1Muscular 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 Becker2) Congenital (MEB, FCMD, LGMD, CMD) - involve mutations in several genes - autosomal recessive inheritance - defects in extracellular molecules, i.e components of the extracellular matrix2Clinical 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 involvementOverview 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 contractionprocesses3Fusion of myoblasts in cell cultureblue color (DAPI) - nuclear staininggreen color (myosin) - marker of differentiated myoblasts cells fuse and contain multiple nucleiSatellite 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 muscle4muscles 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 dystrophinDystroglycan-glycoprotein complexmain function: to provide structural stability to muscle cell membraneduring cycles of contraction and relaxation5Dystrophin - 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 complexSarcoglycans- 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 DGC6Dystrobrevin 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 yetDysbindin- 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)7Dystroglycan- 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 dystrophinno mutations in the dystroglycan gene have been found Fate of DGC components in various forms of muscular dystrophymerosin = laminin2 (DGC intact)loss of sarcoglycans = variable DGC lossloss of dystrophin = DGC destabilization8How 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 role9satellite 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