Muscular dystrophy: basic facts

- heterogenous group of inherited disorders characterized by progressive

muscle weakness and wasting (regeneration of muscle tissue fails)
- most apparent or symptomatic in skeletal muscle but heart and
diaphragm muscle often involved (most patients die of heart failure
or respiratory problems)
- incidence: 1 in 10,000 (worldwide)

Two groups of muscular dystrophy:

1) Duchenne (DMD) and Becker (BMD)
- involve mutations in the dystrophin gene
- X-linked inheritance
- defects in intracellular muscle cell proteins
- the dystrophin is missing in Duchenne, reduced expression
or partially functional dystrophin in Becker
2) Congenital (MEB, FCMD, LGMD, CMD)
- involve mutations in several genes
- autosomal recessive inheritance
- defects in extracellular molecules, i.e components of the
extracellular matrix

Clinical features
- Duchenne: age of onset (4-6 years)
- severe, progressive muscle degeneration
- loss of ability to walk by age 9-12
- death by 14-20 of respiratory failure/cardiomyopathy
- Becker: age of onset (after 16)
- milder form than Duchenne; muscle pain, dilated cardiomyopathy
- Limb-Girdle (LGMD)
- similar to DMD and BMD, involves primarily shoulder and pelvic
girdle muscles
- Muscle-Eye-Brain (MEB) and Fukuyama (FCMD)
- most severe forms of MD
- hypotonia, neurological involvement

Overview of muscle development and function

- new skeletal muscle fibers form by fusion of myoblasts
myoblasts will continue to proliferate as long as certain growth factors
such as FGF or HGF are present
once the growth factors are removed, the myoblasts rapidly stop
dividing, differentiate, and eventually fuse to form fibers
these muscle fibers contain intracellular and extracellular components
that are responsible for providing support to the fibers during contraction
processes

Fusion of myoblasts in cell culture

cells fuse and contain multiple nuclei

blue color (DAPI) - nuclear staining

green color (myosin) - marker of differentiated myoblasts

Satellite cells are myoblasts stored near mature muscle fibers

(stem cells of adult skeletal muscles)
- when muscle is damaged, these cells are activated to proliferate
and their progeny can fuse to repair damaged muscle

How do muscles handle the stress applied to them?

muscles generate large forces between their fibers during contraction
and relaxation (weightlifting, etc)
complexes of proteins at the muscle membrane or sarcolemma are
necessary to transmit force of contraction to connective tissue and
tendons

Campbell and Ozawa made the observation that a large number of

muscle membrane glycoproteins co-purifies with dystrophin

Dystroglycan-glycoprotein complex

main function: to provide structural stability to muscle cell membrane

during cycles of contraction and relaxation

Dystrophin
- cytoskeletal protein localized to the inner surface of the
muscle membrane
- part of a complex with multiple proteins including
sarcoglycans and dystroglycans (binds to -dystroglycan
and F-actin)
- loss of dystrophin results in the destabilization of the
entire dystroglycan-glycoprotein complex

Sarcoglycans
- group of four muscle-specific integral membrane proteins;
function is still unclear (bind to dystrobrevin and related
protein, sarcospan)
- mutations in sarcoglycans lead to forms of LGMD (limbgirdle muscular dystrophy)
- loss of sarcoglycans at the muscle membrane leads to
variable destabilization of the DGC

Dystrobrevin and syntrophins

- intracellular proteins that associate with C terminus of
dystrophin
- dystrobrevin likely acts with syntrophins to recruit signaling
proteins to the DGC (nNOS: nitric oxide synthase)
- no pathogenic mutations found yet

Dysbindin
- binds to dystrobrevin and is associated with the DGC
in muscle
- in the brain, dysbindin is found in axon bundles and axon
terminals of the hippocampus and cerebellum
- mutations in dysbindin are associated with greater risk of
schizophrenia (likely indicates a second function of dysbindin
independent of the DGC)

Dystroglycan
- central protein in the DGC; provides the link between the
cytoskeleton and the basal lamina (ECM)

- contains two subunits:

alpha-dystroglycan:
> completely extracellular
> heavily O-glycosylated in its mucin region
> binds to laminin 2 and other extacellular matrix proteins
with laminin-like domains

beta-dystroglycan:
> transmembrane protein
> binds to dystrophin

no mutations in the dystroglycan gene have been found

Fate of DGC components in various forms of muscular dystrophy

loss of dystrophin = DGC destabilization

loss of sarcoglycans = variable DGC loss

merosin = laminin2 (DGC intact)

How is muscle damaged when DGC components are missing?

no universal agreement on which mechanism is predominant

theories for muscle fiber necrosis:

- mechanical hypothesis: loss of DGC leads to contraction-induced rupture
of muscle cell membranes; noted by cytoplasmic accumulation of serum proteins
in muscle fibers
(exercise in DMD patients likely causes greater damage than in controls)
- calcium hypothesis: influx of calcium into cytosol overwhelms muscle cells ability
to maintain physiologic Ca++ levels which causes programmed cell death via
activation of proteases such as calpains
(overexpression of calpastatin, an endogenous inhibitor of calpains, has been
demonstrated to reduce necrosis in mdx mice)

- gene regulation hypothesis: failure of certain molecules to be localized to the

muscle membrane when DGC components are absent prevents proper signaling
molecules from being recruited
- vascular hypothesis: NO produced in muscle cells by the neuronal form of NO
synthase, nNOS, that is normally tethered to DGC by dystrobrevin and syntrophins;
- in DMD muscle, nNOS becomes delocalized into the cytosol, reducing its stability;
during exercise, the need for oxygen is increased but loss of NO, a vasodilator, can
lead to muscle ischemia (local anemia due to vasoconstriction)
- nNOS knockout mice do not have muscle disease so nNOS may play a direct role

Muscle pathology of muscular dystrophy

satellite cells respond to the damage of muscle cells caused
by loss of dystrophin and other components of the DGC
- regenerative response can not keep pace with the damage;
satellite cells have a limited capacity to divide due to progressive
shortening of their telomeres
- muscle cells are then replaced by connective tissue and fibroblasts;
this prevents further repair (similar to what happens in the elderly)

theories for tissue remodeling:

- inflammatory hypothesis: muscles in DMD patients exhibit coordinated activity of
numerous components of a chronic inflammatory response
- cytokine and chemokine signaling, leukocyte adhesion and complement activation,
in vivo depletion of CD4+ and CD8+ T cells or macrophages reduces pathology in
mdx mice, suggesting that these cells aggravate disease process)
- inflammation appears to cause local overexpression of extracellular matrix genes
in DMD muscle and can contribute to fibrosis
- mdx muscle does not exhibit any fibrosis, indicating that collagen regulation at posttranscriptional stages medaites the extensive fibrosis in human DMD patients

Do Duchenne MD boys have central nervous system impairment

as well as skeletal muscle weakness?
- lower IQ and cognitive impairment
- evidence of disordered CNS architecture, abnormalities in dendrites
and loss of neurons
- not clear why effects on brain arent as drastic as some of the
congenital muscular dystrophies

Therapeutic approaches for muscular dystrophy

- gene therapy represents a major area of research in the muscular
dystrophy field

promising:
nearly all types of muscular dystrophy arise from single-gene mutations
(one target)

challenging:
efficient delivery of the new gene to most of the striated muscle in the
body (>40% of body mass)
design of viral vectors to carry the large genes to be replaced (dystrophin
gene, for example, is 2.4 Mb in size)
muscle transduction with viruses must not trigger toxic or immunological
reactions that can further damage the weakened muscle
tight packing of muscles makes delivery difficult

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expression of smaller forms of the dystrophin gene or dystrophin

mini-genes or the related protein utrophin may be useful alternatives
for gene replacement
- idea came from observation that some mildly affected BMD patients
have deletion mutations that remove large portions of the gene
- in some cases, micro-dystrophins (~ 1/700 of the normal gene: 3.5kb vs.
2.4Mb) can functionally compensate for the loss of dystrophin expression

pharmacological intervention vs. genetic intervention?

- rational design of ligands to adhere components of the

DGC together without correcting the primary defect
- upregulation of analogous genes
- upregulation of growth factors involved in muscle
regeneration

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Methods of dystrophin gene repair

- oligonucleotides are designed that will repair the mutation in the
disease gene
---> for dystrophin, many of the disease-causing mutations are
found in regions of the gene not necessary for normal function
exon skipping
restoration of the open reading frame
oligonucleotide which results in skipping of an exon containing the mutation
was shown to correct some of the muscle weakness in the mdx mouse, a
dystrophin-deficient model for MD

How do you deliver the genes to the muscle tissue?

- crude delivery methods such as hundreds of intramuscular injections
(good for muscles involved in mobility, difficult to use for heart and
diaphragm - these are critical for long-term survival)
- viral vectors are still best choice despite problems with antigenicity

interesting approach: modify viral coat proteins to alter their natural tropism or
selectivity for certain tissues (i.e. so it would bind to muscle tissue rather than liver)

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