanism of damage in this model is uncertain and that the
elevated intracellular calcium that mdx cells often exhibit
might make cells more sensitive to this procedure. In a follow up study, they showed that addition of the Ca2⫹ ionophore A23187 at the time of hyposmotic shock did not
affect the control fibers (283), making this explanation less
likely. Furthermore, they showed that preincubation with
cytochalasin D, which disrupts the actin cytoskeleton, increased the susceptibility of control fibers so that they were
indistinguishable from mdx fibers. These studies suggest
that the combination of the actin cytoskeleton and dystrophin contribute in some way to the integrity of the membrane when exposed to osmotic stress.
While it is possible that mechanical properties of dystrophin/actin contribute to the deleterious effects of osmotic
stress, recent studies have identified Ca2⫹ entry and ROS
production pathways that are involved in this response
(268, 418, 487). These studies will be considered in more
detail in section IIIC.
Another approach to investigate membrane properties is to
suck the membrane into a glass tube; when the suction
pressure is sufficiently great, the membrane fails. In muscle
using conventional patch pipettes (tip diameter ⬃1 ␮m),
membrane failure occurs with a negative pressure of ⬃180
mmHg, equivalent to a membrane stress of ⬃6 mN/m (127,
159, 195). When mdx muscles were compared with WT,
Franco and Lansman (127) found no difference while Hutter et al. (195) found a small but statistically significant
difference (6.0 mN/m for WT vs. 5.2 mN/m for mdx) but
did not believe that this difference was responsible for the
large differences in osmotic sensitivity discussed above. In-
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
stead, they suggested that dystrophin might have an analogous role to spectrin, which maintains the discoid shape of
red blood cells, and in whose absence the more spherical red
blood cells are very sensitive to osmotic swelling and damage. They suggested that dystrophin, through its role in the
costameric connections, might constrain the caveolae and
membrane folds of the skeletal muscle membrane. In its
absence, lack of this folding might lead to less “spare membrane” so that osmotic swelling would lead the membrane
to reach its stretchable limit earlier (194). In later work,
Nichol and Hutter (323) studied the membrane vesicles
obtained by treatment of muscle fibers with collagenase and
hyposmotic solution and showed that increases in the intracellular Ca2⫹ produced by the ionophore A23187 led to a
drastic and rapid decrease in tensile strength. The mechanism of this effect was not explored and the weakening only
occurred above 0.8 ␮M Ca2⫹, but this mechanism might
offer a partial explanation for the greater susceptibility of
the mdx muscle to mechanical perturbations.
Studies of membrane deformation have been extended by
use of simultaneous microscopy to define the shape of the
membrane in the pipette. Suchnya and Sachs (441) found
that the shape of the membrane in the patch pipette was
slightly curved towards the pipette tip in wild-type muscle
in the absence of applied pressure. As negative pressure was
applied, the membrane became curved away from the tip as
expected. Unexpectedly, in mdx muscles, the resting membrane was more strongly curved towards the tip, and this
curvature was eliminated in both wild-type and mdx by
cytochalasin D, which disrupts the ␥-actin cytoskeleton.
These data have important implications for opening of
stretch-sensitive channels in the membrane (which are sensitive to membrane curvature) but might also explain the
small differences in membrane stress at failure noted by
Hutter et al. (195).
Garcia-Pelagio et al. (139) also investigated the deformability of the membrane but used much larger pipettes of ⬃25
␮m diameter (approximately half the fiber diameter). At
low pressures, the membrane and attached contractile machinery were sucked into the pipette. Above the separation
pressure (367 kdyn/cm2, 287 mmHg in wild-type muscles),
the membrane separated from the contractile proteins presumably by failure of the costameres. At still higher negative
pressure (631 kdyn/cm2, 476 mmHg), the sarcolemmal bleb
burst. Both the separation pressure (171 kdyn/cm2) and the
bursting pressure (321 kdyn/cm2) were significantly lower
in mdx mice compared with wild type. These results differ
from the patch-clamp studies discussed above, possibly because the patch-clamp studies used cultured myotubes or
collagenase-treated myocytes which lack the extracellular
matrix. Perhaps when this mechanical support is absent, the
structural role of dystrophin is minimized, which could explain why the membrane stiffness only appears smaller in
mdx fibers when an intact preparation is tested. Another
possibility is that the much larger pipette tips (25 vs. 1 ␮m)
probe different aspects of the mechanical properties of the
membrane, cytoskeleton, and myofibrillar complex.
Indentation stiffness has also been measured with glass
rods that depress the surface of myotubes grown in culture (337). The values obtained in this way for indentation stiffness were 12 dyn/cm for wild-type myotubes
versus 3.4 dyn/cm for mdx fibers. The authors considered
the possibility that elevated [Ca2⫹]i might have influenced the stiffness of mdx fibers, for instance, by activating calpains that proteolyse cytoskeletal proteins, but
attempts to modulate [Ca2⫹]i or inhibit calpains with
leupeptin had minimal effect. They concluded that dystrophin contributes to indentation stiffness. Indentation
stiffness has also been measured in intact muscles using
an atomic force microscope whose conical probe was
indented into the muscle. A Young’s modulus was calculated as 4.2 kPa for wild-type and 1.4 for mdx fibers.
When cytochalasin D was used to disrupt the actin cytoskeleton, stiffness values in wild-type muscles fell to values indistinguishable from mdx fibers while cytochalasin
D only produced small falls in stiffness in mdx fibers.
When either utrophin or dystrophin was expressed in
mdx muscle fibers, stiffness returned to wild-type values.
In summary, the results suggest that under near-normal
conditions, when muscles have their normal complement of
extracellular matrix and myofibrillar proteins, dystrophin
makes some contribution to the radially measured stiffness,
probably through connections with the actin cytoskeleton.
Whether this reduced stiffness has a broader role in the
developing pathology of the disease remains to be established.
C. Increased Membrane Permeability
1. Appearance of CK in the blood
Intense physical activities often result in elevated serum levels of enzymes, such as CK and lactate dehydrogenase
(LDH), which are abundantly expressed inside skeletal
muscle fibers. For normal individuals, the level of these
enzymes can be increased in the blood after strenuous exercises, such as marathon running or resistance training,
particularly when these exercises include eccentric contractions (48). Several inherited myopathies, including many
forms of muscular dystrophy, are also clinically characterized by elevated serum CK levels, which can reach values
100-fold higher than normal levels in DMD patients (531).
On the basis of this evidence, it is widely accepted that high
blood CK levels are due to or associated with muscle damage, either as a result of a genetic neuromuscular disease or
following damaging eccentric contractions. Nevertheless,
studies in animals have shown that the magnitude of CK
increase does not directly correlate with the extent of mus-
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ALLEN ET AL.
cle damage after eccentric contractions, as measured by the
loss of force (130, 261). Furthermore, mutations in musclespecific genes, such as caveolin-3, result in hyperCKemia,
an increased blood CK level without any measurable muscle
weakness, abnormal pathology, or other neuromuscular
disease symptoms. HyperCKemia can also occur from certain drugs and toxins, as well as alcoholism and malignant hyperthermia (48). Therefore, while elevated blood
CK levels are clearly a very useful clinical marker of
myopathy in patients presenting with other symptoms of
neuromuscular disease, its use as an accurate, quantitative measure of muscle damage and muscle function in
animal and human studies should be evaluated with some
caution. On this point, it is known that skeletal muscle
CK is susceptible to oxidative modification by ROS and
reactive nitrogen species (RNS), which reduces its enzymatic activity and targets it for proteolysis (325). Therefore, in mdx and DMD muscles, under conditions of
oxidative stress, only CK not significantly modified by
ROS is likely to be detected by the standard CK activity
assays. This may further complicate the interpretation of
blood CK activity levels and their relationship with muscle damage under conditions of increased ROS production in dystrophic muscles. Quantitative measurements
of CK protein levels by western blotting would be a useful complement to measurement of CK activity.
As in humans, rodents also have large increases in serum
CK and LDH after eccentric contractions and in animal
models of DMD such as the mdx mouse. In addition, animal studies have shown that eccentric contractions can
cause an influx of extracellular proteins, such as albumin
(275), or membrane-impermeable dyes, such as procion orange and Evans Blue dye (162, 354, 504, 505) into muscle
fibers, the magnitude of which is significantly greater in
mdx muscle than WT. Intuitively, the apparent free movement of molecules into (albumin, Evans Blue dye) and out
of (CK, LDH) muscle fibers would be consistent with one
common membrane damage pathway leading to enhanced diffusion of these molecules across the sarcolemma. Over the past 20 years or so, a popular idea to
explain the increased membrane permeability, and the
subsequent damage and fiber necrosis in dystrophic muscles, was mechanical injury of the sarcolemma as a result
of high force eccentric contractions (295, 354) and the
subsequent influx of extracellular Ca2⫹ which stimulated
proteolysis and muscle degradation. This idea was based
on the rationale that during muscle contraction, dystrophin acts as a mechanical shock absorber, dissipating
force laterally from the contractile filaments, via the actin
cytoskeleton, across the sarcolemma to the extracellular
matrix. Thus dystrophin was thought to provide protection against sarcolemmal tears or lesions developing during high force contractions, particularly eccentric contractions (354). While this theory is plausible, it has, to
our knowledge, not been supported by direct experimen-
268
tal evidence. It is important to emphasize that the presence of dye positive fibers and elevated blood CK after
eccentric contractions in mdx mice does not per se provide any information about the physiological pathway(s)
leading to the increased sarcolemmal permeability. Furthermore, despite the popular theory that molecules diffuse in or out of muscle fibers via mechanically induced
sarcolemmal tears, evidence from DMD patients indicates that high force muscle contractions are not necessary for increased membrane permeability, since blood
CK rises significantly soon after birth, before the onset of
high force muscle activity and peaks between ⬃1 and 6 yr
of age (531). Thus the dystrophic disease process, independent of high force contractions, is sufficient to raise
blood CK levels in DMD.
Based on the current knowledge of membrane repair processes in mammalian cells, including mdx skeletal muscle,
mechanically induced membrane holes, even very large laser-induced lesions up to 5 ␮m (26) are rapidly repaired in
seconds, a process that requires extracellular Ca2⫹ and intracellular vesicles (377). Thus, if membrane micro-tears
occurred during eccentric contractions, they would be expected to quickly reseal so that any dye entry would occur
during and within seconds after the contractions and then
remain trapped inside the fiber after the membrane was
rapidly repaired. In other words, the membrane tear theory
would predict a constant level of dye inside fibers, regardless of the time at which the muscle was analyzed after the
eccentric contractions (see FIGURE 3). Moreover, the number of tears would be predicted to be greatest during the first
eccentric contraction, when peak force is greatest, and least
during the last, when peak force is lowest, given that the
absolute force produced by the muscle would be the main
factor leading to micro-ruptures (354). Thus muscles collected for analysis 30 or 60 min after eccentric contractions
would not provide any insight into the very early time
course of dye entry. To test this hypothesis, a time course
study is required, with the first measurements made immediately after the eccentric contractions. McNeil and Khakee
(275) measured the uptake of albumin in eccentrically exercised rats at 0 and 90 min post-exercise and found dyepositive fibers at both time points. However, the eccentric
exercise duration was 90 min in this study, so this does not
provide any information regarding the early time course or
pathway of albumin uptake during the first few contractions. Similarly, studies of dye uptake in mdx mice in vitro
are typically measured 60 –90 min post-eccentric contractions (295, 325).
2. Role of Ca2⫹ influx through mechanosensitive
channels and ROS on membrane permeability
To our knowledge, the study by Whitehead et al. (505) is
the only paper to investigate the early time course of dye
uptake in mdx muscle, from immediately after eccentric
contractions up to 60 min. In this study, muscle force fell
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
A “Membrane tear” theory
Experimental results
Extracellular dye
B
mdx
mdx
mdx
(ecc con 0 min) (ecc con 60 min) (ecc strep 60 min)
Extracellular dye
Pre ECC
Intracellular
Intracellular
500 μm
mdx (ecc con)
mdx (ecc strep)
mdx (no ecc con)
mdx (no ecc strep)
wt (ecc con)
wt (ecc strep)
20
Procion orange
positive area (%)
During ECC
Dye entry
Membrane repair
Seconds
after ECC
15
*
*
10
5
*
0
Dye entry
0
C
30 minutes
after ECC
30
60
Time after eccentric contractions (min)
wild type con
mdx con
mdx NAC
Dye entry
60 minutes
after ECC
500 μm
Dye entry
NAC
Streptomycin
Gsmtx-4
ECC with
NAC or
SAC blockers
ROS
ROS
Ca2+ Ca2+
Evans Blue Dye
positive area (%)
12
10
8
6
4
2
0
*
*
wt con
mdx con
mdx NAC
FIGURE 3. The mechanism of stretch-induced membrane permeability in dystrophic muscle. A: according to
the original “membrane tear” theory, the increased dye entry into dystrophic muscle fibers was attributed to
micro-tears in the sarcolemma, caused by eccentric contractions. The membrane tear theory predicts that
extracellular dyes would rapidly enter the muscle fiber immediately after the mechanically induced tear was
produced, and within seconds the membrane would reseal and trap the dye inside the fiber. Therefore, the
amount of dye inside the fiber would remain constant at all times after the eccentric contractions. However,
experimental results indicate a different mechanism of dye entry. Extracellular dye uptake is very small
immediately after the eccentric contractions and progressively increases up to at least 60 min post eccentric
contractions. Moreover, both mechanosensitive channel blockers (streptomycin and Gsmtx-4) and the antioxidant N-acetyl cysteine can prevent almost all of the dye uptake, suggesting pathways involving Ca2⫹ entry
through mechanosensitive channels and ROS produced by NOX2 are responsible for the increased membrane
permeability (see text for details). B: experimental results showing the time course of procion orange dye
uptake after eccentric contractions in mdx EDL muscle and the inhibitory effect of the MSC blocker streptomycin. [From Whitehead et al. (505).] C: experimental results showing the inhibitory effect of the antioxidant
NAC on Evans Blue dye entry in mdx EDL muscle following eccentric contractions caused by downhill treadmill
running. [From Whitehead et al. (504).]
significantly in mdx muscles to ⬃35% of the original
force at 30 min post-contractions, indicating that considerable muscle damage had occurred. However, despite
this, procion orange dye uptake immediately after the
eccentric contractions was minimal, only a few percent
higher than nonstimulated mdx control muscles. Interestingly, the area of dye-positive fibers increased progressively at 30 min and further at 60 min, to reach a value
⬃15% higher than nonstimulated mdx muscles at this
time point. These data indicate that dye uptake in severely damaged mdx muscles is a relatively slow process
occurring gradually over at least an hour after the eccentric contractions. The data also suggest that up to 60 min,
most fibers do not take up dye and those that do have a
heterogeneous time course of dye uptake. In the same
study, we also investigated if blocking mechano-sensitive
channels (MSC) could reduce the number of dye positive
fibers. Importantly, the dye uptake that occurred in this
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ALLEN ET AL.
study was significantly reduced by two MSC blockers,
streptomycin and Gsmtx-4, which also reduced the force
loss from stretched contractions. Interestingly, the relatively slow, progressive increase in dye uptake over 60
min is similar to the time course of Ca2⫹ influx and raised
[Ca2⫹]i in mdx fibers after stretched contractions. Taken
together, these data argue against membrane tears as the
main route of dye entry after eccentric contractions. Instead, it strongly indicates that MSC-induced Ca2 influx
triggers a pathway that leads to increased membrane permeability and dye entry.
In considering the pathway(s) causing increased membrane
permeability in dystrophic muscle, it should be emphasized
that eccentric contractions carried out in vitro generate considerably higher forces and strains than those produced in
vivo, where contractions are generated by nonsynchronous,
submaximal stimulation, and muscle strains are much
lower than those used in vitro. Since the micro-tear theory is
based on the concept that mechanically induced membrane
lesions are caused by high muscle forces (354), the lack of
direct evidence for micro-tears in vitro makes it very unlikely that these lesions occur in vivo. In support of this idea,
a recent paper (366) did not find any reduction in Evans
Blue dye positive fibers in eccentrically exercised mdx mice
injected with Poloxamer 188, a membrane sealant used for
stabilizing and repairing membrane breaches. In contrast,
Evans Blue uptake in mdx mice could be prevented by administering the MSC blocker streptomycin before the eccentric exercise (505). This result is also supported by a
recent study wherein mdx mice expressing a dominant negative TRPV2 mutant protein in skeletal muscle had significantly reduced Evans Blue uptake and blood CK levels in
vivo (198). TRPV2 forms a Ca2⫹-permeable channel,
which can be activated by stretch and store depletion (198).
Furthermore, lowering [Ca2⫹]i by overexpression of
SERCA1 in skeletal muscles of mdx mice dramatically reduced blood CK levels (149). Therefore, the current data on
intact mdx mice also argue against mechanically induced
micro-tears as the mechanism for stretch-induced dye uptake, while providing strong evidence that Ca2⫹ influx
through SACs and increased [Ca2⫹]i is a major mechanism
for triggering the increased membrane permeability.
In addition to Ca2⫹ entry through MSC, ROS also play an
essential role in membrane permeability of dystrophic muscles. Following eccentric contractions of mdx EDL muscles
in vitro, Evans Blue uptake was significantly reduced by the
antioxidant N-acetylcysteine (NAC), with similar levels to
WT controls (504). A recent study using the antioxidant
ascorbic acid also showed a reduction in Evans Blue uptake
in mdx muscle in vivo (460). Moreover, serum CK levels
were significantly reduced in mdx mice by intraperitoneal
delivery of the antioxidants ␣-lipoic acid and L-carnitine
(182) and oral administration of green tea extract (59).
Based on these data, although the precise sequence of events
270
remains to be elucidated, both Ca2⫹ influx through SACs
and excessive ROS production are primary pathways for
mediating dye entry and CK efflux from dystrophic muscles.
3. Possible Ca2⫹- and ROS-mediated membrane
permeability pathways
In nondystrophic mouse diaphragm muscles, procion yellow dye uptake was stimulated by BAY K 8644-mediated
Ca2⫹ influx, which was mediated by cytosolic, Ca2⫹-dependent phospholipase A2 (PLA2) activity and ROS, particularly the hydroxyl radical (OH⫺). Cytosolic phospholipase
A2 (cPLA2) is a Ca2⫹-dependent enzyme that targets membrane phospholipids, giving rise to lysophospholipids and
free fatty acids (FFA), both of which can cause further membrane damage (354). The activity of cPLA2 was found to be
10 times greater in DMD muscles compared with control
muscles (248). Moreover, mass spectrometry lipid imaging
studies of mdx and DMD muscles using Time-of-Flight Secondary Ion Mass Spectrometry found increased FFA and
arachadonic acid levels in degenerating areas of muscle
(447). Together these results indicate that increased PLA2
activity occurs in dystrophic muscle and is a likely mechanism of sarcolemmal lipid disruption and increased permeability. ROS such as hydroxyl (OH⫺) and peroxinitrite
(ONOO⫺) initiate lipid peroxidation, which can also disrupt the chemical structure and fluidity of membranes and
increase permeability. A recent study has shown that DMD
muscles have threefold greater levels of lipid peroxidation
than control muscles, which is accompanied by a 50% reduction of the endogenous antioxidant glutathione (GSH)
(379). Studies in mdx mice have also shown evidence of
ROS-mediated lipid peroxidation (44, 105), which can be
prevented by antioxidants (286). It is also possible that
PLA2 might work in conjunction with ROS to permeabilize
the membrane, since PLA2 is very effective at removing
oxidized lipids (416, 417). With this in mind, it is interesting to note that PLA2 can also directly increase mitochondrial ROS production in muscle, possibly by effects on the
electron transport chain (324).
4. Role of increased membrane permeability on
muscle function
It is clear that the relatively small number of dye positive
fibers (5 to 20%) (295, 505) does not, by itself, explain the
large, immediate force drop (often 70% or more) that commonly occurs from eccentric contractions in mdx muscle.
Assuming the most extreme case scenario, even if all the
dye positive fibers failed to generate any force, which is
unlikely, this would contribute up to 20% or so of the
force deficit. However, as discussed earlier, the stretchinduced increase in membrane permeability is negligible
immediately after the contractions and increases progressively for at least 60 min in mdx muscle (505), whereas
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the force loss is instantaneous, becoming greater with
each successive eccentric contraction. This difference in
the time course of development suggests that the immediate loss of force is not directly caused by the increased
membrane permeability. However, the same pathways
that mediate the increased permeability, ROS and Ca2⫹
entry through SACs, also contribute to the force loss after
eccentric contractions (see below).
A more likely outcome to explain the apparent selectivity of
membrane-permeable fibers is that these fibers are the most
vulnerable to eccentric contraction-induced damage. This
could occur because some fibers are already partially damaged before the eccentric protocol, as a result of the normal,
dystrophic process in vivo. For example, if the sarcolemma
of these fibers was already undergoing lipid peroxidation or
lipid damage due to PLA2 activity, the additional Ca2⫹
influx and ROS production from the eccentric contractions
could accelerate the membrane permeability process, leading to dye uptake within the first 60 min or so following the
contractions. Another possibility is that certain fibers may
be more susceptible due to anatomical differences such as
cross-sectional area, changes in signaling protein expression or activity, or fiber type differences. Some support for
this last hypothesis was provided by a study in which mdx
EDL (a predominantly fast glycolytic muscle) but not soleus
(a slow, oxidative muscle) was susceptible to eccentric contraction-induced membrane permeability and force loss
(295). This also supports the finding that fast fibers are
preferentially damaged and lost in muscles of DMD patients (494) and in the mdx diaphragm (355), the most
severely affected muscle in these mice. Therefore, Evans
Blue dye uptake might reflect the normal degenerative process in a subset of susceptible fibers, and this process can be
accelerated by eccentric contractions.
Another reason for the selective uptake of dye into a relatively small subset of fibers was provided by the studies of
Head and colleagues (see sect. IVA3). They showed that
following eccentric contractions, localized Evans Blue accumulation occurred preferentially in branched EDL fibers
from mdx mice, at the junction of the normal to branched
fiber region (174). Also, they found that nonbranched fibers
were mostly protected from Evans Blue uptake. These findings could account for the relatively low numbers of Evans
Blue positive fibers found in cross-sections of mdx muscles
after eccentric contractions. Interestingly, it was shown that
in 2-mo-old mdx mice, the EDL muscle contained 17%
branched fibers (64), which is very similar to the percent
Evans Blue positive fibers found after eccentric contractions
of mdx mice of the same age (505). In older mdx mice, the
number of branched fibers increases to 87%, and this was
accompanied by a commensurate rise in Evans Blue positive
fibers and greater force loss after eccentric contractions
(64). Interestingly, branched fibers have reduced SR Ca2⫹
release and increased Ca2⫹ spark activity at branch points
following osmotic stress than normal, unbranched mdx fibers (250). On the basis of the evidence already presented, it
is possible that increased mechanical stress at the branched
region activates more ROS and Ca2⫹ influx through mechanosensitive channels (MSC, also known as stretch-activated
channels), which leads to localized membrane permeability
and dye uptake in these branched regions.
IV. PROTEIN CHANGES IN THE ABSENCE
OF DYSTROPHIN
As described in section II, the loss of dystrophin from the
sarcolemma causes significant changes in the expression
and localization of many structural and signaling proteins
associated with the dystrophin complex. Recent studies using proteomics have also shown protein expression changes
dystrophic muscles that involve a wide range of cellular
pathways including metabolism, ion channel regulation,
extracellular matrix composition, and cellular stress response (107, 312, 375). These studies highlight the complex
nature of the disease and are useful for identifying new
mechanistic pathways and novel therapeutic targets. In this
section, we have focused on the expression changes and
localization of proteins relating to NO, ROS, and Ca2⫹
signaling pathways in dystrophic muscle, and how these
proteins contribute to muscle damage in DMD.
A. NOS and NO
Nitric oxide is an important signaling molecule in many
aspects of skeletal muscle physiology (431), particularly
those relating to adaptation from exercise (442). NO modulates glucose uptake, regulates vascular perfusion during
contraction, maintains mitochondrial function, and modulates excitation-contraction coupling (ryanodine receptor
function) in normal muscle. It is not surprising, then, that
abnormalities in NO bioavailability are associated with
muscle diseases.
Skeletal muscle tissue expresses all three distinct gene products of NOS: NOS1 (nNOS), NOS2 (inducible NOS,
iNOS), and NOS3 (endothelial NOS, eNOS). All NOS isoforms are active in the dimeric form (FIGURE 4). The predominant form of NOS in skeletal muscle fibers is one isoform of nNOS. Alternative splicing of the NOS1 gene product yields four major isoforms: nNOS␣, nNOS␮, nNOS␤,
and nNOS␥ (50). nNOS␮ is the predominant form in skeletal muscle and differs from nNOS␣ by the inclusion of a
muscle-specific exon that encodes a 34-amino acid insert
located in the catalytic domain (419). The function of the ␮
insert is not known. nNOS␤ lacks the amino-terminal PDZ
domain present in nNOS␮. nNOS␥ lacks part of the catalytic domain and its expression at the protein level has not
been convincingly demonstrated. Since nNOS␣ is not expressed in skeletal muscle, this discussion of nNOS in skeletal muscle will be limited to nNOS␮ and nNOS␤.
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Structurally, nNOS and eNOS differ primarily in the amino-terminal region. eNOS lacks a PDZ domain but is dually
acylated by myristoylation and palmitoylation near the
amino terminus. In skeletal muscle, eNOS is expressed primarily in endothelial cells in the vasculature, although some
reports show low levels of the enzyme on the sarcolemma
and in association with mitochondria. Unlike nNOS and
eNOS, iNOS is constitutively active, due to its high affinity
for calcium/calmodulin. Resting levels of Ca2⫹ are sufficient to maintain iNOS in the active state. Like eNOS,
iNOS is present in only low levels in skeletal muscle fibers,
but in dystrophic muscle tissue, iNOS present in inflammatory cells contain high levels of the enzyme. For reviews of
NOS in skeletal muscle, see References 90, 272, 273, 372,
431.
1. nNOS␮ on the sarcolemma
In skeletal muscle, nNOS␮ is largely associated with the
sarcolemma and is enriched at costameres and myotendious
junctions (66). This localization, and presumably its function, is determined by the interaction of nNOS␮ with several proteins, most notably the DPC. For purposes of this
review, we focus on those interactions that are relevant to
muscular dystrophy.
Association of nNOS␮ with the DPC requires ␣-syntrophin
(6, 49). The PDZ domains of nNOS␮ and ␣-syntrophin are
both required. Analysis of the structure of this complex
revealed an unexpected interaction. The PDZ domain of
␣-syntrophin binds to a pseudo-SXV sequence in a ␤-turn
structure in nNOS␮ that lies just downstream of the
nNOS␮ PDZ domain (180). In this arrangement, the PDZ
domain of nNOS␮ is available to bind other proteins.
In studies of muscle from patients with BMD, nNOS␮ is
absent from the sarcolemma in some cases, despite the
presence of ␣-syntrophin (67). Subsequent studies identified the repeats 16 –17 of the dystrophin rod domain as
necessary for nNOS association with the DPC (500).
These results are consistent with the demonstration that
dystrophin constructs used for AAV-mediated expression in mdx muscle need the spectrin-like repeats 16 –17
in the rod domain to restore nNOS to the sarcolemma
(229). The docking site has been further delineated to
certain ␣-helices within the rod region (230). Some studies suggest that utrophin is unable to restore nNOS to the
sarcolemma (244), a potential problem for utrophin replacement therapy. Nevertheless, sarcolemmal localization of nNOS is considered a reliable diagnostic tool for
Becker MD (461, 500) and a marker for complete restoration of the dystrophin complex (500).
In addition to ␣-syntrophin and dystrophin, nNOS␮ also
interacts with other proteins that are important for muscle
function. Phosphofructokinase-M (PFK-M) is an important
regulatory enzyme in glycolysis. Humans that are genetically deficient in PFK-M have muscle weakness (172), experience activity-induced fatigue (482), and exhibit reduced
glycogen metabolism and increased serum levels of CK
(315). PFK-M binds via its carboxy-terminal tail to the PDZ
of nNOS␮ (125). Further studies are needed to fully understand the metabolic and physiological role of the nNOS␮PFK-M interaction (496).
The activity of sarcolemmal nNOS␮ is modulated by its
interaction with several other proteins, including caveolin-3, heat shock protein 90 (HSP90), and protein inhibitor
of NOS (PIN). PIN is a small, highly conserved protein,
A
nNOSμ
B
Oxygenase
PDZ
CaM
μ
Reductase
COOH
nNOSβ
Oxygenase
CaM
Reductase
Oxygenase
CaM
Reductase
Fe
H4B ARG
Myr
eNOS
iNOS
Oxygenase
domain
AM
C
Palm
Oxygenase
CaM
Calmodulin
binding
domain
Reductase domain
FMN
FAD
NADPH
Reductase
FIGURE 4. Nitric oxide synthase (NOS). A: Different isoforms of NOS are generated by 3 genes. All have
three domains in common: the oxygenase domain, the reductase domain, and the calcium/calmodulin binding
domain (CaM). The muscle-specific form of nNOS contains an insert (␮) that is absent from the brain isoform,
nNOS␣. The PDZ domain in the NH2-terminal region of nNOS␮ binds numerous signaling proteins. The nNOS␤
NH2-terminal domain likely targets this isoform to the Golgi elements. eNOS is cotranslationally myristoylated
(Myr) on the NH2 terminus and posttranslationally by palmitoylation (Palm) at two nearby sites. B: All isoforms
are active as homodimers formed by interaction of the oxygenase domains. Arg, arginine binding site; BH4,
tetrahydrobiopterin binding site; FMN, flavin mononucleotide binding site; FAD, flavin adenine dinucleotide
binding site; NADPH, binding site for reduced form of nicotinamide adenine dinucleotide phosphate. [The figure
of the NOS dimer (B) was kindly provided by Dr. Philip Dash, Univ. of Reading, UK.]
272
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
now known to be dynein light chain 8 (181), that blocks
nNOS dimer formation (200, 458). Caveolin-3, also an inhibitor of nNOS (475), is upregulated in mdx and DMD
muscle (380, 468). Furthermore, mutations in caveolin-3
result in LGMD1C, hyperCKemia, rippling muscle disease,
and distal myopathy (141). HSP90 regulates nNOS turnover via ubiquitination (340). These interactions illustrate
the complexity of the mechanisms of nNOS regulation in
normal and dystrophic muscle.
The loss of nNOS from the sarcolemma is not limited to
DMD or BMD. In several other muscle diseases, sarcolemmal nNOS is absent or markedly reduced. These include
LGMD 2C, 2D, and 2E and inflammatory myositis (85) and
several forms of inherited and acquired myopathic conditions (122). Also, diseases in which denervation occurs,
such as amyotrophic lateral sclerosis and spinal muscular
atrophy or in which transmission at the neuromuscular
junction is defective, such as myasthenia gravis, have reduced sarcolemmal nNOS (122, 277). The loss of sarcolemmal nNOS in these diseases is surprising, since at least in
one case, dystrophin and ␣-syntrophin are present at normal levels (277). Understanding the mechanism of this defect in these diseases may shed new light on the regulation of
nNOS␮ association with the sarcolemma.
2. nNOS␮ in the cytoplasm
In skeletal muscle fibers, ⬃50% of nNOS␮ is found in the
cytosol (450). In mdx muscle, the total amount of nNOS␮ is
markedly reduced, but the remaining enzyme is almost entirely in the soluble fraction. Muscle from mice lacking
␣-syntrophin also has a higher level of soluble nNOS␮
(451). The function of soluble nNOS␮ is not known, although transgenic expression of nNOS␣ in skeletal muscle
resulted in reduced amounts of inflammatory cells, fibrosis,
and serum CK in mdx (495) and dystrophin/utrophin DKO
mice (497).
Carboxy-terminal binding protein (CtBP), a transcriptional
corepressor activated by NADH, may be involved in nNOSmediated improvements in skeletal muscle function. CtBP
binds via its carboxy terminus to the nNOS PDZ domain
(383). As NADH levels are depleted by muscle activity, the
repressive effects of CtBP are reduced, thereby activating
transcriptional modulators of mitochondrial biogenesis
(503). Binding of CtBP via its carboxy-terminal tail to
nNOS␮ shifts the location of CtBP from the nucleus to the
cytosol (383), a process that could boost mitochondrial
biogenesis.
The association of nNOS with microtubules may have implications for the early events leading to the dystrophic
pathology. Recent studies demonstrated that nNOS (440)
and ␣-syntrophin (133) bind to microtubule-associated
proteins 1A and/or 1B (MAP1A/1B). Growing evidence
suggests that MAPs are involved in signal transduction by
modulating microtubules and through interaction with signaling proteins, such as nNOS. Microtubules interact with
dystrophin (361), and the highly organized lattice structure
characteristic of healthy muscle is disrupted in dystrophic
muscle (345). Furthermore, the lattice structure is largely
restored by rAAV-microdystrophin (345). Abnormalities in
microtubules may be a critical step in dysfunction in calcium and ROS signaling (213 and see sect. IVD6).
3. nNOS␤ on the Golgi apparatus
In mice, nNOS␤ is sufficient to mediate penile erection, even
though it generates only a small fraction of NO production
(193). As in other tissues, the nNOS␤ isoform is expressed
in skeletal muscle at much lower levels than nNOS␮, but its
localization is highly restricted. The discovery that nNOS␤
is localized on skeletal muscle Golgi elements, along with
soluble guanylate cyclase and protein kinase G, is strong
evidence that it mediates an NO-cGMP signaling pathway
(343). This and other studies prompted examination of
phosphodiesterase (PDE) inhibitors as potential therapeutic
agents in mdx mouse skeletal and cardiac muscle (341).
Inhibition of PDE5 showed improvement in mdx skeletal
(18, 217, 347) and cardiac function (4, 19, 212). Very recently, a trial of tadalafil was conducted in men with BMD
in which muscle oxygenation was reduced due to impaired
modulation of the exercise-induced vasoconstrictor response (267). The results demonstrated that tadalafil alleviates muscle ischemia and restores normal blood flow.
These results provide further evidence for nNOS and NOcGMP as essential components of normal muscle function
and as promising therapeutic targets.
The mechanism(s) by which PDE5 inhibitors alleviate the
dystrophic phenotype are not fully understood. Defects
in mitochondrial localization and ATP synthesis in mdx
muscle are important in the development of the dystrophic pathology, but these abnormalities are not corrected
by sildenafil administration (346). Vascular dysfunction
in mouse models (217) and in humans (267) probably
contributes to, but is not the primary cause of, muscular
dystrophy.
Two genetically engineered mouse models have proven to
be very helpful in understanding nNOS function in skeletal
muscle (160, 191). The KN1 mouse lacks nNOS␮ but
nNOS␤ is retained. All isoforms of nNOS are absent in the
KN2 mouse. A comparison of these models on the same
genetic background (C57BL/6) has provided evidence that
nNOS␮ and nNOS␤ may have different functions in skeletal muscle (343). Tibialis anterior muscle from the KN1
mouse analyzed in situ exhibited normal force output and
post exercise fatigue. This is in contrast to earlier studies
showing a modest increase in fatigue on the B6129 strain,
emphasizing the importance of proper genetic-background
controls in these studies (342). The KN2 mouse, which
lacks both nNOS␮ and nNOS␤, displays a more pro-
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nounced functional phenotype. KN2 tibialis anterior muscle exhibited dramatically increased contraction-induced
fatigue and force deficits after exercise. Also, the rate of
recovery after fatigue was greatly slowed. Finally, KN2
muscle generated markedly reduced maximum isometric
force and was significantly weaker, based on reduced mean
specific force.
The role of nNOS isoforms in the dystrophic pathology of
DMD has received considerable attention, in part because
of their potential as therapeutic targets. Two general approaches have been used: 1) elimination of one or both
forms of nNOS␮ in mdx mice and 2) expression of nNOS as
a soluble or membrane-targeted protein in mdx muscle.
Interpretation of the studies from different labs is compromised by the use of different genetic strains, methodologies,
ex vivo versus in situ physiological tests, muscle type, and
the nNOS isoform. Nevertheless, some general conclusions
can be gleaned from the studies.
Genetic deletion of nNOS␮ from the mdx mouse (dystrophin/nNOS␮ DKO) improved force generation by the EDL
measured ex vivo (246). This result strongly suggests that
the level of cytosolic nNOS␮ has a marked impact on muscle function. In a tail suspension model of unloading-induced muscle atrophy, nNOS␮ became dislocated from the
sarcolemma to the cytosol (445). Atrophy was much milder
in nNOS␮ null mice subjected to the same treatment. The
mechanism appears to involve cytosolic NO production by
delocalized nNOS␮ resulting in Foxo transcription factor
translocation to the nucleus, which then activates muscle
atrophy genes.
In addition to causing the loss of nNOS␮ from the sarcolemma, dystrophin deficiency also affects nNOS␤ associated with skeletal muscle Golgi elements (132). In the mdx
mouse gastrocnemius muscle, the Golgi levels of nNOS␤
are reduced. Furthermore, the “beads on a string” organization characteristic of Golgi elements in normal skeletal
muscle is also disrupted, even in 2-wk-old mdx mice (132,
345). Thus the absence of dystrophin disrupts signaling by
nNOS␤ by reducing levels of the Golgi-associated enzyme
and altering its organized distribution, even before the onset
of muscle necrosis.
Complete elimination of nNOS in mdx mouse muscle was
accomplished by crossing the mdx mouse with the KN2
mouse that lacks both nNOS␮ and nNOS␤ (mdx-NOS
DKO) (132). These mice have smaller body mass than WT
or mdx mice and lack the skeletal muscle hypertrophy characteristic of mdx mice. Central nucleation is also reduced,
suggesting that regeneration may be impaired in mdx-NOS
DKO muscle. Also, macrophage infiltration was dramatically increased (⬎10-fold) compared with mdx muscle.
Muscle weakness was markedly worse as was susceptibility
to eccentric contraction-induced muscle damage in the
274
mdx-NOS DKO mouse. However, mdx muscles lacking
both forms of nNOS do not exhibit increased muscle fatigue. TA muscle from mdx and mdx-NOS DKO mice show
a similar force decrement during repetitive stimulation in
situ. This surprising result suggests that the absence of dystrophin has a compensatory effect on muscle lacking both
nNOS␮ and nNOS␤, which is very susceptible to fatigue.
These results revealed unappreciated complexity in the role
of nNOS in dystrophic muscle.
Restoration of nNOS␮ to dystrophic muscle could potentially correct physiological defects associated with DMD.
Transgenic expression of nNOS in the mdx mouse has been
reported to improve the histopathology of skeletal muscle,
without targeting of the enzyme to the sarcolemma (495).
Immune cell infiltration and fiber central nucleation were
both decreased. Similar experiments on the mdx:utrophin
double knockout showed a small increase in life span and
reduced fibrosis, cellular infiltration, inflammation, and
procion red uptake (497). While these results are consistent
with a positive impact of nNOS overexpression on the dystrophic histopathology, the approach had several limitations. First, these studies used nNOS␣, the brain isoform
rather than the muscle-specific nNOS␮. Second, the enzyme
was expressed in skeletal muscle at very high levels (455). In
addition, the high levels of nNOS resided entirely in the
cytosol (or at least, not on the sarcolemma), where excess
production of NO could be detrimental. Finally, no contractile physiological studies were performed to determine
if nNOS␣ expression had a functional effect.
To force expression on the sarcolemma, nNOS␮ was modified by introduction of a RAS sequence at the carboxy
terminus. Earlier studies had shown that ␣-dystrobrevin
containing a RAS sequence is targeted to the sarcolemma
when transgenically expressed in the mdx mouse (7). As
predicted, nNOS␮-RAS was targeted to sarcolemma in
mdx muscle. In situ contractile physiological studies revealed that nNOS␮-RAS markedly reduced both muscle
fatigue and eccentric contraction-induced force loss, in mdx
TA muscle (Rebolledo et al., unpublished data). Unmodified nNOS␮ did not associate with the sarcolemma and
failed to improve the contractile properties of mdx TA muscle, highlighting the importance of sarcolemmal localization of nNOS for improving physiological function of mdx
muscle. The beneficial effects of nNOS␮-RAS may be due,
in part, to increased sarcolemmal expression of utrophin,
␣-syntrophin, ␣-dystrobrevin, and ␤-dystroglycan. Therefore, AAV-mediated expression of this novel, sarcolemmal
targeted form of nNOS␮ may be therapeutically useful in
diseases in which sarcolemmal nNOS is lacking.
4. eNOS
This form of NOS has received little attention in the DMD
field. In one interesting study, eNOS levels in resistance
arteries were reduced in the mdx mouse, compared with
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control mice (249). Importantly, NO-dependent vascular
function and vascular density were also reduced. Treatment
with gentamycin to promote readthrough of the early stopcodon in dystrophin restored both parameters. These results indicate that eNOS may play a significant role in the
pathological development. Additional studies of vascular
eNOS are warranted.
5. iNOS
iNOS is expressed at low levels in normal muscle fibers, but
in dystrophic muscle, infiltration by inflammatory cells containing iNOS leads to much higher levels of this isoform in
skeletal muscle tissue. However, cultured mdx myotubes
express higher levels of iNOS than wild-type myotubes, a
result of Ca2⫹ induced gene expression (14) and exhibit
increased NO production. Reduction of iNOS levels by
siRNA treatment markedly reduces the levels of iNOS protein and NO production. As discussed above, iNOS has
been proposed as the source of NO that nitrosylates RyRs,
thereby increasing calcium leak at the triad (30). This proposal has been challenged by the finding that genetic ablation of the iNOS gene fails to improve the histopathology or
contractile function of mdx muscle (245). In addition, the
level of nNOS in the cytosol of BMD muscle correlates with
RyR1 hypernitrosylation and disease severity (144). Finally, association of nNOS with RyR1 in skeletal muscle
has been observed (397, 397, 519). The relative roles of
nNOS and iNOS in promoting Ca2⫹ leak from RyR1
clearly require further study.
B. Caveolae and Caveolin-3 as Signaling
Platforms in Muscular Dystrophy
Caveolae are 60- to 80-nm vesicular structures of the
plasma membrane, broadly defined by the presence of the
structural protein caveolin, and their unique lipid composition, rich in cholesterol and sphingolipids. A number of
excellent reviews have described the numerous structural,
signaling, and trafficking functions of caveolae and the
caveolin proteins in many cell types, under a wide variety of
cellular conditions (78, 335). The current review will instead focus on the role of caveolae and caveolin-3 in skeletal
muscle and, in particular, recent evidence that caveolae are
mechano-signaling platforms for Ca2⫹, ROS, and NO production that contribute to the pathophysiology of dystrophin-deficient muscles.
There are three caveolin isoforms: 1, 2, and 3, which are
differentially expressed in a wide range of cell types. Caveolin-3 (Cav-3) is the muscle-specific isoform, expressed in
skeletal, smooth, and cardiac muscle. In addition to caveolin, recent studies have shown that another protein family,
the cavins, is required for correct formation and stabilization of caveolae (335). The long-standing view was that
caveolae are omega/flask-shaped membrane vesicles; how-
ever, recent evidence has shown that they can also be cupshaped, depending on the method in which the cells are
fixed (406). Caveolae formation in muscle requires the oligomerization of caveolin, as highlighted by the lack of caveolae in muscle of Cav-3 KO mice (78). Interestingly, it has
been estimated that between 100 and 200 caveolin proteins
can oligomerize to form a single caveola. This provides a
remarkable repertoire of possible proteins, including ion
channels, kinases, and enzymes, which can associate within
a single caveola to orchestrate signaling cascades in response to conditions such as muscle contraction, stretch,
and neurotransmitter or hormonal stimulation. The scaffolding domain of Cav-1 and Cav-3, a 20-amino acid residue region, is thought to bind and sequester many signaling
proteins within caveolae and to maintain them in an inactive conformation until activated by various stimuli. Of
particular relevance for DMD, many proteins involved in
the pathophysiological production of Ca2⫹, ROS, and NO
in dystrophic muscle have been shown to localize in caveolae, and some can also directly bind to caveolin. These
include TRPC1 and 4, NADPH oxidase 2 (NOX2), nNOS,
src kinase, and rac1. Thus there is good evidence that caveolae act as a signaling platform for Ca2⫹, ROS, and NO
pathways and therefore may regulate these deleterious
pathways in dystrophic muscle.
1. Caveolin-3 expression and localization in muscular
dystrophy
During skeletal muscle development, Cav-3 is expressed
during the process of myoblast fusion to form myotubes.
The loss of Cav-3 interferes with normal myogenesis, leading to abnormal growth of myotubes and abnormal t-tubular structural development (136). In mature muscle, Cav-3
is localized along the sarcolemma but is also highly expressed in t-tubules (310, 370), with “hot spots” of expression at the necks of the t-tubules (310). Cav-3 has a key role
in the formation of the t-tubular system, and certain Cav-3
mutations cause improper formation of the t-tubules leading to abnormal conduction of action potentials within
muscle fibers. In humans this condition is referred to as
rippling muscle disease (78).
While Cav-3 is not considered an integral component of the
dystrophin complex (87), it does co-immunoprecipitate
with members of the DGC (420), and caveolae are in close
proximity to dystrophin and ␤-dystroglycan in rat ventricular muscle (102).
Regulation of Cav-3 expression and/or localization is associated with many forms of muscular dystrophy. Mutations
in Cav-3 result in absence or significant reduction in sarcolemmal Cav-3 expression and cause four different types
of muscle disease: limb girdle muscular dystrophy 1C, rippling muscle disease, distal myopathy, and hyperCKemia
(for review, see Ref. 140). In contrast, mdx and DMD muscle have significantly increased Cav-3 expression levels
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compared with normal muscle (145, 380, 468). In addition,
muscle freeze-fracture studies of the sarcolemma have demonstrated increased numbers of caveolae in DMD, which
are smaller in size and abnormally distributed (42). Interestingly, in mice that transgenically overexpress Cav-3 in
skeletal muscle, a “Duchenne-like” dystrophy develops,
with many features similar to mdx mice including raised
CK, muscle necrosis, and central nuclei (137). A key finding
of this study was that Cav-3 transgenic mice had no dystrophin expression and very low levels of ␤-dystroglycan on
the sarcolemma, essentially the same as mdx mice. This
result can be explained by the finding that Cav-3 binds to
the WW domain of ␤-dystroglycan and therefore effectively
competes with dystrophin for this interaction (425). Thus
high expression of Cav-3 competitively inhibits dystrophin
from binding to ␤-dystroglycan and essentially creates a
dystrophin-deficient muscle phenotype. The binding of dystrophin and Cav-3 to ␤-dystroglycan is also regulated by
phosphorylation of dystroglycan. As discussed above,
phosphorylation of a tyrosine residue (Y890) within the
WW domain of ␤-dystroglycan, by src kinase, prevents dystrophin and utrophin binding to ␤-dystroglycan but allows
Cav-3 binding to the same site (197). Furthermore, ␤-dystroglycan that is phosphorylated by src is internalized from
the sarcolemma to an internal membrane vesicular structure and coimmunoprecipitates with src at this location
(423). Recently, it was shown that crossing mdx mice with
a ␤-dystroglycan knock-in mouse, with a phenylalanine residue substituting the tyrosine residue (Y890F), led to reduced muscle damage and provided some protection
against eccentric contraction-induced force loss (290).
Therefore, increased src expression and activity in dystrophic muscle (145) is likely to play an important role in
linking Cav-3, and its associated signaling proteins, with
the DPC, through ␤-dystroglycan. This might be a point of
consideration for DMD gene therapy and exon skipping
approaches (see sect. VIA), since the high expression of
Cav-3 in dystrophic muscles might restrict the amount of
dystrophin that can bind ␤-dystroglycan and therefore limit
the effectiveness of these therapies.
2. Caveolin-3 and TRPC channels
TRPC1, a putative MSC responsible for the increased
stretch- and/or store-operated Ca2⫹ influx in mdx muscle,
requires Cav-3 for sarcolemmal localization and stretchinduced activity in C2 myoblasts (145). In the same paper,
it was shown that src, also a Cav-3 binding protein, triggers
Ca2⫹ influx through TRPC1, in response to H2O2 exposure
(145). While the precise mechanism by which src activates
TRPC1 channel activity is unclear, there is evidence that all
TRPC channel members, including TRPC1, contain a src
homology domain and are able to bind src (211), suggesting
that src-mediated tyrosine phosphorylation might regulate
the activity of TRPC channels. TRPC1 has also been shown
to form heterodimers with TRPC4 in dystrophic muscle,
and the activity of these SAC/SOCs is reduced by binding to
276
␣-syntrophin (394). These findings suggest another possible
mechanosignaling link between Cav-3 and the dystrophin
complex, via TRPC1/4 and syntrophin. In addition to interactions between TRPC1 and Cav-3, the lipid composition
of caveolae, in particular cholesterol, can also influence
MSC activity. A recent study showed that MSC activity,
measured by patch clamping, was greatly enhanced in WT
myotubes following knockdown of Cav-3 or depletion of
cholesterol from caveolae by methyl-␤-cyclodextrin (190).
This result is interesting, since the membrane cholesterol
content of myotubes derived from DMD muscle is 35%
lower than nondystrophic control cells (237). Further studies will be required to determine if this membrane cholesterol reduction impacts the activation of MSC in dystrophic
muscles, particularly during periods of mechanical stretch.
3. Caveolae and Nox2
In some nonmuscle cells, the ROS-producing enzyme NADPH oxidase (Nox2) and its subunits (described in more
detail in sect. IVD) have been found to assemble and localize in lipid rafts and caveolae, which can serve as lipid
environments for activation or inhibition of Nox2 (165,
480, 521). In skeletal muscle, Nox2 is located along the
sarcolemma (507) and in the t-tubules (178), both of which
are enriched with Cav-3. Immunostaining of isolated mouse
fibers indicates colocalization between Nox2 and Cav-3
(396) and between the Nox2 subunit p22phox and Cav-3
(507). Furthermore, the Nox-2 activator protein rac1 has
been shown to localize in caveolae and bind Cav-3 in cardiac muscle (210), thereby providing another link between
Nox2 and caveolae. Thus there is some data suggesting that
Nox2 and its subunits localize in caveolae in muscle, although the mechanisms regulating Nox2 activation by
caveolae and Cav-3 during muscle stretch remain to be
studied.
Src kinase is another Cav-3 binding protein that is involved
in activating Nox2. In other cells, Src regulates the activation of Nox2, via two main mechanisms. First, in smooth
muscle it phosphorylates the Nox2 organizer subunit
p47phox, stimulating its translocation from the cytosol to
the membrane complex (462). Second, it can activate rac1
by regulating the guanylyl exchange factors Vav2 and
Tiam1 (415). As discussed in section IVD7, Src phosphorylation of p47phox also occurs in mdx muscle and is an
important step in Nox2 activation (324). Given that Src is
activated by ROS and can stimulate TRPC1 channel opening, we postulate that this kinase might provide an important link between Nox2-mediated ROS and subsequent
TRPC activity and Ca2⫹ influx.
As discussed in section IVD6, in order for stretch to activate
Nox2 in mdx muscles, an intact microtubule network is
required (213). Microtubules can associate with caveolae in
cardiac muscle cells and regulate the expression of Cav-3
(173). Furthermore, several studies have shown that micro-
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tubules are required for the trafficking of caveolae from the
Golgi to the plasma membrane (173, 305, 508). Given the
abnormal distribution of caveolae on the sarcolemma of
dystrophic muscles, it is tempting to speculate that the irregular microtubule lattice structure at the membrane in
mdx muscle (345, 361) might contribute to this disorganized spatial arrangement of caveolae. Microtubules can
also bind src and rac1 (33, 313), suggesting that muscle
stretch might lead to activation and translocation of these
proteins to stimulate Nox2 activity. In support of this idea,
it has recently been shown that src can be very rapidly
activated by an integrin-mediated mechanotransduction
pathway (⬍300 ms) and travel large distances (15– 60 ␮m)
along microtubules following activation (313).
4. Cav-3 and nNOS
As discussed previously, nNOS binds to ␣-syntrophin,
which localizes it to the dystrophin complex at the sarcolemma. It has also been shown that nNOS can bind to the
Cav-3 scaffolding domain in vitro, which inhibits its activity (475). However, while these two proteins can bind together, it is unclear whether this interaction is very prevalent in dystrophic muscle. For example, the loss of dystrophin or ␣-syntrophin causes mislocalization of nNOS from
the sarcolemma to a cytosolic location. This occurs despite
increased sarcolemmal expression of Cav-3 expression in
the case of mdx or DMD muscles. Therefore, while Cav-3
might regulate the activity of nNOS, it alone is not sufficient
to target nNOS to the sarcolemma. One possibility is that
nNOS can simultaneously bind ␣-syntrophin and Cav-3,
through the nNOS ␤-finger domain binding ␣-syntrophin
(180) and part of the nNOS catalytic domain binding Cav-3
(404). In this way, nNOS activity could potentially be regulated by the interaction between the dystrophin, via ␣-syntrophin, and Cav-3 mechanosignaling pathways. Another
potential reason why nNOS binds to Cav-3 is to regulate
the activity of other proteins within the caveolae by NOmediated S-nitrosylation of cysteine groups. Of particular
relevance to dystrophy, it was recently shown that Nox2
activity is inhibited by S-nitrosylation of a cysteine residue
(Cys 89), which is evolutionarily conserved in plants and
animals (527). Therefore, loss of nNOS from the sarcolemma might enable Nox2 activation to be enhanced in
dystrophic muscle due to the absence of this NO-mediated
inhibitory mechanism.
C. Mechanosensitive Channels, StoreOperated Channels, SERCA, and Ca2ⴙ
It has long been appreciated that total muscle calcium was
greatly increased in damaged DMD fibers (39), but there
has been very variable literature on whether myoplasmic
free [Ca2⫹] ([Ca2⫹]i) is elevated in mdx fibers prior to obvious damage (for review, see Ref. 147). Many studies have
found increases in resting [Ca2⫹]i in mdx compared with
wild-type muscle (189, 465, 466, 523), but others have
failed to detect any such difference (134, 174). Gillis (147)
pointed out that collagenase-treated fibers generally did not
show an increase in [Ca2⫹]i, whereas manually dissected
fibers did. This is consistent with the concept that mechanical stresses involved in dissection could trigger channel
activity leading to the elevated [Ca2⫹]i. Given that [Ca2⫹]i
can be elevated in mdx fibers, how does this arise? In this
section, we review the substantial evidence that activation
of Ca2⫹-permeable channels contribute to this rise in
[Ca2⫹]i. An alternative view, that mechanically induced defects in the membrane (micro-tears) cause increased
[Ca2⫹]i, has been considered in section IIIC. We also briefly
review the role of the SR Ca2⫹ pump (SERCA) in modulating Ca2⫹ and consider the pathways by which elevated
[Ca2⫹]i contribute to the pathology of DMD.
1. Mechanosensitive channels
Mechanosensitive channels (MSC) were first described by
Guharay and Sachs (159) in chick embryonic muscle. They
patch-clamped channels on the surface membrane and identified a class of channels whose activity increased when
negative pressure was applied to the interior of the patch
electrode. The application of negative pressure to the pipette sucks membrane into the patch electrode and is assumed to stretch channels therein (441). These MSC were
permeable to a range of cations and thus allow Na⫹ and
Ca2⫹ entry when activated. MSC were shown to be more
prevalent and active in mdx fibers (127, 128) and in DMD
myotubes (473). Yeung et al. (523) showed that a series of
eccentric contractions caused a large rise in [Ca2⫹]i in mdx
fibers that lasted for at least an hour. This rise in [Ca2⫹]i
could be prevented by the known blockers of MSC (Gd3⫹,
streptomycin, and GsMTx-4) and is therefore likely to be
caused by activation of MSC. Mdx mice that were exercised
on a treadmill with downhill running, which provokes severe muscle damage, had reduced force loss and membrane
permeability when treated before the exercise with streptomycin. This result suggests that elevated [Ca2⫹]i associated
with activation of MSC has a role in the muscle damage of
dystrophinopathies.
Unfortunately, the identity of the gene that encodes MSC is
still uncertain. Maroto et al. (263) originally suggested that
TRPC1 encoded the muscle MSC. This was based on 1)
purification of the 80-kDa protein, the size predicted; 2)
demonstration that an antibody to TRPC1 bound the protein; 3) expression of mammalian TRPC1 in frog oocytes
and mammalian COS cells produced a 10-fold increase in
MSC activity; and 4) that siRNA to TRPC1 reduced MSC
expression in oocytes. Later, the same group partially retracted this conclusion on the basis that TRPC1 expression
in COS cells no longer reliably increased MSC activity and
the expressed protein did not traffic to the surface membrane (150). Further doubt about the role of TRPC1 in
MSC has been raised by studies of muscles from a TRPC1
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ALLEN ET AL.
KO mouse, which still contained MSC activated by negative
pressure in the patch pipette (529). Instead, a smaller conductance and pressure-insensitive channel was absent in the
TRPC1 KO muscle. However, a study of cytoskeletal damage induced by eccentric contractions in WT muscle found
that the damage was reduced in TRPC1 KO mice, suggesting that TRPC1 has some role in Ca2⫹ entry associated with
damage (534).
Other candidates for the MSC include TRPC6, which appears to be responsible for the shear strain-activated current
in ventricular myocytes (109), and TRPV2, which encodes
stretch-sensitive channels in vascular smooth muscle (307)
and appears to be the source of Ca2⫹ entry in the ␦-sarcoglycan deficient mouse model of muscular dystrophy
(198).
Given the possibility that TRPC1 has some role in MSC and
in store-operated channels (see next section), several studies
have investigated the binding partners of TRPC1. Gervasio
et al. (145) showed that TRPC1 and Cav-3 coimmunoprecipitated and appeared to be close binding partners, based
on FRET between coexpressed TRPC1-CFP and Cav-3YFP. TRPC1, Cav-3, and src kinase are all upregulated in
mdx mice and may form a complex which allows pathological, unregulated Ca2⫹ entry. Homer-1, a scaffolding protein, has been shown to bind to TRPC1 (526), and Stiber et
al. (436) showed that a Homer 1 KO had abnormal Ca2⫹
influx and developed a skeletal myopathy. The Ca2⫹ influx
and myopathy was ameliorated by gene silencing of
TRPC1. TRPC1 also binds to ␣-syntrophin, and Vandebrouck et al. (472) showed that dystrophin, ␣-syntrophin,
and TRPC1 form a macromolecular complex based at the
costamere. In mdx muscle, the loss of expression of dystrophin and ␣-syntrophin is associated with abnormal Ca2⫹
influx that can be restored by expression of a mini-dystrophin and ␣-syntrophin. Subsequently, this group showed
that silencing of ␣-syntrophin in normal muscle was sufficient to cause enhanced Ca2⫹ entry. The above studies all
suggest that a macromolecular complex including dystrophin, ␣-syntrophin, Cav-3, TRPC1, and src-kinase is located at the costamere and allows regulated Ca2⫹ entry. In
DMD, dystrophin is absent and ␣-syntrophin is severely
downregulated, but the other members of this complex are
upregulated and allow unregulated Ca2⫹ entry (for review,
see Ref. 393).
In theory, MSC can be activated by a variety of pathways
(74; for review, see Ref. 224): 1) intrinsic bilayer forces
acting upon the channel within the membrane and modulated by curvature of the membrane or insertion of lipids
into the bilayer; 2) tethered proteins connecting the channel
to the cytoskeleton (the cellular cytoskeletal is ubiquitous
and connects to many proteins in the membrane); and 3)
indirect activation by cell signaling pathways. Although
MSC were discovered by their pressure sensitivity, this is
278
not necessarily the mechanism by which they are activated
in vivo. Pathways that cause posttranslational modification
of a channel may change the mechanism of activation. Alternatively, expression of proteins involved in the macromolecular complex that regulates the channel may change.
The activation of MSC in mdx mice after eccentric contractions appears to be an example of indirect activation. In
patch-clamp experiments, the muscle MSC is turned on and
off within 100 ms of the instigating pressure change (163,
523). In contrast, Yeung et al. (523) could detect no change
in Ca2⫹ during the period when the muscle was stretched.
However, they observed a persistent (30 min) increase in
myoplasmic Ca2⫹ following eccentric contractions, even
though the muscle was no longer being stretched during this
period. Thus activation of the MSC appears after a substantial delay, suggesting that activation is indirect. A considerable body of evidence points to the involvement of ROS,
produced by NADPH oxidase, which subsequently activate
MSC. This will be discussed in section VD6.
The hypothesis that the activation of MSC in dystrophic
muscles has a central role in dystrophic pathology has been
questioned based on studies in which increased utrophin
was expressed at various ages (429). When utrophin expression was increased in utero or at birth, both MSC function
and dystrophic pathology were corrected. However, when
utrophin expression was increased at 30 days, MSC function was corrected but the pathology was not. An alternative interpretation of these findings is that the pathology
caused by elevated intracellular calcium eventually becomes
self-sustaining.
2. Store-operated Ca2⫹ channels
Extracellular Ca2⫹ entry into cells following store depletion
was first described in nonexcitable cells (365) and is now
recognized to occur in skeletal muscle (228, 235). The functional role of these channels is not entirely clear. They appear to have roles in enhancing Ca2⫹ influx during repeated
stimulation leading to fatigue, when stores become depleted, and in regulating myotube development and gene
expression (236; for recent review, see Ref. 435). Several
studies have established that store-operated Ca2⫹ entry
(SOCE) is more active in mdx muscle and may well contribute to the increased [Ca2⫹]i (104, 111, 474).
There has been a prolonged debate about the identity of
genes that encode SOCE. TRP channels have long been
candidates for SOCE, and Vandebrouck et al. (474) showed
that antisense downregulation of TRPC1 and 4 reduced the
SOCE in mdx fibers. In a different approach, Millay et al.
(289) overexpressed TRPC3 in mouse skeletal muscle and
found an approximately sixfold increase in SOCE and the
induction of a dystrophic-like myopathy. Thus these studies
are consistent with the idea that TRPC channels contribute
to SOCE.
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However, since the discovery that Orai1 constitutes the
surface membrane channel in lymphocytes (121), studies in
muscle have implicated Orai1 as the membrane channel of
SOCE. Lyfenko and Dirksen (254) showed that in normal
muscle, siRNA knockdown of STIM1, the putative Ca2⫹
sensor in the SR, or replacement of Orai1 by an impermeant
version both eliminated SOCE. These findings led to a
model of SOCE in which STIM1, situated in the junctional
SR membrane, interacts with Orai1, located in the junctional t-tubular membrane. The implication of this result is
that, if a component of SOCE depends on a TRPC channel,
it also requires Orai1. In the mdx mouse, both STIM1 and
Orai1 are upregulated, which may explain the increased
SOCE in mdx muscle (111). Furthermore, siRNA knockdown of Orai1 can return SOCE in the mdx muscle to
wild-type levels (535).
While the demonstration of increased SOCE in mdx is of
interest, it does not necessarily provide an explanation
for the increased [Ca2⫹]i. SOCE is activated only when
Ca2⫹ within the SR falls below the level required to activate STIM1. Current estimates are that SOCE is first
activated when SR Ca2⫹ falls below 225 ␮M and is maximally activated below 100 ␮M (236). Whether these
reductions ever occur under physiological conditions is
debatable. However, in mdx muscle, these activation levels are higher (111), so a smaller degree of depletion
would activate a greater fraction of the available SOC
channels. The magnitude of the SOCE would presumably
also be increased by the greater number of STIM1 and
Orai1 proteins (111). Several studies suggest that Ca2⫹
leak from the SR is greater in mdx muscle (30, 386), and
the combination of reduced SR Ca2⫹ content and raised
activation threshold for SR Ca2⫹ could provide a stimulus for SOCE. Another class of possibilities is that some
other factor in the myoplasm activates the SOCE process
without changing the threshold for SR Ca2⫹. One possibility is that the products of Ca2⫹-independent PLA2,
such as lysophosphatidylcholine, can activate SOCE
(41). A further possibility follows from the observation
in cell lines that H2O2 can stimulate SOCE through activating inositol trisphosphate (IP3) receptors in the SR and
causing SR Ca2⫹ depletion (158). Given that ROS production is increased in muscular dystrophies (see sect.
VD), this could provide another pathway for SOCE activation.
3. SR Ca2⫹ pump
The primary function of the SR Ca2⫹ pump (SERCA1) is
to return Ca2⫹ from the myoplasm to the SR and thus
cause relaxation. Whether its function is affected in mdx
muscle is controversial with increased pump rate (386),
unchanged pump rate (358, 448), and decreased pump
rate (99, 143, 207) all being reported. Despite these diverse findings, overexpression of SERCA in both the mdx
mouse and the ␦-sarcoglycan-null mouse produced a ro-
bust improvement of the dystrophic phenotype with substantial reductions in serum creatine kinase, central nuclei, fibrosis, and improvements in the histological appearance of muscle and an increase in running time (149).
SERCA overexpression also caused a concurrent reduction in resting [Ca2⫹]i, accelerated decay of the Ca2⫹
transient, and rescued the dystrophic phenotype caused
by TRPC3 overexpression. Goonasekera et al. (149) proposed that accelerated SR uptake reduces resting [Ca2⫹]i
and mitochondrial Ca2⫹ and that, as a consequence, calpain activation was reduced. These experiments support
the concept that elevated [Ca2⫹]i is the “final common
pathway” in several dystrophic diseases and that increased SERCA activity can correct this. While this is an
intuitively attractive concept, there is a theoretical difficulty that changes in internal buffering of [Ca2⫹]i within
a cell, such as provided by the SR, should have no longterm effect on the resting [Ca2⫹]i which is determined by
the balance of Ca2⫹ influx and efflux at the surface membrane (384). To paraphrase the situation, if it is true, as
observed experimentally, that enhanced SR leak is associated with increased myoplasmic [Ca2⫹]i, then there
must be a concomitant rise in surface membrane Ca2⫹
influx or a decrease in surface membrane Ca2⫹ efflux.
The presence of SOCE might conceivably represent this
link, but it seems hard to believe that a small SR Ca2⫹
leak could deplete the SR to the point at which SOCE is
activated. Goonasekera et al. (149) report that SR Ca2⫹,
judged by 4-CMC Ca2⫹ efflux, was unaffected by SERCA
overexpression in the disease model. For a recent review
of the role of Ca2⫹ handling in DMD, see Reference 471.
An interesting study by Gehrig et al. (143) provides further
support for the benefits of improving SERCA performance
in mdx muscle and in the more severe dystrophic model in
which both dystrophin and utrophin are not expressed (dystrophin/utrophin DKO). The study focused on Hsp72, a
heat shock protein that acts as a molecular chaperone and
can prevent the degradation of proteins, including SERCA,
under conditions of cellular stress. Gehrig et al. (143)
showed that in mdx muscles, SERCA activity was reduced
and that this could be improved by overexpression of
Hsp72 in the muscles. Simultaneously, a large range of indicators of muscle pathology were also improved. Of
course, Hsp72 might be protecting other proteins, but the
value of improving SERCA function has been independently demonstrated by Goonasekera et al. (149). An important aspect of this study (143) is that a pharmacological
agent, BGP-15, causes increased expression of Hsp-72, and
this agent was shown to produce more modest improvements in the dystrophic phenotype. Both Hsp-72 overexpression and BGP-15 treatment were also effective in the
mdx/utrophin dko mouse model, which has a much more
severe phenotype and arguably is a better model for establishing the efficacy of treatment for DMD. For further dis-
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cussion of the role of molecular chaperones in DMD, see
Reference 51.
The SR, in addition to Ca2⫹ release and reuptake, has a role
in protein folding. In a range of conditions, collectively
known as SR stress, Ca2⫹ homeostasis and protein folding
are abnormal and cause an unfolded protein response
which can trigger apoptosis. There is some evidence that SR
stress occurs in DMD (164, 302) and contributes to the
phenotype.
4. Calpains
Calpains are Ca2⫹-activated proteases of which three
forms, ␮-calpain, m-calpain, and calpain-3, are expressed
in muscle (for comprehensive review, see Ref. 148). Given
that resting [Ca2⫹]i can be elevated in mdx muscle, it might
be expected that calpain activity would increase and make a
contribution to the pathology. Turner et al. (465) first
showed that resting [Ca2⫹]i was increased in mdx compared with wild-type muscle fibers and that this was associated with an increased rate of protein degradation. In
subsequent studies, this group established that the elevated
[Ca2⫹]i arose through an unidentified leak channel and
could be blocked by a calpain inhibitor (11). Many groups
have subsequently shown that calpain activity is higher in
mdx muscles (for example, Ref. 427). Moreover, overexpression of the endogenous calpain inhibitor calpastatin
could at least partly ameliorate some of the features of the
dystrophic pathology (426).
Recent studies have given more detailed insights into the
extent and pathways of calpain activation. Gailly et al.
(135) used a membrane-permeant fluorescent calpain substrate and could modulate calpain activity over a 30-fold
range by lowering or raising [Ca2⫹]i. Fast transient changes
of [Ca2⫹]i, such as caused by normal tetani, did not increase
activity, whereas slow increases in [Ca2⫹]i by depleting the
SR or by activating store-operated Ca2⫹ entry produced
moderate (50 or 100%) increases in calpain activity. Hyposmotic solutions, which cause swelling and should activate mechanosensitive channels, also produced increased
calpain activity, which was blocked by GsMTx-4, a specific
inhibitor of stretch-activated channels. The collagenase-isolated fibers used in the present studies did not show an
elevated [Ca2⫹]i in mdx versus WT; nevertheless, they had a
50% higher calpain activity. Gailly et al. (135) showed that
⬃10% of the ␮-calpain was present in the autolysed form
(78 vs. 80 kDa for the un-autolysed form). Autolysis is
required for activation, and the autolysed form is more
sensitive to [Ca2⫹]i and is expected to provide the calpain
activity measured in their experiments.
Recent experiments on skinned fibers with strongly buffered [Ca2⫹]i provide further insights into the distribution of
calpains and the [Ca2⫹]i required to activate them. Murphy
et al. (311) showed that ␮-calpain is mostly inactive at rest
280
and requires a concentration of ⬎2 ␮M for ⬎2 min to
produce significant autolysis and activity. However, the autolysed ␮-calpain can have its activity maintained by much
lower levels of [Ca2⫹]i. Thus the elevated Ca2⫹ levels noted
by Gailly et al. (135) (⬃300 nM for 10 min) would appear
insufficient to cause the autolysis of ␮-calpain but might
allow the continuing activity of preautolysed calpain. However, the appearance of calpain activity at rest in mdx fibers
with normal resting [Ca2⫹]i is not explicable on this basis
and requires some additional explanation. These could include elevated [Ca2⫹]i near the channels in the membrane or
in vivo situations that increase the sensitivity of calpains to
calcium, such as phospholipids or phosphorylation (for review, see Ref. 148).
While there is good agreement that calpains, and probably
␮-calpain, are activated in mdx and DMD muscle, the targets and possible therapeutic benefits of inhibiting calpain
are less clear. Goll et al. (148) list many proteins that are
targets for calpain, but this list has to be modified by the
knowledge that most calpain-3 is tightly bound to titin or
the triad region (309), whereas ␮-calpain, while freely diffusible at rest, rapidly binds when activated, much of it to
the surface membrane (311). One of the most likely targets
is the reduction in Ca2⫹ release from the SR, which is often
a feature of muscles subject to prolonged elevations in
[Ca2⫹]i (13). This is particularly clear in skinned fibers
where Ca2⫹ release from the SR can be shown to decline in
a Ca2⫹- and time-dependent fashion after elevation of
[Ca2⫹]i (476). It has recently been shown that junctophilin,
a structural protein that maintains the correct separation
between the t-tubules and SR, is degraded after eccentric
contractions (82). Murphy et al. (308) subsequently
showed that junctophilin is an activated calpain target, and
its proteolysis is associated with a decline in SR Ca2⫹ release. Thus this Ca2⫹-calpain activated process may explain
an important element of the weakness in DMD and mdx
muscle.
While the study of Spencer and Mellgren (426) established
that elevated calpastatin could reduce the histological features of dystrophy and the number of regenerating fibers in
mdx muscle, it required an overexpression of calpastatin of
more than 300-fold to achieve this outcome. Subsequent
studies with pharmacological inhibitors of calpains have
given quite variable results. Badalamente and Stracher (21)
used intramuscular injections of the membrane-permeant
tripeptide calpain inhibitor leupeptin and showed that calpain activity was reduced while histological signs of dystrophy (cell size, central nuclei, and fibrosis) were all improved.
However, several other studies of leupeptin-based agents
found minimal benefit either in the canine model of DMD
(72) or in mdx mice after either 1 or 6 mo treatment, testing
a range of functional, histological, and biochemical markers (410).
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
Thus current evidence suggests that calpain inhibition is of
limited value as a treatment option in DMD. This view is
reinforced by the fact that mutations in calpain-3, which
lead to lack of proteolytic activity, are the cause of limb
girdle muscular dystrophy 2A (382, 422). Thus it seems that
the main physiological role of calpain-3 is sarcomeric remodeling (221).
D. ROS and NADPH Oxidase
1. Physiological and deleterious effects of ROS in
skeletal muscle
In a biological system, ROS are oxygen-containing molecules that can chemically react with lipids, DNA, and
proteins to modulate their structure and function. At low
to moderate concentrations, ROS play an important role
in many diverse cell signaling pathways but at high levels
can cause reversible or irreversible damage. The cellular
effects of ROS are complex and depend on a number of
factors including the expression, localization, and regulation of the ROS source; the type of ROS produced and
its reactivity; and the effectiveness of the antioxidant
defense system.
Oxidative stress is a general term that characterizes pathological conditions in which cellular damage is induced
by ROS. It is manifest by a complex series of pathways
that cause dysregulation of the normal redox state in cells
and tissues. Oxidative stress is commonly defined as an
elevated ROS concentration due to increased ROS production and/or a reduced capacity for antioxidant pathways to scavenge excessive ROS; however, it has been
more recently defined as a disruption of redox signaling
and control (206). Oxidative stress can be acute or
chronic and is associated with a wide range of common
human diseases, including cardiovascular disease, diabetes, neurological disorders, and cancer (470). In skeletal
muscle, oxidative stress is produced under pathophysiological conditions such as hypoxia, metabolic disorders,
aging, and various myopathies. Oxidative stress can result in damage to muscle leading to impairment of muscle
function, reduced regenerative capacity, atrophy, and ultimately muscle weakness. However, in recent years, this
view has been modified to incorporate many important
physiological functions of ROS in muscle, including muscle regeneration, mitochondrial function, metabolism,
and muscle hypertrophy (27, 199). In addition, muscle
function can be positively regulated by low levels of ROS
during exercise, although at higher levels, ROS can impair force production and contribute to muscle fatigue
(199, 378). Therefore, it is important to take into consideration the beneficial functions of ROS under normal
physiological conditions when studying the deleterious
effects of ROS in muscle undergoing oxidative stress.
2. Antioxidant clinical trials in DMD
Three decades ago, oxidative stress was hypothesized to
contribute to muscle damage in DMD (for review, see Ref.
373). Initially, this hypothesis was based on similarities in
muscle pathology of DMD and vitamin E-deficient individuals and animals (398, 459). Vitamin E is an abundant and
important lipid-soluble antioxidant, and its deficiency in
humans produces muscle myopathy with preferential damage to type IIB muscle fibers (459), as is the case in DMD
(494). These findings prompted a series of clinical trials in
DMD boys using antioxidants such as superoxide dismutase, vitamin E, and selenium (20, 138, 434). At the time,
these trials were considered to be unsuccessful. In one trial,
a superoxide dismutase mimetic was administered to DMD
boys over an 18-mo period and changes in muscle strength
were used as the main outcome measure. While muscle
force improvement was not statistically significant overall,
a wide range of muscles did show a positive improvement
trend. This raises some important questions for consideration. First, was the dosing regimen appropriate? A single
dose was used and apparently not optimized before the
study was designed. Second, superoxide dismutase mimetics act extracellularly, and therefore will not effectively target ROS produced intracellularly. Third, even if most of the
ROS is produced extracellularly, for example, by NADPH
oxidase (see below), the SOD mimetic will catalyze the reaction of superoxide to H2O2. This would favor the production of H2O2 instead of other superoxide-derived ROS
or RNS. While this may provide some benefits, by reducing
the levels of damaging radicals such as peroxinitrite or superoxide itself, it would instead favor the accumulation of
the H2O2, a relatively stable and membrane permeable
ROS, which can modify a wide range of intracellular and
extracellular molecules. For example, mdx myotubes are
much more susceptible than WT myotubes to cell death by
H2O2 (374), and this oxidant also directly increases Ca2⫹
influx in mdx fibers through SACs (145). In other words,
converting one ROS (superoxide) to another (H2O2) may
not be an effective antioxidant strategy. In further support
of this idea, a recent study in mdx mice showed that overexpression of catalase, which decomposes H2O2, improved
muscle function (411). Unfortunately, the lack of observable improvement in these original antioxidant trials has
thus far diminished subsequent interest in ROS scavengers
as a treatment for DMD. However, with renewed interest in
the role of ROS in the pathogenesis of dystrophy from studies in the mdx mouse and DMD (see below), hopefully this
will pave the way for established or novel ROS scavengers
to be tested in DMD patients.
3. Evidence of ROS-induced damage in dystrophic
muscle
Despite the unsuccessful clinical trials in DMD, numerous
studies in the intervening years have provided compelling
evidence that ROS contribute to the pathophysiology of
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muscular dystrophy. In DMD muscle biopsies, there is evidence of oxidative stress as measured by the twofold increased levels of protein carbonyl groups, a measure of
protein damage by ROS (171). Oxidative damage to lipids
has also been found in DMD muscle as determined by the
increased levels of isoprostanes, a measure of lipid peroxidation (155). Similar results have been shown in mdx mice,
with increased levels of muscle protein oxidation (170) and
lipid peroxidation (286) compared with WT mice. In recent
years, a plethora of different antioxidants have been administered to mdx mice, and many have been shown to significantly reduce muscle damage and improve muscle function, strongly supporting the role of oxidative stress in muscular dystrophy. These studies have been described in detail
in a number of recent review papers (214, 373, 506) and
therefore will not be further discussed here. Rather, the next
sections will focus on the susceptibility of mdx muscle to
ROS-induced damage and the sources of ROS in dystrophic
muscles.
4. Increased susceptibility of dystrophic muscle to
ROS
An important finding from Rando et al. (374) was that
cultured dystrophic muscles have an increased susceptibility to muscle damage and cell death from oxidative stress.
They showed that mdx myotubes underwent cell death with
much lower concentrations of ROS such as H2O2 and the
superoxide generating agent paraquat compared with WT
myotubes (374). On the other hand, the rate of cell death
was the same for mdx and WT myotubes after exposure to
several metabolic toxins, suggesting a selective vulnerability
to oxidative and not metabolic stress for mdx myotubes.
Interestingly, undifferentiated myoblasts from mdx and
WT muscles were equally susceptible to oxidative stress.
Since dystrophin is not found in myoblasts but is expressed
in WT myotubes, this finding indicated that the absence of
dystrophin in mdx myotubes was the primary cause of the
increased susceptibility to oxidative stress. This hypothesis
was subsequently tested in another study, in which mdx
myotubes expressing various truncated isoforms of dystrophin were exposed to oxidants (98). Here, it was shown
that myotubes expressing the Dp71 isoform were more susceptible to oxidative stress than those expressing a Becker
dystrophy isoform, which, in turn, were more susceptible
than myotubes expressing full-length dystrophin. These results suggested that the susceptibility to ROS-induced cell
death in myotubes positively correlates with the severity of
the dystrophy in transgenic mice expressing these various
dystrophin isoforms. This draws a direct link between dystrophin and the sensitivity to oxidative damage in skeletal
muscle. Interestingly, this group also showed that overexpression of nNOS in mdx myotubes did not affect the susceptibility to oxidative stress, suggesting an NO-independent mechanism for the enhanced sensitivity to ROS-induced cell death (538). However, whether or not nNOS
contributes to the sensitivity of ROS-induced oxidative
282
stress in mature mdx muscle fibers, particularly during muscle contraction, is not known.
The mechanisms underlying the increased susceptibility to
oxidative stress in dystrophic muscle have not been fully
elucidated. However, as discussed in other sections, there is
good evidence that ROS-induced Ca2⫹-influx through
SACs is one pathway that contributes to muscle damage in
mdx muscle and that mdx muscles are much more sensitive
than WT to activation of this pathway (145, 418, 504,
507). Recent evidence demonstrates that depletion of endogenous antioxidant defense systems, such as glutathione
(GSH), increases oxidative stress in DMD muscles (379).
Therefore, it is likely that both an increased susceptibility of
ROS-activated damage pathways, as well as reduction in
endogenous antioxidants, contribute to the greater susceptibility of dystrophic muscles to oxidative stress. Research
from our laboratory has shown that ROS enhance the influx
of Ca2⫹ following stretched contractions (145, 507). We
also found in resting, uncontracted fibers that H2O2 increased Ca2⫹ in mdx but not WT, again highlighting the
greater susceptibility of dystrophic muscle to ROS-induced
Ca2⫹ influx (145). Moreover, the increased [Ca2⫹]i in mdx
was reduced to baseline levels by addition of streptomycin,
suggesting that the exogenous H2O2 was somehow regulating the opening of MSC (145).
5. NOX2 is a major source of ROS in normal muscle
For a number of years, mitochondria were considered to be
a major source of ROS production in a many cells and
tissues, including skeletal muscle, produced as a byproduct
of oxidative metabolism. Early studies estimated that between 2 and 5% of the oxygen consumed by mitochondria
produced superoxide; however, subsequent results predict
much lower values of 0.1– 0.15% (166, 430). This evidence
suggests that other sources of ROS might play an important
role in muscle under both normal and pathophysiological
conditions. Indeed, recent evidence indicates that during
repetitive contractions of isolated mouse muscle fibers, mitochondrial ROS production is not increased despite an
almost 100% increase in cytosolic ROS levels (288). In
addition, this study provided evidence that NADPH oxidase (NOX) was a major source of ROS during contractile
activity.
NOX is a ROS-producing (superoxide or H2O2), multiprotein enzyme complex. It was first identified in inflammatory
cells, such as neutrophils and macrophages, and plays a key
role in the body’s host defense system by generating high,
most likely molar, concentrations of ROS which kill invading microbes. The importance of NOX to innate immunity
is highlighted by the inherited condition chronic granulatomous disease, where NOX mutations lead to reduction or
inhibition of NOX activity in inflammatory cells, which
compromises the host defense system. Paradoxically, increased NOX activity in other organs and tissues is associ-
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
ated with many common human diseases (234). Therefore,
a tight regulation of NOX expression and activity is essential to provide cells with both the beneficial ROS-mediated
signaling pathways and protection from the deleterious effects of excessive and chronic ROS production. There are
several isoforms of the catalytic subunit of NOX: NOX1,
NOX2 (also known as gp91phox), NOX3, NOX4, and
NOX5, which are differentially expressed, and regulated, in
a wide range of mammalian tissues and organs (for a comprehensive review, see Ref. 29).
Until recently, little was known about NOX expression and
activity in skeletal muscle. However, in the last few years
several studies have investigated the expression, localization, regulation, and function of NOX in skeletal muscle
under various physiological conditions. Skeletal muscle of
mice and humans expresses both RNA and protein for the
NOX2 and NOX4 isoforms, and their activity is important
for regulating myoblast proliferation (296). Both NOX2
and NOX4 constitutively bind to the p22phox subunit
within cell membranes. However, unlike NOX4, which is
an inducible isoform regulated by increased transcription
(412), NOX2 requires several regulatory proteins to become activated; in mouse skeletal muscle, these subunits are
p47phox, p67phox, and rac1 (201, 507). An additional subunit, p40phox, found in phagocytes, was not found in rat
skeletal muscles but was expressed in muscle of a cancer
cachexia mouse model (201). Therefore, this subunit may
be differentially expressed depending on the muscle type,
species, and biological stimulus. NOX2 and its associated
subunits are localized at the sarcolemma and t-tubules in
skeletal muscle fibers (178, 507). The t-tubule fraction of
NOX2 was found to play a role in regulating increased
Ca2⫹ release from RyR1 via S-glutathionylation in isolated
triads from mouse muscle (178). NOX4 is localized at the
SR in skeletal muscle and is regulated by the partial O2
pressure (PO2), with higher PO2 increasing its activity (443).
This study also showed that H2O2 produced by NOX4 can
regulate RyR1 activity and enhance muscle force production.
6. NOX2 is stretch-activated in dystrophic muscle
and causes muscle damage and weakness
As discussed earlier, stretched contractions of mdx fibers
increased ROS production which triggered Ca2⫹ entry
through SACs and reduced force production (145). Since
NOX is rapidly activated in smooth muscle during cyclic
stretch (70, 156), it seemed plausible that it might also be
stretch-activated in skeletal muscles. To test this hypothesis, mdx fibers were stretched after incubation with the
NOX inhibitor DPI. Blocking NOX activity significantly
reduced the [Ca2⫹]i and improved muscle force after
stretched contractions compared with control, untreated
mdx fibers (507). These data suggested that in mdx fibers,
stretch-induced activation of NOX2 increased ROS levels, which subsequently led to the opening of SACs and
Ca2⫹ influx. The same study also demonstrated increased
NOX2 activity in mdx muscle compared with WT and
two- to threefold higher expression of NOX2 and its
subunits, p67phox and rac1, in mdx muscle. Importantly,
these proteins were increased in young adult mdx mice
but also in muscles of prenecrotic, 19-day-old mdx mice
(507), suggesting that increased ROS production from
NOX2 might be a key source of the oxidative stress at
this time (98) and therefore a mediator of the subsequent
muscle fiber damage and necrosis. Similarly, other studies have also reported increased protein expression of
gp91phox, p67phox (213, 418), and rac1 (213) in adult
mdx muscle. In the first of these studies (418), isolated
mdx muscle fibers were subjected to hyposmotic stress,
and both intracellular and mitochondrial ROS and Ca2⫹
were measured with fluorescent indicators. Here, it was
shown that both basal ROS and stress-induced ROS levels were much higher in mdx than WT fibers and were
inhibited by blocking NOX with DPI. Furthermore, they
showed that stress-induced Ca2⫹ influx was blocked by
an antioxidant and DPI. Interestingly, it was also reported that basal mitochondrial ROS production was not
higher in mdx muscles; however, stress-induced Ca2⫹
influx led to increased mitochondrial Ca2⫹ content,
which then triggered increased mitochondrial ROS production. Together, these results suggest that during
stretch of dystrophic muscle, NOX2 rapidly produces
ROS, which triggers Ca2⫹ entry through SACs, leading to
increased mitochondrial Ca2⫹ and additional ROS production (FIGURE 5), causing muscle damage and weakness (FIGURE 6).
Recently, a new insight into the pathway by which stretch
increases NOX2 activity in mdx muscle was reported (213).
This study found that a dense network of microtubules
provided a mechanosensitive pathway for NOX2 activation in mdx muscle. The paper showed that the microtubule
lattice is denser and grossly disorganized in mdx muscle,
supporting findings of earlier studies in which dystrophin
was shown to directly bind microtubules (361) and microdystrophin expression in mdx muscle restored the normal
microtubule lattice structure (345). In the study of Khairallah et al. (213), muscle fibers were attached to micro-tweezers with an adhesive (MyoTak) and passively stretched by
10% of resting sarcomere length. Intracellular ROS and
Ca2⫹ were measured using standard fluorescent indicators.
The role of microtubules on NOX-induced ROS production in mdx fibers was determined by using microtubule
depolymerizing agents, which prevented, or a microtubule
polymerizing agent, which increased, stretch-induced ROS
production. These results were recapitulated by in vivo administration of these agents or a NOX2 inhibitor, which
subsequently reduced stretch-induced force loss in vitro.
The same effect was found after inhibiting the activity of
rac1, a key regulator of the NOX2 complex, which is increased in mdx muscle (507).
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ALLEN ET AL.
Interestingly, actin cytoskeleton depolymerization had no
effect on ROS production, suggesting that the actin cytoskeleton is not directly involved in the mechanosignaling
leading to NOX2 activation. These data are supported by a
study in which knockout of cytoskeletal ␥-actin, which is
substantially upregulated in mdx muscle, did not affect the
dystrophic phenotype or the susceptibility to stretch-induced damage compared with regular mdx mice (362). Another important result from the study of Khairallah et al.
(213) was that a NOX2 inhibitor, a microtubule depolymerizing agent, and the MSC blocker GsMTX-4 all prevented the influx of Ca2⫹, supporting the idea that mechanical stretch initiates a mechanosignaling pathway involving
microtubules that lead to activation of NOX2 and subsequent influx of Ca2⫹ through MSC (418, 507). Finally, it
was shown that several NOX subunits, microtubule proteins, and putative MSC candidates, TRPC1 and TRPV2,
were all increased by microarray RNA analysis (213), supporting the involvement of the microtubule-NOX-SAC network in DMD muscle (summarized in FIGURE 6).
7. Nox2 and impaired autophagy in dystrophic
muscle
Autophagy is an important cellular pathway in which damaged intracellular proteins, organelles, and protein aggregates are degraded and then recycled by the cell for energy
use. The autophagy process involves several, highly regulated steps, involving two main organelles, the autophagosome and the lysosome (for review, see Refs. 231, 294).
Impaired autophagy can lead to cell death, and there is
growing evidence that it is implicated in a number of human
diseases, including neuromuscular disorders (for review, see
Refs. 222, 294). In skeletal muscle, proper regulation of
autophagy is essential for normal cellular and physiological
function, with high autophagic activity contributing to
muscle atrophy and low activity causing muscle degeneration and weakness (91). Two key protein markers of impaired autophagic flux are protein sequestosome-1 (p62)
and microtubule-associated protein 1A/1B light chain 3
Ca2+
ROS
Cav-3
NOX2 p22
src
GTP
P
Mitochondria
P
rac1
src
P
GTP
src
p67 p47
P
P
p67
P
p47
rac1
SAC/SOC
Cav-3
T-tubule
Caveola
NOX2
P
src
P
src
p22
P
Microtubules
ROS
NO
iNOS
nNOS
CaM
Sarcoplasmic reticulum
Ca2+
RyR1
Ca2+
FIGURE 5. Mechanisms of stretch-induced reactive oxygen species (ROS) production and Ca2⫹ influx in
dystrophic muscle. This schematic highlights some of the key deleterious pathways that lead to impaired
muscle function, resulting from the loss of dystrophin. Particular focus is given to the pathways relating to ROS,
Ca2⫹ and NO. During eccentric contractions, NADPH oxidase 2 (NOX2) is stimulated by a pathway involving
microtubules and activation of rac1 and src kinase (src). This leads to phosphorylation of Nox subunits and
activation of Nox2. The ROS produced by Nox2 further activates src, which triggers the opening of stretchactivated or store-operated channels (SAC/SOC), and influx of Ca2⫹. Increased Ca2⫹ uptake by mitochondria
can also stimulate additional ROS production. Both ROS and NO, produced by mislocalized nNOS and/or iNOS,
increase the opening of the RyR, leading to Ca2⫹ leak. See text for further details.
284
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
Dystrophin loss
Microtubule dysfunctions
Autophagy
Src
NOX2
Loss of sarcolemmal nNOS
ROS
SAC/SOC
RyR1leak
RNS
SGK1
Membrane
permeability
PLA2
Ca2+
Mitochondrial dysfunctions
Muscle Damage
Impaired metabolism
CTGF
Fibrosis
Inflammation
TGF-β
Impaired regeneration
Osteopontin
Branched fibers
Impaired muscle function
FIGURE 6. Mechanisms of damage and functional impairment in dystrophic muscle. This schematic highlights some of the key deleterious pathways that lead to impaired muscle function, as a result of the loss of
dystrophin. Particular focus is given to the pathways relating to ROS, Ca2⫹, NO, and fibrosis. See text for
details.
(LC3), which has a cytosolic form (LC3A) and a lipidated
form (LC3B). When autophagy is reduced, p62 binds
ubiquinated protein aggregates which accumulate in the
autophagosome membrane and therefore p62 levels increase (294). Impaired autophagy is also measured by the
ratio of LC3A to LC3B, with lower LC3B relative to LC3A,
indicative of reduced autophagic flux. In both mdx and
DMD muscles, p62 levels are increased and LC3B levels
reduced, highlighting a significant impairment of autophagy. Also, persistently increased Akt/mTOR signaling
in mdx muscle is associated with the impaired autophagic
flux (93, 334). Treatment of mdx mice with the AMPK
activator AICAR enhanced autophagy and improved diaphragm health and force production (339). In addition,
long-term feeding of a low-protein diet also triggered autophagy in mdx muscle, and this reduced inflammation and
fibrosis and improved muscle function (93).
Recent evidence shows that impaired autophagy in mdx
muscle is mediated by a pathway involving NOX2-derived
ROS and Src kinase (334). Here it was shown that ROS
produced by NOX2 activates Src, which in turn stimulates
the Akt/mTOR pathway, and inhibits autophagolysosome
formation, both contributing to impaired autophagy. In
addition, Nox2 activity was further enhanced by Src-mediated tyrosine phosphorylation of the Nox2 organizer subunit p47phox, thereby providing a positive feedback loop for
NOX2 activation. Importantly, genetic knockout of
p47phox in mdx mice led to enhanced autophagy, which was
accompanied by inhibition of NOX2 activity and Ca2⫹ influx, reduced muscle fibrosis, and improved diaphragm
force production. Therefore, together these data highlight a
pathway by which NOX2 and Src perturb normal autophagic flux, which leads to accumulation of damaged
proteins and organelles, and this contributes to muscle
damage, fibrosis, and impaired physiological function in
dystrophic muscle (see FIGURE 6).
8. Nox and cardiac muscle dysfunction in mdx mice
While not the focus of this review, it should be mentioned
that NOX-induced ROS also plays a role in the pathogenesis of dystrophic cardiac muscle (364, 510). Interestingly, a
stretch-induced microtubule signaling pathway underlies
the activation of NOX2 in cardiac muscle as well (364).
Here, it was shown that NOX2 is localized at the sarcolemma and t-tubule in close proximity to junctional SR in
mdx cardiomyocytes and stretch causes increased ROS pro-
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ALLEN ET AL.
duction from NOX2, which is dependent on an intact microtubule network. The ROS produced by NOX2 sensitizes
the RyR2, leading to Ca2⫹ sparks which trigger arrhythmogenic Ca2⫹ waves. The authors suggested that this pathway
would likely be activated in vivo during each diastolic
stretch and could contribute to the progressive cardiomyopathy found in mdx mice and DMD. In support of this
idea, administration of the antioxidant NAC to mdx mice
improved cardiac function and reduced fibrosis and inflammation (510).
V. IMPAIRED MEMBRANE REPAIR AND
MUSCLE REGENERATION
The muscular dystrophies are characterized by activity-induced membrane damage and muscle necrosis which, eventually, exceed the repair capacity of the muscle. Here, we
briefly review the substantial evidence that both membrane
repair and regenerative capacity can be impaired in muscular dystrophies. For fuller reviews on muscle repair and
regeneration in muscular dystrophies, see Wallace and McNally (486) as well as Ciciliot and Schiaffino (75).
Skeletal muscle has two repair processes that might potentially be defective and contribute to the muscular dystrophies.
A. Repair of Small Membrane Breaches
Membrane integrity is critical to the maintenance of cellular
function and, consequently, cells have mechanisms for repairing membrane lesions. Membrane breaches can occur
in skeletal muscle as reviewed in section IIIC. Much of the
early work on membrane repair has been performed on
large cells, such as the sea urchin eggs, and the various
mechanisms involved in small and large membrane lesions
have been defined (for review, see Ref. 276). Lesions in the
membrane (⬎1 ␮m diameter) are rapidly repaired by an
extracellular, Ca2⫹-dependent process that involves the recruitment and fusion of intracellular membrane vesicles in a
process that appears similar to exocytosis and involves the
SNARE proteins.
Bansal et al. (26) developed a membrane damage and repair
assay for isolated skeletal muscle fibers utilizing a laser
which damaged a 5-␮m-diameter region of the membrane.
Damage and repair were assessed with the dye FM1-43
which is soluble in water and lipids but does not cross
membranes. It fluoresces in lipids so that before damage
there is minimal fluorescence from the surface membrane
and t-tubules. When the membrane is breached, the dye has
access to the intracellular membranes (SR and ER), which
have much greater area and produce a large spreading fluorescence. In the presence of extracellular Ca2⫹, normal
fibers after damage showed a rapid increase in fluorescence
286
over 1 min and fluorescence then stabilized, showing that
the membrane breach had been sealed. In the absence of
Ca2⫹, fluorescence continued to rise. These data are consistent with the membrane vesicle sealing hypothesis. Importantly, the rate of membrane repair was similar in mdx
fibers, suggesting that absence of dystrophin and the dystrophin-associated proteins do not markedly influence
membrane repair.
In contrast, a dysferlin KO mouse was constructed and
exhibited major defects in membrane repair, which were
unchanged in the presence or absence of Ca2⫹ (26). Dysferlin is a synaptotagmin-like protein, which together with the
SNARE proteins cause vesicle fusion and release. Mutations in this protein cause the type IIB limb girdle muscular
dystrophy and Miyoshi myopathy. The dysferlin KO mouse
showed a slowly developing dystrophy with characteristic
disrupted sarcolemmal lesions, which showed underlying
vesicles. Eccentric muscle exercise caused Evans blue uptake, but the distribution differs from that in the mdx
mouse with individual fibers showing uptake whereas in the
mdx muscles the damaged fibers are characteristically
grouped in bundles. The authors proposed that dysferlin is
a Ca2⫹-sensitive protein necessary for the fusion of membrane vesicles and that in its absence, membrane lesions that
occur normally fail to heal leading to necrosis and degeneration. Thus dysferlinopathy appears to be a dystrophy in
which the primary mechanism is inability to repair membrane defects.
B. Regeneration of Damaged Muscle
A critical feature of skeletal muscle is its ability to regenerate after major or minor damage. The general features of
the regenerative process are well known, and here we provide only a very brief outline as a background to considering how the process might be modified in muscular dystrophies (for review, see Ref. 68).
Regeneration depends on satellite or muscle stem cells located outside the surface membrane but within the basal
lamina. Severe muscle damage leads to break-down of the
muscle membrane, allowing entry of Ca2⫹ and activation of
proteases with resulting disruption to myofibrils and other
cellular structures. This necrotic process triggers an inflammatory response characterized by the entry of neutrophils
and macrophages. Some aspect of the inflammatory process, probably involving a range of trophic factors (for review, see Ref. 68), leads to activation of satellite cells and
they start to divide (proliferation phase). Importantly, some
of the early divisions of satellite cells are asymmetric, meaning that some products of the division remain as satellite
cells so that the overall number of satellite cells remains
roughly constant, while others become “committed myogenic precursors,” initiating a differentiation phase leading
to myoblasts. Myoblasts continue to divide and at some
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
point begin to fuse forming myotubes, characterized by expression of muscle proteins. Finally myotubes fuse with
existing, damaged myofibers. The fact that multiple myotubes fuse to form muscle cells explains the central nuclei
that are one of the hallmarks of regenerated fibers. Failure
of complete fusion of myotubes is thought to explain the
frequent appearance of split fibers in dystrophic muscle
(63). Details of the marker protein characterizing each of
these cell types and the signaling pathways involved in each
step have been recently reviewed (75).
It has long been recognized from histopathological studies
that very early in the disease (DMD boys aged 2–3 yr)
regenerating fibers are evident; however, as the disease progresses (age 6 –7), when fibrosis and atrophy are apparent,
regenerating fibers are less frequent (490, 493). Early evidence of deficiencies in myoblast activation and proliferation was provided by Blau et al. (38), who studied myoblasts grown in culture from biopsies of normal and DMD
muscles. Dystrophic muscles provided only 5% of the number of myoblasts per weight of muscle compared with normal muscles, and these myoblasts grew more slowly and
ceased division earlier. Fibroblasts obtained in the same
way from dystrophic muscle were present in normal numbers and divided normally, so the defect appears limited to
myoblasts. Quantification of satellite cells assessed by histological methods generally shows increased numbers in
dystrophic muscle (220, 491), so Blau et al. (38) suggest
that many of the cells counted in intact tissue may already
be senescent and incapable of proliferation. Later, Webster
and Blau (493) extended this work by systematically studying the number of myoblast doublings before replicative
senescence occurred. In muscle biopsies from normal 5 yr
olds, myoblasts were capable of 56 doublings (providing
1017 cells from the initiating cell), and this declined in older
subjects (44 doublings at 32 yr and 22 doublings at 48 yr).
In DMD patients, at the age of diagnosis (2–3 yr), myoblasts could only achieve 20 – 40 doublings, whereas by the
age of 14 –16, doublings were down to about 10. Agerelated falls in doubling capacity have frequently been reported in other cell lines, and as a general biological rule,
doubling capacity falls in proportion to the number of previous doublings the cells achieved in vivo. Thus, Webster
and Blau (493) proposed that the reduced replicative potential of DMD myoblasts reflected the increased doublings
previously expended in fiber regeneration.
In the last decades of the 20th century, the role of telomeres
in chromosomal degradation, replicative senesence, and
proliferative capacity of cells has gradually emerged. Telomeres are DNA repeats at the end of the chromosome that
protect the chromosome at division from abnormal fusion,
recombination, etc. At each cell division, telomeres are
shortened and thus provide both a record of cellular turnover and an indication of future proliferative capacity. Decary et al. (95) isolated human myoblasts and showed that
telomere length was shorter in clones that had undergone
greater numbers of divisions. In addition, myoblasts from
older subjects had reduced telomere length. Subsequently,
Decary et al. (94) extended this approach to dystrophic
patients and estimated telomere lengths from muscle biopsies. For this analysis, they used the minimal telomere length
because whole muscle includes some nuclei that have undergone very few divisions (quiescent myonuclei), whilst
some of the satellite cells may have completed many divisions and thus have shorter telomeres. The main finding was
that minimal telomere lengths were shorter at comparable
ages in dystrophic patients compared with controls and the
rate of reduction of telomere length with age was greater
(by a factor of 14) in the dystrophic samples than the normal controls. Thus these data indicate that dystrophic muscle has undergone more rounds of proliferation and regeneration than normal muscle and that consequently its capacity for division is restricted. This was confirmed by the
observation that amongst the oldest dystrophic patients
(ages 11–13), regeneration was not visible in the biopsies.
Telomere lengths have been further explored by Sacco et al.
(395) in a mouse model that is a double KO for dystrophin
and telomerase, the enzyme that can restore telomeres by
synthesis of the repeated DNA sequences. In-bred strains of
mice have longer telomeres (⬎40 kb) than humans (5–15
kb), and Sacco et al. postulate that this difference contributes to the milder phenotype of the mdx mouse compared
with the DMD patients. In keeping with this hypothesis,
Sacco et al. (395) show that the phenotype of the dystrophin/telomerase KO is much more severe than the mdx with
higher creatine kinase levels, more rapid exhaustion on a
treadmill, more central nuclei, more severe histology,
greater muscle atrophy, worsened scoliosis, and increased
severity of symptoms with age. They showed that these
features were caused by telomere shortening by demonstrating more chromosomal free ends and showed that the
myoblasts from the double KO had impaired proliferation.
Importantly, the proportion of myoblasts that developed
muscle specific markers was unchanged, indicating that differentiation of the myoblasts was unimpaired. They also
showed that the double KO myoblasts did not proliferate
well when engrafted in a host muscle. These data strongly
support the concept that excessively shortened telomeres, as
a result of repeated regeneration, are an important factor in
the failure of regeneration in older DMD patients. Furthermore, they provide an animal model that more closely simulates the human disease and offer an explanation for the
mild phenotype in the mdx mouse.
In summary, there is compelling evidence that failure of
regeneration contributes to the pathology of muscular dystrophies. The best-explored mechanism is the reduced proliferative potential of myoblasts associated with repeated
episodes of regeneration and reflected in the telomere
lengths. However, other possible mechanisms including
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ALLEN ET AL.
failure of activation of satellite cells and reduced fusion
ability, possibly related to the changing muscle environment, remain to be explored in detail.
suggests a marginally significant increase present in the low
dose but not the high dose. Other drugs in the same class
with higher read-through potential are under investigation.
VI. THERAPEUTIC DIRECTIONS
Perhaps the most promising direction under development is
the use of drugs that promote exon skipping. 60 – 80% of
DMD patients have a frame-shifting deletion. In such patients, if the exon with the mutation is skipped, then expression of a shortened dystrophin is possible. Drugs in this
class include anti-sense oligonucleotides that are designed
to hybridize to the complementary section of the premessenger RNA and lead to exclusion of the relevant exon. A
difficulty inherent in this approach is that mutations in different exons will require alternative drug designs. The current drugs are targeted to frame-shifting mutations within
exon 51 which encompasses ⬃13% of DMD boys. One
drug in this class, drisapersen, which is in the 2’-O-methylphosphorthioate class, has been successfully used in several
small scale open-label trials and was shown to induce some
dystrophin expression and produce benefit in the 6-min
walk test. However, a recent press release from the manufacturer revealed that a phase 3 double blind trial with 186
patients failed to show improvement in the 6-min walk test,
the primary endpoint. A second drug, eteplirsen, with morpholino chemistry, has also been successful in small scale
open-label trials, but again the dystrophin expression was
variable and only reached 20% of normal levels in the best
expressing patients (281). For a recent analysis of the current status of exon-skipping as a treatment for DMD, the
reader is referred to Hoffman and McNally (186).
A. Gene-Based Therapies
After many decades in which steroids were the only approved treatment that slowed progression of the disease, the
situation is changing rapidly with a range of treatments in
clinical trials (for recent reviews, see Refs. 1, 119, 185,
387). All of these treatments have been extensively trialled
in the mdx mouse so that proof of principle is established.
Most of these treatments focus on replacement of the dystrophin gene or repair of the mutation and are dependent on
the rapid growth in knowledge about manipulation, modification, and repair of genes.
Viral gene therapy has been attempted or considered in
many genetic diseases with mixed results. A fundamental
problem for the treatment of DMD is that the very large size
of the dystrophin mRNA (14 kb) exceeds the carrying capacity of most viral vectors, including the adeno-associated
virus with a carrying capacity of ⬃5 kb. Based on the observation of a relatively mild clinical phenotype in a Becker
MD patient with a deletion of exons 17– 48 (115), dystrophin mini genes have been designed, which lack many of the
spectrin repeats but retain much of the functionality (168)
including the nNOS binding site (229). Such mini-dystrophins can be packaged in an adeno-associated virus (AAV)
and have been successfully used in mdx mice, where they
induce expression of mini-dystrophin and ameliorate symptoms (168, 488). In a small trial, six boys with DMD were
injected with an AAV containing mini-dystrophin into one
biceps muscle. Only minimal and transient expression of
the mini-dystrophin was detected (278). However, an immune response to the AAV virus was generated in four of
the six boys, possibly because a generalized CMV promoter
was used as opposed to a muscle-specific promoter (280,
387).
Mutations causing stop codons are present in ⬃15% of
DMD cases. In principle, if the stop codon can be read
through by the ribosome, a full-length dystrophin could be
expressed. A drug, ataluren, formerly called PCT124, has
this property and in the mdx mouse generated dystrophin
expression and protection against eccentric muscle damage
(499). Multiple small clinical trials have been published,
and a very recent trial (124) demonstrated a small but statistically significant increase in dystrophin expression.
Whether the small and variable dystrophin expression (average increase of 11% above pretreatment level) will lead to
a clinically useful outcome remains to be seen. Results of the
6-min walk test in a double blind trial with 174 boys over
48 wk have not been published, but a company press release
288
A fourth approach, which is in active development, is the
use of small molecules that cause increased expression of
utrophin. The protective role of utrophin, a dystrophin analog, in mdx mice was introduced in section IC. As a result
of these studies, a systematic search was undertaken to find
drugs that could increase utrophin expression. In mdx mice,
SMT-C1100 increases utrophin expression and reduces the
dystrophic phenotype (457). Small-scale toxicity and doseranging studies are commencing in humans.
The fact that four different gene-based approaches to treatment have reached the stage of clinical trials in the last few
years is an enormously exciting and significant development. None has yet achieved the current gold standard,
which is an increase in the 6-min walk time in a doubleblind clinical trial. The most obvious alternative marker is
the appearance of dystrophin (or additional utrophin) in the
muscles, but this is a highly invasive test, difficult to quantify and subject to a large sampling error not suited to large
scale use (for discussion, see Ref. 185). Several of these
approaches to therapy will lead to the expression of a shortened dystrophin, and this has triggered renewed interest in
the clinical history of BMD patients with equivalent mutations (1). It is still unclear how much dystrophin needs to be
expressed to improve the disease, and it is likely that all the
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
current methods to produce dystrophin will need to be more
effective. Furthermore, the importance of spectrin-like repeats in several aspects of DPC function must be taken into
account when large segments of the dystrophin repeat region are deleted. A combinatorial approach of genetic manipulation and drug administration to correct signaling deficiencies caused by the absence of a fully functional dystrophin will likely be required for successful treatment.
B. Targeting Damage Pathways: Nongenetic
Therapeutic Approaches
As an alternative or complementary approach to dystrophin or utrophin gene or cell-based therapies for DMD,
numerous drugs and recombinant protein therapies have
been tested in dystrophic animal models over the last decade
or more (262, 390; for recent reviews, see Ref. 536). Some
of these drugs have been chosen based on their known im-
provement of signaling pathways that are deficient in dystrophic muscle. Others have been found by unbiased
screens, for example, using the zebrafish model of DMD
(209). The next section discusses some of the most promising therapeutic approaches aimed at targeting key damage
pathways in DMD (FIGURE 7).
1. Drugs targeting ROS, Ca2⫹, and NO
As discussed in previous sections, antioxidants have shown
efficacy in the mdx mouse (214). Of these, NAC is particularly attractive based on its improvement of muscle function
and amelioration of muscle damage in mdx mice (92, 223,
504). NAC has been extensively studied in a wide range of
human diseases and acute conditions and has only mild side
effects (400). The antioxidant properties of NAC involve
direct scavenging of ROS, reducing disulfide bonds in proteins and boosting intracellular GSH synthesis, via its cysteine group (400). This later pathway is likely to be partic-
NO
Tadalafil
Sild
Sildenafil
fil
Blood flow
ow
Myofibro
ob
blast
Myofibroblast
Vasoc
Vasoconstriction
TGF-β
TGF
β
Functional ischemia
HT-100
M2a
M2
2a
Osteopontin
Collagen/fibronectin
FG-3019
Fibrinogen
Remicade
Etanercept
TNF
M1
Fibrosis
CTGF
Latent
TGF-β
TGF-β
NAC
EGCg
ROS
SAC
SGK1
Biglycan
ROS
PLA2
MG53
βDG
NOX2
Dystrophin/Utrophin
MMP2/9
Syn
nNOS
NDB
NF-κB
ROS
NAC
EGCg
Dystrophin
Exon skipping
Syn
Utrophin
upregulation
Dystrophin/utrophin
gene therapy
Sarcoplasmic reticulum
SMTC-1100
RyR1 leak
Rycals
FIGURE 7. Therapeutic pathways for Duchenne muscular dystrophy (DMD). This figure highlights some of
the key therapeutic targets for DMD, which include both muscle-specific and extracellular pathways. A wide
range of therapeutic approaches are being evaluated for DMD (red boxes). These include gene-based therapies, pharmacological agents, and recombinant proteins (see text for details).
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289
ALLEN ET AL.
ularly important in DMD, since skeletal muscle GSH concentrations are decreased by 50% compared with normal
muscle, and this is accompanied by oxidative muscle damage (379). Another compound with antioxidant properties,
epigallocatechin gallate (EGCG), a component of green tea,
has shown efficacy in ameliorating muscle damage in mdx
mice (101, 314). As a result of these preclinical studies,
DMD patients are currently being recruited for a phase 2
clinical trial to test the safety and efficacy of EGCG (clinicaltrials.gov). As discussed earlier, antioxidants are also
beneficial at improving intracellular Ca2⫹ homeostasis, by
inhibiting Ca2⫹ influx via MSC activation and preventing
increased membrane permeability (145, 504). In addition,
compounds that stabilize the binding of the RyR1 to calstabin, termed Rycals, have been shown to reduce RyR1 Ca2⫹
leak and improve muscle function in mdx mice (30). While
still in the preclinical stage, these compounds are being
developed for future clinical trials in DMD. The NO-cGMP
pathway is another therapeutic target for DMD. Sildenafil
and Tadalafil, PDE-5 inhibitors that enhance the NOcGMP signaling pathway, have both shown efficacy in mdx
mice (18, 347). Sildenafil improved diaphragm force production and reduced fibrosis and inflammation (347).
Tadalafil was found to reduce muscle damage in mdx mice
following muscle stimulation during ischemia (18). Improved muscle blood flow is thought to be one mechanism
by which PDE-5 inhibitors improve dystrophic muscle, and
Tadalafil was shown to reduce ischemia and improve blood
flow in muscles of BMD patients (267). A phase 3 clinical
trial to test the efficacy of Tadalafil in DMD is currently in
the recruitment stage (clinicaltrials.gov). Sildenafil also increased hemeoxygenase 1, a recently discovered therapeutic
target involved in mitigating disease progression (208).
2. Drugs targeting inflammation and fibrosis
Inflammation and fibrosis are major pathogenic processes
affecting muscle function in mdx mice and DMD (see FIGURE 6). They are both regulated by complex interactions
involving many different cell types, and as such, a detailed
description is beyond the scope of this review. Several recent
reviews provide a very good overview of the underlying
mechanisms and current therapeutic approaches for targeting inflammation and fibrosis and should be referred to for
a more detailed understanding of these processes (262, 413,
414, 536). Here, we will briefly outline the major pathways
and the most promising, current therapeutic approaches for
targeting inflammation and fibrosis in DMD.
Inflammation has diverse, regulatory effects on the pathophysiology of DMD. On one hand, inflammatory cells such
as macrophages are required for effective muscle regeneration following muscle damage (409) and for improved myoblast survival in dystrophic mice (243). However, an excessive, chronic inflammatory response, as occurs in DMD,
exacerbates muscle damage, impairs effective regeneration,
and promotes excessive extracellular matrix deposition, re-
290
sulting in fibrosis (for a review, see Ref. 414). Therefore,
targeting the deleterious pathways of inflammation, while
preserving its beneficial effects, is a key criterion for effective anti-inflammatory approaches for DMD. Proinflammatory macrophages, often termed M1 macrophages, produce cytokines such as TNF-␣ and IL-1␤. Studies in mdx
mice have tested two TNF-␣ inhibitors, Etanercept (183,
356) and Remicade (116, 157), which are both used in
humans to treat autoimmune diseases such as rheumatoid
arthritis. These drugs have shown some benefits in mdx
mice, including reduced muscle necrosis and inflammation
(157, 183), improved muscle function and decreased fibrosis (116), and reduced collagen and TGF-␤1 levels (356).
Therefore, these drugs have potential to attenuate muscle
damage and fibrosis in DMD. The transcription factor
NF-␬B is chronically activated in mdx mice and during the
early stages of DMD. It is a key regulator of the inflammatory response to injury, but chronic activation likely contributes to ongoing muscle degeneration. NEMO binding
domain (NBD) is a small peptide designed to block activation of NF-␬B, and after systemic administration in mdx
mice, there is improvement in pathology (376) and diaphragm function (353). NBD is currently in development
for clinical studies in DMD. However, one note of caution
regarding NF-␬B inhibition as a potential treatment for
DMD relates to the time course of NF-␬B activation. It was
shown that NF-␬B nuclear activity peaks very early in
DMD, up to ⬃2 yr of age, and then progressively decreases
to low levels by 9 –10 yr of age (71, 287). This implies that
these drugs would need to be administered very early in the
disease to have maximum benefits. Another approach is the
use of antioxidants such as NAC, which have been shown
to effectively inhibit NF-␬B translocation to the nucleus in
mdx muscle (219, 490).
Fibrosis also occurs at an early age in DMD patients and is
a major cause of progressive muscle weakness (97), as depicted schematically in FIGURE 6. Therefore, therapeutic
approaches aimed at preventing fibrotic deposition and/or
reversing preexisting fibrosis are a major priority for effectively treating DMD, at all stages of the disease. Several
studies have shown that TGF-␤ is a key activator of fibrosis
in mdx mice, is upregulated in DMD, and its activity is
influenced by modifier genes (for review, see Ref. 61).
TGF-␤ is produced by a number of cells associated with
muscle repair, including fibroblasts, macrophages, and neutrophils (61). Inactive TGF-␤ resides in the extracellular
matrix as a latent complex; however, upon cleavage by
serine proteinases such as MMP2 and 9, active TGF-␤ can
bind to the TGF-␤1/2 receptor complex. This initiates phosphorylation of the transcription factors SMAD2 and 3,
which translocate to the nucleus and increase expression of
profibrotic proteins such as collagen I and fibronectin (240).
Recently, the cytokine osteopontin was shown to be an
important inducer of TGF-␤ in mdx and DMD muscle, and
its deletion improved mdx muscle function and reduced
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EARLY DAMAGE PATHWAYS IN MUSCULAR DYSTROPHY
fibrosis (477). A number of drugs have been tested in mdx
mice for their ability to reduce fibrotic deposition by inhibiting the TGF-␤ pathway (348). Currently, one of the most
promising candidates is halofuginone, a natural alkaloid
compound with known antifibrotic effects. Halofuginone
reduced fibrosis and improved muscle function in mdx
mice, via inhibition of the TGF-␤/SMAD signaling pathway
(192, 464). DMD boys are currently being recruited for a
phase II clinical trial to test the safety and pharmacokinetics
of Halofuginone (HT-100) (clinicaltrials.gov).
Connective tissue growth factor (CTGF) is another key
stimulator of fibrosis in dystrophic muscle. CTGF overexpression in tibialis anterior muscles of WT mice produces a
dystrophic-like muscle disease, with muscle damage and
regeneration, increased fibrosis, and reduced specific force
production (303). The same group more recently showed in
mdx mice that genetic reduction of CTGF levels or treatment with a neutralizing monoclonal antibody against
CTGF (FG-3019), both reduced muscle fibrosis and increased muscle function (304). These data suggest that FG3019 might have therapeutic potential in DMD.
Recent studies have identified novel pathways that could
lead to new therapeutic approaches for reducing fibrosis in
DMD. Genetic ablation of the serum and glucocorticoidinducible kinase (SGK1) dramatically improved muscle
function and reduced fibrosis in mdx muscles (433). Since
SGK1 has been shown to increase CTGF levels, this could
provide a new therapeutic target for reducing fibrosis. Another recently discovered pro-fibrotic pathway, mediated
by increased fibrinogen, is evident in muscles of mdx mice
and DMD (478, 479). Fibrotic deposition in dystrophic
muscles is accompanied by accumulation of fibrinogen, and
its depletion by genetic and pharmacological approaches
reduces diaphragm fibrosis in mdx mice. The findings indicate that fibrinogen binds to the Mac-1 receptor on alternatively activated, M2a macrophages, which are considered to play an important role in dystrophic muscle fibrosis
(262a). The activation of M2a macrophages by fibrinogen
triggers TGF-␤1 release that, in turn, stimulates secretion of
fibrotic proteins from fibroblasts (478, 479). Therefore,
drugs targeted to reduce fibrinogen levels and/or block fibrinogen binding to macrophages could provide a novel
therapeutic approach for reducing fibrosis in DMD.
3. Protein therapies
An alternative to replacing dystrophin via gene therapy or
exon skipping is to improve the function of the dystrophin
complex by exogenous delivery of proteins associated with
the dystrophin complex. Perhaps the best example of such
an approach is the work of Fallon and colleagues, using
biglycan (16, 45, 284, 367). Biglycan is upregulated in the
mdx mouse and has been shown to bind to several members
of the dystrophin complex, notably ␣-dystroglycan and ␣and ␥-sarcoglycan (45, 367). Importantly, systemic delivery
of recombinant, nonglycanated biglycan (rNG-BGN) in
mdx mice increases sarcolemmal nNOS and utrophin expression, and this is associated with improved muscle health
and function (16). Currently, rNG-BGN is under preclinical
development as a potential therapeutic approach for DMD
(525). Another protein, mitsugumin 53 (MG53), has recently been shown to reduce membrane permeability in
mdx mice when injected as a recombinant protein
(rhMG53) (498). While these results are encouraging, further preclinical studies, particularly testing muscle function,
are required to determine if rhMG53 is a viable protein
therapy for DMD.
VII. CONCLUDING REMARKS
Mutations in the dystrophin gene lead to a complex disease
affecting many aspect of muscle function. There is still no
clear definition of the normal function of dystrophin because absence of dystrophin leads to multiple changes in
membrane properties, signaling pathways, and many aspects of muscle function. Almost three decades after the
identification of the mutated gene and its protein product,
gene-based treatments now appear to be a therapeutic possibility with multiple clinical trials in progress utilizing a
variety of approaches. In the mdx mouse, an extraordinary
range of interventions have produced some therapeutic benefit, and it seems likely that, if and when gene-based approaches become a possibility, some of these interventions
will be helpful in minimizing those aspects of the disease
that are not improved by the gene therapy.
The literature on muscular dystrophy continues to grow at
an astonishing rate. If we define the literature by papers that
include “dystrophin,” “muscular dystrophy,” or “mdx
mouse” in their title, abstract, or key words, then prior to
1940 there was on average only 0 or 1 paper per year. By
1960 this had grown to around 100 per year, and by 1980
to 400 per year. Since then, the rate of publications has
continued to increase linearly, and in 2013 there were 1,100
papers per year and still steadily growing. This huge interest
reflects the still unmet clinical need coupled with the insights into so many aspects of muscle function that have
been triggered by trying to understand the role(s) of dystrophin.
It is still widely considered that the initial consequences of
dystrophin’s absence are the mechanical defects associated
with dystrophin’s structural role and the membrane damage that results directly from this mechanical role. While
this sequence of events may play some minor role, it is
increasingly clear that dystrophin and the DPC have multiple signaling roles and disruption of these functions leads to
the abnormal regulation of Ca2⫹, ROS, and NO that seem
to cause many of the pathological effects of the disease. We
believe that as molecular and pharmacological approaches
to therapy start to minimize some of these disruptions, a
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ALLEN ET AL.
more sophisticated understanding of the mechanisms regulating these signaling pathways will provide additional therapeutic avenues for DMD and related neuromuscular diseases.
ACKNOWLEDGMENTS
We thank Dr. Philip Dash, University of Reading, UK, for
kindly granting us permission to use the figure of the NOS
dimer shown in FIGURE 4.
Address for reprint requests and other correspondence: D.
Allen, Sydney Medical School & Bosch Institute, Univ. of
Sydney, NSW 2006, Australia (e-mail: [email protected]
edu.au).
GRANTS
D. G. Allen gratefully acknowledges support from a Program Grant of the National Health and Medical Research
Council of Australia. S. C. Froehner and N. P. Whitehead
gratefully acknowledge current and past support from the
National Institutes of Health (Grants R01 NS33145, R01
AR056221, R21 NS088691, and P01 NS04678), Parent
Project Muscular Dystrophy, and the Raymond and Beverly
Sackler Foundation. The contents of this review are solely
the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared
by the authors.
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