1. Introduction
Muscular dystrophy is a deadly genetic disorder which represents a group of
complex muscular diseases. Patients with this disease generally suffer from
muscle weakness as well as loss of ambulation, and mostly die in their early
twenties due to respiratory and cardiac complications [1, 2]. The first
historical account of muscular dystrophy was given in 1830s by Conte and Gioja
that this disease causes progressive weakness of multiple muscle groups [3];
however, in 1852, Edward Meryon suggested that a disorder in the cell membrane
causes muscular dystrophy but the disease was mistaken for tuberculosis [4]. It
was suspected that this disease is genetically transmitted by females and it
affects males only, in addition to causing progressive muscle damage which is
replaced with connective tissue. Furthermore, it was pointed out that the
development of this skeletal muscle disorder is not only age and sex dependent
but it also affects cardiac muscle in advanced stages [4]. Later on in 1868, this
disease was called Duchenne muscular dystrophy (DMD) after the name of a French
neurologist, Guillaume Duchenne, due to his significant contributions in
understanding its mechanism [5]. Most of the diagnostic criteria of this
disorder, established at that time, are still being used and these include: (a)
weakness in legs; (b) hyperlordosis; (c) hypertrophy of weak muscle; (d) reduced
muscle contractility on electrical stimulation; (e) absence of bladder or bowel
dysfunction; and (f) sensory disturbances and febrile illness. In addition, there
occurs cardiomyopathy and mental retardation at advanced stages of this disease
and results in death at early age. The progressive nature of these
characteristics of muscular dystrophy is depicted in Fig. 1 [6, 7, 8, 9]. It is pointed
out that in 1878, Gowers was the first to report the genetic basis of muscular
dystrophy [10] but it was not until 1891, when the concept of histological
alterations in muscle was outlined and the classification of muscular dystrophies
such as infantile and juvenile types along with many other subtypes was described
[11].
Fig. 1.
Schematic representation of different stages of muscular
dystrophy.
Currently, there are approximately 50 muscular dystrophy causative genes and
more than 40 types and subtypes of these are associated with genetic mutations.
These various forms of muscular dystrophies have been characterized by patterns
of their inheritance, symptoms, origin of gene mutation, age at onset, rate of
progression and level of severity [12]. The spectrum of mutations varies as it
ranges from complete deficiency of a gene product to a decrease in gene
expression and/or expression of an abnormal molecule with complete or partial
loss of functionality. Most of the major types of muscular dystrophies are
categorized on the basis of X-linked, autosomal dominant and autosomal recessive
genes; the location of altered gene product lies in muscle fiber but is linked to
other proteins, enzymes and extracellular matrix [12, 13, 14]. While DMD, Becker
muscular dystrophy and Limb-Girdle dystrophies are the common forms of this
disease, it is noteworthy that DMD is the most prevalent and severe form among
various types of muscular dystrophies. In fact, over 80% of cases of muscular
dystrophy worldwide are associated with DMD, whereas most of the other types are
fairly rare [12]. The global prevalence of DMD is 19.8 per 100,000 male births
[15]. However, there are variations in disease etiology; the common physiognomies
of muscular dystrophy include primary genetic defects and mechanisms based on
repetitive cycles of muscle degeneration, necrosis and impaired regeneration
resulting in muscle fibrosis, muscle wastage and muscle dysfunction [8, 16]. The
evaluation of family history and physical symptoms such as contracture, muscle
stiffness, and weakness are the basic diagnostic procedures. In addition to
mutation screening in the predicted gene, the assessment for muscular dystrophies
is carried out from the determination of muscle weakness, serum creatine kinase
(CK) levels, muscle biopsy examination, muscle magnetic resonance imaging,
neurological evaluation, electromyographic and electrocardiographic analysis as
well as exercise tolerance examination [16].
In spite of extensive preclinical and clinical research efforts over the past 50
years, the exact pathogenesis and therapies of muscular dystrophy remain to be
poorly understood. It should also be emphasized that muscular dystrophy is not an
entity but represents a group of various muscle disorders, which differ from one
another with respect to the location of defects in plasma membrane, as well as
trans-membrane, extracellular matrix, nuclear membrane, nucleus and cytosol
proteins. Since clinical phenotype and pathophysiology of muscle degeneration are
different in each type of muscular dystrophy, this article is planned to deal
with biochemical and metabolic alterations in dystrophic muscles from all forms
of this disease in a general way rather than in any specific manner. It is
noteworthy that several molecular and biochemical defects have been identified in
dystrophic muscle and their modulation has been shown to slow down the disease
progress [12, 17, 18, 19, 20, 21, 22, 23, 24, 25]. Abnormalities in dystrophic muscle include increased
membrane permeability, Ca-handling defects, depressed energy production,
oxidative stress and myocyte necrosis as well as apoptosis. Impairment of the
blood flow to skeletal muscle is invariably associated with dystrophic muscle
dysfunction. It is also pointed out that irrespective of differences in the
pathogenesis of different types of muscular dystrophy, dystrophic muscles from
all types of diseased subjects show similar metabolic defects and impaired
function. This article is therefore intended to provide a comprehensive and
updated information about metabolic and biochemical alterations in dystrophic
skeletal muscle during the development of muscular disorder in general.
Derangements for abnormal Ca-entry as well as Ca-handling in
myocytes from dystrophic muscle will also be described in some details and in
particular, the role of store-operated Ca-channels in the development of
intracellular Ca-overload will be outlined. Mechanisms of
Ca-handling abnormalities and metabolic defects in dystrophic muscles from
different experimental models of muscular disease will also be described. Some
changes in membrane activities and metabolic status of the hind leg muscle from
myopathic hamsters will be discussed to show if there is any relationship with
impaired muscle performance. Since dystrophin, an important component for
anchoring different proteins and enzymes in the membrane, the function of
sarcolemma in several forms of muscular dystrophy will be evaluated [13, 14, 20].
The significance of dystrophin gene mutations leading to dystrophin deficiency in
the development of this disease will also be highlighted. In addition, attempts
will be made to describe some of the therapeutic strategies, including gene
therapy and pharmacologic interventions, which are used for the management or
treatment of muscular disorder.
2. Biochemical defects in dystrophic muscle
Different mechanisms including impaired metabolism [8, 26, 27, 28, 29, 30, 31], structural and
biochemical membrane defects [32, 33, 34, 35, 36, 37, 38, 39] and Ca regulatory abnormalities
[40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52] have been described to understand the pathogenesis of weakness in
dystrophic muscle. An increased activity of enzymes, such as CK in the serum is
considered to reflect damage to the muscle cell membrane and serves as a
sensitive marker for the progression of this disease [53, 54, 55, 56, 57, 58, 59]. Recent studies
with circulating proteins and metabolites have validated different biomarkers for
patients with DMD [60, 61, 62, 63, 64]. It was demonstrated that defects of the plasma
membrane lead to leakage of several cellular constituents such as myoglobin,
glycogen, potassium, ATP and creatine from muscle fibres in addition to producing
increased influx of Ca in myocytes [33, 43, 65, 66, 67, 68, 69]. Ultrastructural
examination of dystrophic muscle revealed segmental fiber breakdown and
Ca-deposits in myocytes with intact basement membrane as significant
features [65, 66, 70, 71, 72]. Several changes indicating biochemical abnormalities
in the sarcolemma (SL) membrane were observed in different types of dystrophic
muscle [73, 74, 75, 76]. Marked alterations in lipid and electrolyte content were also
seen in dystrophic muscle indicating abnormal function of the cell for
maintaining appropriate levels of intracellular components [73, 77]. Thus, it has
become evident that the increased permeability of skeletal muscle is reflected by
defective SL membrane during the development of muscular disease [43, 70, 78].
Alterations in the integrity of dystrophic muscle membrane [79, 80] were
associated with changes in different SL enzyme systems such as Na-K ATPase, Ca/Mg ecto-ATPase, and adenylyl cyclase [74, 75, 76, 81, 82, 83, 84, 85].
Since both cholesterol and phospholipids are known to exert membrane stabilizing
effects [86, 87] and the ratio of cholesterol/phospholipids was elevated in
dystrophic muscle [88], it has been suggested that the observed changes in enzyme
activities are a consequence of alterations in the SL lipid composition. Studies
from dystrophic muscle fibroblasts and skin fibroblast cultures have also shown
different alterations such as cytoplasmic inclusion bodies, defective collagen
incorporation as well as membrane defects [89, 90, 91, 92, 93]. Furthermore, derangements in
the function of intracellular membrane systems such as the sarcoplasmic reticulum
(SR) [40, 76, 88] and mitochondria [41, 76, 94, 95, 96] were reported in dystrophic
muscle. Although no alterations in myofibrillar ATPase activity and contractile
proteins in myopathic hamster muscle were observed [30, 76], some investigators
have shown defective myosin in a chicken model of muscular dystrophy [97].
Nonetheless, marked changes in metabolism [26, 27, 28, 29, 30, 98] indicating the impaired
performance of skeletal muscle in different types of muscle disorders may be
associated with subcellular defects and metabolic abnormalities. In addition,
maldistribution of electrolytes in dystrophic myocytes [66, 77], there occurs the
development of intracellular Ca-overload [41, 42, 43, 44, 99], which activates
different proteolytic enzymes [100, 101] and leads to muscle breakdown as well as
structural defects dystrophic muscle of DMD patients [102, 103].
Since the discovery of the DMD gene and identification of the role of
dystrophin-deficiency in DMD in 1980s, various genetically engineered animal
models have been developed to understand the biology, biochemistry and
pathophysiology of different types of muscular dystrophies [104, 105, 106]. The most
widely used is a dystrophin-deficient mouse (mdx), which is considered to be an
excellent model for studying DMD [107, 108, 109, 110, 111, 112, 113, 114, 115]. The other animal models of
dystrophin-deficiency have employed pigs [116, 117], rats [118, 119], dogs
[120, 121, 122] and cats [123, 124]. It is pointed out that marked changes in protein,
lipids, carbohydrate and energy metabolism have been observed in skeletal muscle
from mdx dystrophic mouse [125, 126, 127, 128, 129]. Furthermore, impaired mitochondrial
oxidative phosphorylation and increased Ca content due to
dystrophin-deficiency have been reported in mdx mouse skeletal muscle [130, 131, 132, 133, 134].
Treatments of mdx dystrophic mouse with Ca-antagonists, verapamil and
diltiazem, as well as with creatine were observed to reduce skeletal muscle
degeneration and mitochondrial dysfunction [135, 136]. It is noteworthy that
skeletal muscle from DMD patients to have also been found to exhibit
mitochondrial dysfunction and sarcolemmal alterations due to
dystrophin-deficiency [137, 138]. It is also pointed out that
sarcoglycan-deficiency models have also been developed in different animals such
as hamsters, mice and chickens to examine metabolic and cellular defects in
various muscular disorders [139, 140, 141, 142, 143, 144, 145].
3. Metabolic and subcellular defects in skeletal muscle of myopathic
hamster
In order to examine the functional significance of metabolic and subcellular
alterations for impaired performance of myopathic muscle, hind leg muscles from
two experimental models (BIO 14.6 and UM–X7.1) of Syrian hamster
(-sarcoglycanopathy) at different stages of development were used [30, 40, 74, 75, 76]. It may be noted that the clinical signs of muscle impairment in BIO
14.6 strain of hamsters start developing at the age of 100 to 150 days (early
stage) whereas moderate and severe stages of myopathy become apparent at 180–210
days and 260–275 days of age, respectively [30, 146]. On the other hand,
UM–X7.1 strain of myopathic hamsters develop degenerative lesions as early as 20
to 30 days whereas these animals at the age of about 60 days and about 150 days
were considered to be at moderate and severe stages of muscular disorder,
respectively [74, 147]. Although different stages of myopathy in both models of
hamsters have been categorized on the basis of pathological lesions in the hind
leg, it is understood that these stages are arbitrary and reflect the progression
of muscular impairment with respect to the age of animals.
By employing the BIO 14.6 hamster model of myopathy, Sulakhe et al.
[74] were the first to show increases in the activities of SL Mg-ATPase
and Na-K ATPase in skeletal muscle. This observation showing defect in the
SL membrane at the biochemical level was confirmed by Peter and Fiehn [148], who
reported increased activities of Na+-K+ ATPase and Ca-ATPase in
myotonic muscles of rats treated with diazacholesterol. Although no changes in
skeletal muscle SL ATPase activities were detected at early stages in BIO 14.6
myopathic hamsters, marked increases in the activities of SL Na-K ATPase, Mg-ATPase and Ca-ATPase were seen in both moderate and
severe stages of muscular disorder (Table 1) [75]. Likewise, marked increases in
these SL enzyme activities in skeletal muscle were observed in UM–X7.1 strain of
myopathic hamsters at both moderate and severe stages except that Na-K ATPase and Mg-ATPase activities were not altered at the moderate stage
(Table 1) [76]. Similar increases in the hind leg skeletal muscle SL
Na-K ATPase, Mg-ATPase and Ca-ATPase were seen in rats
on vitamin E deficient diet [75], which is considered to be a good model for
studying the pathogenesis of muscular weakness [34]. However, preliminary studies
with dystrophic skeletal muscles from human showed that SL Na-K ATPase activity was depressed but both Ca-ATPase and Mg-ATPase
activities were increased [75]. Decreased Na-K ATPase and increased
Mg-ATPase activities were also observed in SL preparations from dystrophic
muscles of Bar Harbor strain of 129/Rej mice [149]. These observations suggest
that alterations in SL Na-K ATPase may depend upon the type and stage
of muscular disorder; the increased activity of this enzyme may play a
compensatory role in maintaining the electrolyte composition of the myopathic
muscle whereas the depressed Na-K ATPase may be associated with
increased entry of Ca through Na-Ca exchange system.
Furthermore, the increased activities of skeletal muscle SL Ca-ATPase and
Mg-ATPase, which have been shown to represent Ca/Mg-ecto
ATPase [150], may promote Ca-influx for the occurrence of intracellular
Ca overload and thus play a pathogenic role for the impaired performance
of myopathic muscle. It is pointed out that SL Ca/Mg-ecto ATPase,
which is activated by millimolar concentrations of Ca or Mg, has
been suggested to serve as a “gating mechanism” for the entry of Ca into
the cell [150, 151, 152, 153]. Although the status of SL Na-Ca exchange system
was not examined in hamster myopathic muscle, the activity of Na-Ca
exchange was increased in dystrophic muscle from patients with DMD as well as mdx
model of muscular dystrophy in mice [154, 155].
Table 1.ATPase activities of sarcolemma from skeletal muscles of
control and myopathic hamsters of different ages.
|
|
|
Na-K ATPase (µmoles Pi/mg/hr) |
Mg-ATPase (µmoles Pi/mg protein/hr) |
Ca-ATPase (µmoles Pi/mg/hr) |
A. BIO 14.6 myopathic hamsters |
|
|
|
|
a. Control: (100–272 days old) |
20.4 1.2 |
33.1 1.6 |
31.5 2.9 |
|
b. Myopathic |
|
|
|
|
|
(i) 100–150 days old (Early stage) |
19.7 1.9 |
31.5 2.4 |
32.4 2.5 |
|
|
(ii) 180–210 days old (Moderate stage) |
27.5 1.5* |
43.3 2.1* |
42.4 1.4* |
|
|
(iii) 260–275 days old (Late stage) |
38.5 3.3* |
59.2 4.2* |
56.2 3.7* |
B. UM-X7.1 myopathic hamsters |
|
|
|
|
(i) 60 days old |
|
|
|
|
|
Control |
6.6 0.7 |
30.6 2.6 |
10.0 1.3 |
|
|
Myopathic (Moderate stage) |
6.5 0.4 |
28.7 1.7 |
16.8 1.0* |
|
(ii) 150 days old |
|
|
|
|
|
Control |
13.8 1.1 |
25.0 1.9 |
27.6 2.8 |
|
|
Myopathic (Late stage) |
19.1 0.8* |
50.5 4.2* |
58.0 5.1* |
The data for BIO 14.6 strain of myopathic hamsters are taken from our paper,
Dhalla et al. Res Commun Chem Pathol Pharmacol 6, 643–650, 1973 [75] whereas the data for UM-X7.1 strain of myopathic hamsters are taken from our
paper Dhalla et al. Clin Sci Mol Med 49, 359–368, 1975 [76]. * Significantly (P 0.05) different from the corresponding control
values. |
Since the SR plays a critical role in regulating the intracellular Ca concentration in myocytes, Ca-binding and Ca-transport activities
of this subcellular organelle were examined in skeletal muscles of both BIO 14.6
and UM–X7.4 strains of myopathic hamsters at moderate and severe stages of
muscular disorder [40, 76]. The results in Table 2 show that ATP-dependent
Ca-binding activities (studied in the absence of oxalate) of SR from
skeletal muscles of myopathic animals were not altered at both moderate and
severe stages. Although no changes in the ATP-dependent Ca-uptake
activities (studied in the presence of oxalate) of SR of myopathic muscle were
detected at moderate stage, these Ca-transport activities were depressed
in both models of myopathy at severe stages. It may also be noted from Table 2
that the SR Ca-pump ATPase activity was decreased markedly at severe
stages of myopathy. Several investigators have also reported defects in the
Ca-transport activities in the SR preparations from different myopathic
animals as well as in DMD patients [156, 157, 158, 159]. However, others have denied the
occurrence of such abnormalities in muscular disorder [160, 161, 162], which may be
due to the use of skeletal muscles at early or moderate stage of the disease.
Thus, it appears that the reduced ability of SR to accumulate Ca at the
late stage of muscular disease may be secondary to other mechanisms leading to
impaired performance of skeletal muscle.
Table 2.Ca-transport activities of sarcolplasmic reticulum from
skeletal muscles of control and myopathic hamsters of different ages.
|
|
|
ATP-dependent Ca- binding (nmoles/mg protein/5 min) |
ATP-dependent Ca- uptake (µmoles/mg protein/5 min) |
Ca-pump ATPase (µM Pi/mg protein/5 min) |
A. BIO 14.6 myopathic hamsters |
|
|
|
|
(i) 220 days old |
|
|
|
|
|
Control |
153 22 |
3.6 0.20 |
– |
|
|
Myopathic (Moderate stage) |
136 11 |
3.4 0.15 |
– |
|
(ii) 260 days old |
|
|
|
|
|
Control |
142 5 |
3.8 0.45 |
2.76 0.31 |
|
|
Myopathic (Late stage) |
135 7 |
2.3 0.62* |
1.32 0.19* |
B. UM-X7.1 myopathic hamsters |
|
|
|
|
(i) 60 days old |
|
|
|
|
|
Control |
118 13 |
3.8 0.52 |
1.44 0.15 |
|
|
Myopathic (Moderate stage) |
95 11 |
3.6 0.21 |
1.07 0.19 |
|
(ii) 150 days old |
|
|
|
|
|
Control |
145 12 |
5.2 0.39 |
2.56 0.27 |
|
|
Myopathic (Late stage) |
109 14 |
2.8 0.26* |
1.45 0.21* |
The data for BIO 14.6 strain of myopathic hamsters are taken from our paper,
Dhalla & Sulakhe. Biochem Med 7, 157–168, 1973 [40] whereas the data for
UM-X7.1 myopathic hamsters are taken from our paper Dhalla et al. Clin
Sci Mol Med 49, 359–368, 1975 [76]. * Significantly (P 0.05) different from the corresponding control
values. |
The data in Table 3 show the status of mitochondrial Ca-binding and
Ca-uptake activities as well as oxidative phosphorylation in UM–X7.1
strain of hamsters at moderate or severe stages of myopathy [76]. No changes in
mitochondrial Ca-accumulation and ATPase activities as well as different
parameters of oxidative phosphorylation were observed in 150 days old myopathic
animals. On the other hand, 60 days old hamster myopathic muscle showed
depressions in both mitochondrial Ca-uptake and oxidative phosphorylation
rate, unlike Ca-binding and ATPase, P:O ratios and RCI values (Table 3).
The inability to detect changes in mitochondrial Ca-transport and
oxidative phosphorylation in myopathic muscle at late stages of the disease in
UM–X7.1 strain of hamsters was not attributed to the method used for the
isolation of mitochondria because the same procedure was employed for obtaining
mitochondrial preparation from muscles at moderate stage of muscular disease.
Since the mitochondrial defects in myopathic muscle have also been shown to be
due to the occurrence of mitochondrial Ca-overload [41], it is likely that
the excessive amount of Ca from mitochondria at late stages of myopathy
may have been lost during the isolation procedure. Nonetheless, other
investigators have shown defects in mitochondrial oxidative phosphorylation
activities in myopathic muscles of BIO 14.6 strain of myopathic hamsters
[94, 95, 96]. Such abnormalities in the mitochondrial oxidative phosphorylation can
be seen to impair the ability of myopathic muscle to generate energy for the
function of skeletal muscle myocytes. In fact, marked changes in the high energy
phosphate content and other metabolic processes showing impaired energy
production in skeletal muscles from BIO 14.6 strain of hamsters at late stages of
muscular disease have been reported [30]. Some of these data shown in Table 4
indicate that both creatine phosphate and ATP were depressed without any
significant changes in ADP and AMP content of dystrophic hamster muscle.
Furthermore, lactate, NADH and NADPH were increased indicating marked alterations
in muscle metabolism without any changes in pyruvate content of myopathic muscle
(Table 4). These observations support the view that reduction in the high energy
phosphate stores due to impaired process of energy production may play an
important role in dysfunction of skeletal muscle in muscular disorder.
Table 3.Mitochondrial Ca-transport and oxidative phosphorylation
activities of skeletal muscles from UM-X 7.1 strain of myopathic hamsters of
different ages.
|
60 days old |
150 days old |
|
Control |
Myopathic (Moderate stage) |
Control |
Myopathic (Late stage) |
Ca- binding (nmoles/5 min/mg) |
84 7 |
79 5 |
116 14 |
113 11 |
Ca- uptakes (nmoles/5 min/mg) |
552 64 |
353 58* |
596 73 |
571 49 |
ATPase activity (µmol/5 min/mg) |
3.9 0.63 |
4.3 0.71 |
4.5 0.96 |
5.2 1.08 |
P:O ratio |
2.8 0.07 |
2.9 0.05 |
2.9 0.04 |
3.0 0.06 |
Phosphorylation rate (µmol ADP phosphorylated/min/g protein/min) |
110 8.8 |
81 5.6* |
127 19.4 |
112 26.0 |
RCI |
5.3 0.52 |
6.3 0.21 |
8.1 1.4 |
7.6 1.1 |
The data are taken from our paper, Dhalla et al. Clin Sci Mol Med 49,
359–368, 1975 [76]. * Significantly (P 0.05) different from the corresponding control
values. |
Table 4.High energy phosphate stores, glycolytic intermediates and
pyridine nucleotides in skeletal muscle of 215 day old BIO 14.6 strain of
myopathic hamsters.
|
|
Control |
Myopathic (Late stage) |
(i) Creatine phosphate (µmol/g muscle) |
13.3 0.26 |
6.13 0.49* |
(ii) Adenine nucleotides (µmol/g muscle) |
|
|
|
ATP |
5.97 0.21 |
4.04 0.27* |
|
ADP |
0.35 0.04 |
0.39 0.02 |
|
AMP |
0.17 0.02 |
0.21 0.02 |
(iii) Glycolytic intermediates (µmol/g muscle) |
|
|
|
Lactate |
1.19 0.02 |
7.18 0.14* |
|
Pyruvate |
0.050 0.002 |
0.051 0.003 |
(iv) Pyridine nucleotides (mµmol/g muscle) |
|
|
|
NADH |
139 5.0 |
168 3.1* |
|
NADPH |
45 3.1 |
67 5.7* |
The data are taken from our paper, Dhalla et al. Can J Biochem 50,
550–556, 1972 [30]. * Significantly (P 0.05) different from the corresponding control
values. |
4. Role of dystrophin deficiency in muscular dystrophy
It is now well known that stability of the cell membrane is very critical for
appropriate function of skeletal muscle and thus any defect in the plasma
membrane can be seen to induce muscular abnormality [14, 15, 23, 25]. Since
dystrophin based cytoskeleton has been shown to anchor various proteins and other
constituents of the SL membrane, dystrophin deficiency has been associated with
the development of muscular dystrophy [14, 15, 20, 46, 73, 163]. Advances in
molecular biology techniques in late 1980’s have facilitated the discovery of
mutations in dystrophin gene on the chromosome region Xp21, which is the largest
known gene, responsible for the expression of dystrophin protein [164, 165]. This
research was started with the elucidations of 2300 kb dystrophin gene, which
consists of 79 exons and encodes dystrophin protein. Various mutations in this
gene identified thus far, have been shown to reduce the level of dystrophin
protein and lead to the development of muscular dystrophy as well as
cardiomyopathy, respiratory dysfunction and mental retardation [166]. It is also
pointed out that the SL dystrophin protein has been reported to occur as a
dystrophin-glycoprotein complex consisting of dystroglycans, sarcoglycans,
sarcospan, dystrobrevins and syntrophins [167, 168]. Furthermore, this
devastating inherited muscular disorder is considered to be caused by mutations
in dystrophin gene and is known to affect mainly males due to X-linked recessive
mode of inheritance; females are carriers of these gene mutations [169].
A deficiency of dystrophin protein plays a significant role in the pathology of
muscular dystrophy, and in this regard, it is pointed out that more than 1000
mutations have been identified in dystrophin gene. As a part of an incredibly
complex group of proteins, dystrophin allows muscle to function properly as well
as aids in anchoring various components within muscle cells and link them to the
SL membrane [167]. Thus, dystrophin provides a scaffold for holding several
molecules in place near the cell membrane whereas dystrophin deficiency
dislocates these molecules and cause disorders in their function [167]. There are
several reports on structural, functional, biochemical, molecular and metabolic
defects, which are induced by these dystrophin gene mutations [13, 29, 73, 170, 171]. Reduction of dystrophin-glycoprotein complex promotes the occurrence of
structurally unstable SL membrane, which is more permeable to extracellular
environment and thus contribute to muscle fiber damage and wastage of skeletal
muscles [170, 171]. Particularly, repetitive cycles of contraction and relaxation
of dystrophin deficient skeletal muscle produce microtears in the SL membrane,
and result in cellular instability and progressive leakage of intracellular
components including CK. Such a leakage of CK can be seen to reduce the
intracellular level of CK content and thus may impair the storage of energy in
skeletal muscle. In addition, the increased permeability of the SL membrane due
to dystrophin deficiency will promote an excessive entry of Ca into
myocytes to result in the development of intracellular Ca-overload, which
is well known to activate different proteolytic enzymes and produce muscle
wastage. Furthermore, dystrophin deficiency has been reported to induce different
abnormalities in the SL signal transduction pathways, which impair muscle
regeneration and induce weakness in muscle performance [171]. A schematic
representation of dystrophin deficiency related events is shown in Fig. 2.
Fig. 2.
A schematic representation of dystrophin deficiency related
events.
It needs to be emphasized that mutations of genes other than for dystrophin gene have also been reported to induce muscular disorders. For
example, mutations in dysferlin gene have been shown to decrease dysferlin
content and result in progressive development of muscle wasting and myopathic
characteristics [172]. Although deficiency of laminin-alpha2, a protein of the
extracellular matrix and a component of the basal membrane, has also been
reported to induce the SL instability and result in the development of muscular
disorder [23, 24], dystrophin mutations have been recognized as the primary cause
in the development of muscular dystrophy because of a significant decrease in the
expression of dystrophin associated complex proteins. In fact, changes in
dystrophin associated proteins such as sarcoglycan complex as transmembrane
proteins have been shown to promote the production of oxidative stress and lead
to muscle cell death as well as stiffness in myopathic muscle [171]. It should be
mentioned that neuronal nitric oxide synthase, which interacts indirectly with
dystrophin protein, is also influenced markedly due to dystrophin deficiency and
thus affects the performance of dystrophic muscle [173]. In addition, some
studies have indicated that the membrane impairment occurs frequently in the
absence of dystrophin, leading to localized cell damage and leakage of
intracellular constituents [174]. However, a significant increase in total
Ca content is consistent with excessive Ca entry into the cell
through damaged membranes due to dystrophin deficiency. Although other pathways
of Ca entry such as Na-Ca exchange [155] and Ca-gating
system (Ca/Mg- ecto-ATPase) [153] may also be enhanced in muscular
dystrophy as a consequence of dystrophin deficiency, extensive experiments need
to be carried out to make any meaningful conclusion. Nonetheless, it is evident
that dystrophin deficiency may play a critical role in the pathogenesis of
muscular dystrophy due to the occurrence of intracellular Ca-overload
[171, 173, 175].
5. Role of store-operated Ca entry in muscular dystrophy
The store-operated Ca entry (SOCE) is one of the important mechanism for
extracellular Ca influx and is activated by depletion of intracellular
Ca stores [176, 177]. Several studies have been carried out to explore the
status of SOCE regulation in normal and dystrophic skeletal muscles [48, 178, 179]. In fact, SOCE it was found to play an essential role in some physiological
functions including skeletal muscle development, contractile activity and
metabolism. Furthermore, it prevents muscle weakness and serves as a signaling
pool of Ca required to modulate muscle-specific gene expression in
myopathic muscle [180, 181, 182, 183]. Abnormal increases in the SOCE channel activity
result in muscle dysfunction including the activation of Ca signaling
pathways leading to metabolic disorders, irregular protein handling, and
detrimental remodeling phenotype in the pathogenesis of muscular disease [179, 180, 184, 185, 186]. The disruption of SOCE channels has been suggested to cause an
imbalance in the level of intracellular Ca and subsequent
Ca-dependent activation of proteases and muscle degeneration due to
dystrophin deficiency in dystrophic muscle [40, 187, 188]. It has been reported
that the occurrence of an excessive SOCE occurrence is an early event in the
pathology of muscular dystrophy and the altered Ca dynamics in myotubes
result in continued cytosolic Ca transients and increased Ca uptake
by mitochondria [187, 189]. The phospholipase A product,
lysophosphatidylcholine, was found to trigger Ca entry through SL
channels, and acts as an intracellular messenger responsible for the opening of
store-operated channels in dystrophic fibers [190]. Elevation of intracellular
Ca was found to increase phospholipase A2 activity and the overexpression
activity of NADPH oxidase and excessive production of reactive oxygen species
(ROS) were observed to contribute to the pathology in dystrophin deficient
dystrophic muscle [191, 192].
Both STIM1 and Orai1 proteins have been identified as essential
components of SOCE channel in the plasma membrane [193, 194]. Studies from
genetic mouse models with deletion of these components have provided evidence of
impaired skeletal muscle growth, suggesting the physiologic role for SOCE in
skeletal muscle development and contractile function [195, 196, 197, 198, 199]. These molecular
components which skeletal muscle expresses in abundance serve as the main channel
constituents involving SOCE for contributing to the mitochondrial Ca
homeostasis and a range of downstream signaling pathways as well as in the
regulation of several transcription factors [144, 200, 201, 202, 203, 204]. STIM1 is a
multipurpose stress transducer initiated by various stimuli such as oxidation,
temperature, hypoxia, and acidification and may regulate varied downstream
targets including different ion channels, pumps/exchangers, adaptor proteins,
endoplasmic reticulum (ER) chaperones, signaling enzymes, and ER
stress/remodeling proteins. On the other hand, Orai1 serves as a SOCE channel in
skeletal muscle [205]. Furthermore, fast kinetics of the SOCE activation in the
skeletal muscle, depends on the pre-formation of STIM1-protein complexes with the
plasma-membrane, whereas Orai1-mediated Ca influx appears essential to
control the resting Caconcentration and proper SR Ca filling.
Ca influx through STIM1-dependent activation of SOCE from the T-tubule
system may recycle the extracellular Ca loss during muscle stimulation and
thereby maintain proper filling of the SR Ca stores and muscle function
[206, 207]. Various studies have demonstrated that STIM1/Orai1-dependent signals
promote muscle fiber maturation, growth, oxidative process, fatigue-resistant
fibers, and muscle development [195, 207, 208, 209]. There is evidence to suggest that
the STIM1/Orai1-dependent SOCE promotes sustained force generation during periods
of prolonged activity as well as resistance to muscle fatigue [210, 211, 212, 213].
Altered function of essential proteins regulating the SOCE activity, contributes
to or amplifies the pathogenesis of muscle disorders including muscular
dystrophy. A number of studies have provided evidence for a modulatory
contribution of the STIM/Orai1-dependent SOCE in animal models of muscular
dystrophy [178, 214, 215, 216]. Increased STIM1/Orai1 expression as well as SOCE
functionality, including a shift in the threshold for SOCE activation and
deactivation to SR luminal Ca concentrations have been observed in muscle
fibers from dystrophic mice [178, 217]. In another study, although STIM1 levels
were unchanged in muscles from dystrophic mice, an increase was found in both
Orai1 mRNA and protein levels corresponding to the enhanced SOCE activity and SR
Ca storage. Since these augmented activities were reduced by either
shRNA-mediated Orai1 knockdown or treatment of animals with BTP-2 (a potent CRAC
channel inhibitor), it was proposed that increased function of the
STIM1/Orai1dependent SOCE contributes to Ca-mediated muscle fiber
degeneration in dystrophic mice [218]. Enhanced SOCE and increased muscle
inflammation, fibrosis, necrosis, mitochondrial swelling, and serum CK levels,
were noticed due to the muscle-specific STIM1 overexpression in dystrophic mice
[214]. The role of STIM1-mediated Ca signaling for skeletal muscle
hypertrophic growth has also been demonstrated in the skeletal muscle STIM1 knockout mice [195]. Furthermore, a correlation of the overstimulation of SOCE
in dystrophic cells with increased STIM protein content has been reported [188, 207, 208]. Since overexpression of STIM1 stimulates muscle cell differentiation
whereas silencing inhibits this process [208], it is evident that high SOCE and
elevated STIM1 levels in muscular dystrophy are associated with greater
differentiation of dystrophic myoblasts [198].
Increased rate of SOCE activity with high STIM1 protein levels in dystrophic
mice myoblasts has revealed that mutation of dystrophin gene may significantly
impact the cellular calcium response to metabotropic stimulation. It is pointed
out that an aberrant response to extracellular stimuli may contribute to the
pathogenesis of muscular dystrophy and inhibition of such responses might modify
the progression of this deadly disease [188]. Overactivation of SOCE upon
dystrophic cell stimulation may lead to intracellular Ca-overload and
increased susceptibility to cell death as well as progressive muscle degeneration
[219]. It has been demonstrated that Ca-influx across an unstable SL due
to increased activity of the STIM1/Orai1 complex is a major determinant in
muscular dystrophy [214] and STIM1/Orai1 along with TRPC1 are involved in
increasing SOCE in the dystrophin deficient myotubes in both dystrophic patients
and dystrophic mice. Thus, the participation of a specific Ca/PKC/PLC
pathway in increasing the SOCE activity may be due to STIM1/Orai1/TRPC1 protein
interactions, which are regulated by the dystrophin scaffold [215]. These
findings support the functional role of STIM1/Orai1-dependent SOCE in the
pathophysiology of muscular dystrophy and are considered to represent a potential
therapeutic target [216].
6. Mechanisms of subcellular Ca-handling abnormalities in
dystrophic muscle
An excessive amount of Ca entering dystrophic muscle is considered to
play a critical role in raising the intracellular level of Ca as well as
inducing metabolic defects, subcellular remodeling, Ca-handling
abnormalities and impairment of muscular function [15, 20, 25, 47, 155, 171, 180, 185, 217]. Increases in Ca-entry and SL permeability have been shown to be
associated with dystrophin deficiency in muscular dystrophy; such defects in some
other types of this disease are linked to deficiencies of different proteins
including dysferlin and -sarcoglycan [47, 171, 172, 180, 185, 220]. The
role of intracellular Ca-overload in the pathogenesis of muscular disorder
is supported by observations that exercise in dystrophic mdx mice enhanced
Ca-influx, impaired Ca-homeostasis and aggravated this disease [221]. It should be pointed out that the increased Ca-influx in myopathic
muscle may be occurring through SOCE channels [192, 216, 222],
voltage-independent Ca-leakage channels [223], voltage-dependent
Ca-channels [189] and Na/Ca exchange system [154, 155, 224, 225] in the SL membrane. Since the SL phospholipase A[190] and adenylate
cyclase activities [226] are increased in dystrophic muscles, these signal
transduction systems have also been suggested to participate in enhancing
Ca-entry into muscle fiber. The involvement purinoceptor associated
mechanisms has also been reported to induce increased Ca-influx because
the expressions and activities of P2X receptors are increased in dystrophic
muscle [227, 228, 229]. The increased activities of both Mg-ATPase and
Ca-ATPase in the surface membrane of dystrophic muscle were decreased with
age of the animal [230]. Likewise, some investigators have shown an increase
[231] whereas others have observed a decrease in the Na-K ATPase
activity in dystrophic muscle [232]. Such variable changes in the SL enzyme
activities may be due to the type or age of dystrophic animals. In fact, a
variety of changes in other SL components have been identified in dystrophic
muscle [233, 234]. Taken together, all these observations support the view that
increased Ca-influx through the plasma membrane of dystrophic muscle may
be a consequence of SL remodeling during the development of muscular dystrophy.
The impaired function of dystrophic muscle is generally considered to be due to
Ca-handling abnormalities in myocytes. Since the SR is intimately involved
in maintaining the intracellular Ca-homeostasis, different defects in this
intercellular organelle have been identified in muscular dystrophy. Marked
alterations in the structure and function of Ca-release channel or
ryanodine receptor in the SR have been demonstrated in dystrophic muscle
[235, 236, 237]. A progressive increase in the expression of ryanodine receptor and
ryanodine receptor binding in the SR has been shown to occur during the
development of muscular dystrophy [238]. The leaky ryanodine receptors in
dystrophic muscle have been indicated to limit the activation of SOCE channels
and produce changes in Ca-homeostasis [239]. It should also be noted that
drastic reductions in both sarcalumenin and calsequestrin have been reported in
dystrophic muscle to depress Ca-binding in the lumen of SR and are
considered to play a role in abnormal Ca-handling in muscular dystrophy
[240, 241]. Some investigators have shown marked depressions in the SR
ATP-dependent Ca-uptake and Ca-pump ATPase activities in dystrophic
muscle [242, 243, 244, 245], whereas others have failed to observe such defects [246, 247, 248].
Such conflicting results may be due to the stage or type of muscular dystrophy.
However, the depression in the Ca-transport in the SR can be seen to occur
because of the increased expression of sarcolipin, a known endogenous inhibitor
of Ca-pump ATPase, in dystrophic muscle [249, 250, 251]. Although calmodulin
mRNA and content in dystrophic muscle are increased, the stimulation of the SR
Ca-pump ATPase by calmodulin is markedly depressed [252]. Thus, a
reduction of Ca-transport in the SR may also contribute to eliciting
Ca-handling abnormalities in dystrophic muscle. In this regard, it is
noteworthy that an overexpression of the SR Ca-pump ATPase has been
demonstrated to alter intracellular Ca levels in dystrophic muscle and
mitigate muscular dystrophy [181].
The development of Ca-handling abnormalities in dystrophic muscle has
been considered to affect other subcellular organelles such as mitochondria and
myofibrils. In fact, mitochondrial dysfunction has been shown to occur before the
onset of myofiber necrosis, muscle wasting and myofibrillar defects [253].
Impaired substrate utilization and ATP synthesis have been reported in dystrophic
mitochondria [254, 255]. There occurs an uncoupling in the process of
mitochondrial oxidative phosphorylation during the progression of muscular
dystrophy [256, 257]. Although the development of mitochondrial
Ca-overload is considered to induce defects in energy production in
dystrophic muscle [41], abnormalities in ATP synthesis by dystrophic mitochondria
have also been shown to be caused by complex I insufficiency [258], disruption of
mitochondrial protein Mss51 [259] and decreased CK content [260]. It is pointed
out that the impaired performance of dystrophic muscle is not only a consequence
of depressed energy stores but defects in the process of energy utilization have
also been observed during the development of muscular dystrophy. In this regard,
myofibrillar Ca-stimulated ATPase and myosin ATPase activities have been
shown to be decreased in dystrophic muscle [261, 262]. A shift in myosin heavy
chain from alpha to beta isoform as well as changes in troponin-tropomyosin have
been reported to cause alterations in dystrophic myofibrillar ATPase activities
[263, 264]. Increased Ca-activated protease activity in dystrophic muscle
has also been shown to induce myofibrillar defects [100, 101, 265, 266].
It is becoming clear that Ca-handling abnormalities in dystrophic muscle
are associated with various defects in the subcellular organelles. Such
alterations have been suggested to be caused by abnormalities of lipid metabolism
in dystrophic muscle [78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 267, 268]. It may be mentioned that marked changes
inn cholesterol, triglycerides, unsaturated fatty acids and phospholipid content
have been observed in dystrophic muscle [269, 270, 271, 272]. Furthermore, increased
oxidative stress in dystrophic muscle [172, 191, 273, 274] has been suggested to
account for inducing remodeling and Ca-handling abnormalities. It is
pointed out that the expression of NADPH oxidase, which generates ROS, is
markedly increased in addition to mitochondrial oxyradical production in
dystrophic muscle [275, 276]. Furthermore, synergistic interactions of
nitrosative/oxidative stresses due to increased neural nitric oxide synthase in
dystrophic muscle [277, 278], have been indicated to be involved in
Ca-handling abnormalities during the development of muscular dystrophy.
7. Strategies for the treatment of muscular disorder
Corticosteroids therapy was reported for the first time to delay the progression
of muscular disorder and was considered as the gold standard for its treatment
[279]. Because almost all types of muscular dystrophies are caused by a
single-gene mutation [280], gene therapy was also introduced as a potential
intervention to replace, repair or bypass the mutated gene [281, 281, 282, 283]. In order
to restore an appropriate level of dystrophin in skeletal muscle, different gene
strategies have been based on implantation and delivery of naked plasmid DNA for
the dystrophin gene [284, 285, 286, 287, 288], by adeno-associated virus (AAV) [289] as well as
exon-skipping with antisense oligonucleotides [290, 291, 292]. Due to challenges like
targeted delivery of therapeutic molecules, lysosomal setup, enzymatic
degradation as well as low intracellular uptake, successful working with gene
therapy has stimulated the search for different drug delivery systems such as
polymersomes [293, 294], liposomes and lipid-nucleic acid complexes [295, 296, 297],
PMMA nanoparticles [296, 298], and cell-penetrating peptides [290, 298, 299].
While a great progress has been made with gene therapy [12, 300], other
interventions such as injections of myoblasts (derived from healthy donors) did
not induce beneficial effects due to low survivability, migration and immune
rejection of the transplanted cells; thus stem cells based therapies have been
suggested for the treatment of diverse muscular disorders [301, 302].
Furthermore, various molecular pathways have been shown to be most befitting for
the development of novel medicinal products to prevent muscle degeneration and
fibrosis. The genetic modifiers including LTBP4, Jagged1 and osteopontin, which
regulate the disease progression by interfering with pro-fibrotic and
pro-regenerative pathways (TGF-, myostatin and Notch signaling), have
also offered a platform to identify novel pharmacological targets for the therapy
of muscular weakness [303, 304]. Thus, several approaches (Fig. 3) are considered
worthwhile for not only preventing the progression of this disorder but also the
improvement of dystrophic muscle performance.
Fig. 3.
Current strategies for the treatment of muscular dystrophy.
It is noteworthy that, recent data have shown better efficiency of gene therapy
when high doses are administered [287]. Although high doses are fairly tolerated
and achieve adequate transgene expression, these lack adequate balance of safety
and efficacy for the success of gene therapy [305]. Clinical trials have shown a
slight risk of liver toxicity represented by transient elevation in liver enzymes
and bilirubin as side effects in humans subjected to such a gene therapy.
Furthermore, the major obstacle for gene therapy is the pre-existing immune
reaction to AAV, which is used as vehicle; but the use of plasmapheresis was
found to evade the immune reaction to AAV and this was considered to allow safe
administration of gene therapy to patients with impaired muscular function [306].
Although, there is some optimism for utilizing plasmapheresis or T-cell
suppression by agents such as rituximab and rapamycin to overcome the
pre-existing immunity, the success of these approaches remains to be clearly
demonstrated [307]. One of the shortcomings for the use of AAV is its limited
packaging capacity for dystrophin molecule, which necessitated the development of
a shorter protein, mini-dystrophin [308]. The construction of mini-dystrophin was
based on the shortened dystrophin protein as expressed in dystrophic muscle and
found to be highly functional [309]. Although mini-dystrophin gene therapy was
shown to improve the systemic muscle function at early stages, it was only
effective partially in advanced cases of muscular disease [308]. Such
observations suggest that restoring muscle function to near-normal levels with
gene therapy will probably necessitate additional research to improve and enhance
the muscle strength in dystrophic muscle.
Since the intricacy of numerous mutations of dystrophin gene causes several
challenges for the dystrophin gene therapy, a great deal of experimental work is
needed for having an effective treatment of muscular disorder. Furthermore, it
should be recognized that deficiency of laminin—alpha 2 protein has also
been reported in dystrophic muscle and this was shown to be attenuated by
omigapil, an antiplatelet agent [23]. Thus, it is likely that some combination
gene therapy may prove more beneficial in delaying the course of this disease
progression. It is also pointed out that pathological lesions in dystrophic
muscle were found to be associated with marked increase in Ca content but
these changes were not prevented with verapamil treatment, a well known
antagonist of voltage sensitive Ca-channels in the SL membrane [147]. On
the other hand, reduction of Ca-influx through SOCE channels was found to
improve function in diseased muscle [185, 189]. Reintroduction of mini-dystrophin
in dystrophic muscle was also reported to reduce Ca transients as a
consequence of its effect on SOCE channels [192]. In addition, inhibition of
phospholipase A as well as lysophosphatidylcholine production was shown to
depress Ca-entry and prevent the degeneration of dystrophic muscle [190].
It should be mentioned that diapocynin, an inhibitor of NADPH oxidase, which
reduces the production of ROS, was observed to depress the phospholipase A
activity as well as Ca-influx through SOCE channels in dystrophic muscle.
Accordingly, it is suggested that gene therapy in combination with some
inhibitors of Ca-entry and oxidative stress may be more effective for the
treatment of muscular disorder.
8. Conclusions and future perspectives
From the foregoing discussion, it is evident that muscular dystrophy is a group
of complex diseases, which results in skeletal muscle degeneration, loss of
muscle fibers and impaired muscle function. Some forms of muscular dystrophy are
considered to be a consequence of a genetic defect leading to deficiency of
dystrophin for inducing abnormalities in the SL membrane. The progression of this
disease is associated with leakage of intracellular enzymes and other
constituents, maldistribution of electrolyte content, marked metabolic
alterations and development of necrosis, apoptosis as well as fibrosis in
dystrophic skeletal muscle. The depression in the high energy stores as a
consequence of the mitochondrial Ca-overload, and abnormalities in the SR
due to defects in Ca-release channel and Ca-pump ATPase are
considered to explain the impaired muscle performance. On the other hand, the
activation of different proteolytic enzymes due to the occurrence of
intracellular Ca-overload is responsible for dystrophic muscle
degeneration and wastage. Some studies concerning defects in the SL membrane and
other subcellular organelles as well as metabolic status of skeletal muscle have
been conducted by employing a myopathic hamster model of muscular weakness. Other
studies by using genetic mouse and chicken models of muscular dystrophy indicate
that dystrophin deficiency induce the activation of SOCE channels,
Na-Ca exhanger (forward mode) and Ca/Mg ATPase
(Ca-gating system) in the SL memebrane and promote the occurrence of
intracellular Ca and subsequent abnormalities in dystrophic muscle. A
schematic representation of some main events associated with the development as
well as consequence of intracellular Ca-overload due to
dystrophin-deficiency is shown in Fig. 4. Various, treatments including gene
therapy with dystrophin and some pharmocological interventions such as
antioxidants are being attempted for delaying the progression of this disease as
well as improving the performance of dystrohpic muscle. It is suggested that a
combination therapy, by emplying dystrophin gene and some drugs, which
reduce the development of intracellular Ca-overload, may prove more
beneficial for the treatment of impaired muscular performance.
Fig. 4.
A schematic representation of some main events associated with
the development as well as consequence of intracellular Ca-overload in
dystrophic muscle.
Author contributions
NSD conceived, designed and edited the article; SKB searched the literature,
analyzed the data and wrote the first draft; AKS and MN analyzed the data and
wrote the manuscript.
Ethics approval and consent to participate
Not applicable.
Acknowledgment
The authors wish to thank Ms. Andrea Opsima for typing the manuscript.
Funding
There was no grant funding except the infrastructure support for this project
was provided by the St. Boniface Hospital Research Foundation, Winnipeg, Canada.
Conflict of interest
The authors declare no conflict of interest.