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Muscular dystrophy: basic facts

- heterogenous group of inherited disorders characterized by progressivemuscle weakness and wasting (regeneration of muscle tissue fails)

- most apparent or symptomatic in skeletal muscle but heart anddiaphragm muscle often involved (most patients die of heart failureor 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

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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 factorssuch as FGF or HGF are present

once the growth factors are removed, the myoblasts rapidly stopdividing, differentiate, and eventually fuse to form fibers

these muscle fibers contain intracellular and extracellular componentsthat are responsible for providing support to the fibers during contractionprocesses

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Fusion of myoblasts in cell culture

blue color (DAPI) - nuclear staining

green color (myosin) - marker of differentiated myoblasts

cells fuse and contain multiple nuclei

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

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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

How do muscles handle the stress applied to them?

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 membraneduring cycles of contraction and relaxation

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Dystrophin

- cytoskeletal protein localized to the inner surface of themuscle membrane

- part of a complex with multiple proteins includingsarcoglycans and dystroglycans (binds to ß-dystroglycanand 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 (limb- girdle muscular dystrophy)

- loss of sarcoglycans at the muscle membrane leads to variable destabilization of the DGC

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Dystrobrevin and syntrophins

- intracellular proteins that associate with C terminus of dystrophin

- dystrobrevin likely acts with syntrophins to recruit signalingproteins 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)

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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

merosin = laminin2 (DGC intact)loss of sarcoglycans = variable DGC loss

loss of dystrophin = DGC destabilization

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How is muscle damaged when DGC components are missing?

- mechanical hypothesis: loss of DGC leads to contraction-induced ruptureof muscle cell membranes; noted by cytoplasmic accumulation of serum proteinsin muscle fibers (exercise in DMD patients likely causes greater damage than in controls)

- calcium hypothesis: influx of calcium into cytosol overwhelms muscle cell’s abilityto 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)

theories for muscle fiber necrosis:

no universal agreement on which mechanism is predominant

- gene regulation hypothesis: failure of certain molecules to be localized to themuscle membrane when DGC components are absent prevents proper signalingmolecules 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

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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 progressiveshortening of their telomeres

- muscle cells are then replaced by connective tissue and fibroblasts;this prevents further repair (similar to what happens in the elderly)

Muscle pathology of muscular dystrophy

- 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 post- transcriptional stages medaites the extensive fibrosis in human DMD patients

theories for tissue remodeling:

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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 aren’t as drastic as some of thecongenital muscular dystrophies

Therapeutic approaches for muscular dystrophy

- gene therapy represents a major area of research in the musculardystrophy 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 patientshave 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

- 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

pharmacological intervention vs. genetic intervention?

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Methods of dystrophin gene repair

- oligonucleotides are designed that will repair the mutation in thedisease 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 mutationwas 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 anddiaphragm - 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 orselectivity for certain tissues (i.e. so it would bind to muscle tissue rather than liver)