Cytoskeletal and Nuclear Membrane Proteins in Muscle

Muscle Fibre General Structure, ECM, Sarcolemma

• Muscle fibre general structure includes:
  • ECM (Extracellular Matrix).
  • Sarcolemma.
• Main proteins and complexes are crucial for muscle function.
• Muscle diseases include:
  • Dystrophies.
  • Myopathies.
• Nuclear envelope: structure, main proteins, diseases.

Structure of Skeletal Muscle

• Components of skeletal muscle:
  • Myofibre.
  • Fascicle.
  • Perimysium.
  • Epimysium.
  • Capillary.
  • Myonucleus.
  • Myofibril.
  • Mitochondria.
  • Sarcolemma.
  • Basement membrane or Basal lamina (Extracellular matrix).
  • Satellite cell.
• Fascia: connects muscle to muscle.
• Tendon: connects muscle to bone.

The Sarcolemma

• Includes the muscle plasma membrane, surrounding basement membrane, and sub-sarcolemma cytoskeleton.
• Separates outside from inside of the cell, maintaining cell integrity.
• Controls passage of materials into and out of the cell.
• Involved in:
  • Synaptic transmission.
  • Action potential propagation.
  • Excitation-contraction coupling.
• Biological importance of the basement membrane-sarcolemma-cytoskeleton is underscored by inherited muscle diseases caused by mutations in their components.

Muscular Dystrophies

• Disorders characterized by progressive muscle weakness and degeneration.
• At least 34 clinical disorders.
• Heterogeneous in age of onset, severity, and muscle affected.
• Inherited; at least 29 genetic loci involved.
• Types include:
  • Duchenne and Becker.
  • Emery-Dreifuss.
  • Limb girdle.
  • Facioscapulohumeral.
  • Distal.
  • Oculopharyngeal.
  • Myotonic.
  • Congenital.

The Costamere

• A focal adhesion is a large macromolecular complex at the interface between a cell and the extracellular matrix.
• In striated muscle, the costamere serves an analogous function to the focal adhesion, sharing a similar protein composition.
• The costamere bidirectionally and mechanically links the cytoskeleton to the extracellular matrix.
  • Transmits forces from the sarcomere to the extracellular matrix (“inside-out”).
  • Conversely transmits forces on the extracellular matrix to the myocytes (“outside-in”).
• Main components:
  • Vinculin-talin-integrin system.
  • Dystrophin glycoprotein complex.
• Intermediate filaments align the costamere with the M-band and Z-line.

Extracellular Matrix (ECM)

• Includes the interstitial matrix (between cells) and the basement membrane (thin sheet-like deposition of ECM that surrounds muscle cells).
• Three major components:
  • Laminins (link proteins: Fibronectin, Laminin etc.).
  • Collagen type IV (fibrous elements: Collagen, Elastin etc.).
  • Nidogens (entactin) and perlecan or agrin (heparan sulphate proteoglycan): space-filling molecules, connect the laminin and collagen networks to each other.
• Independent networks of Laminins and collagen type IV are connected by nidogens and perlecan and associate with cells through interactions with cell surface receptors (e.g., integrins and dystroglycan).
• Provides a solid scaffold for cells and tissue structure.
• Crucial for survival and differentiation of cells.
• Important barrier that limits bacterial/viral infections or malignant cell infiltration.
• Involved in cell signaling.

Laminin

• A group of high molecular weight glycoproteins, abundant in the basement membrane.
• Mature laminin proteins are cross-shaped, composed of one α, one β, and one γ chain that associate to form a heteromeric molecule.
• The LG 1-5 globular domains bind dystroglycan and integrin, and these interactions are crucial for basement membrane formation, cell differentiation, and cell survival.
• Up to now, five α, three β, and three γ chains have been characterized (at least 15 combinations).
• Laminin211 ($\alpha2$, $\beta1$, $\gamma1$) (= merosin) is the major form of laminin in skeletal muscle; other laminins define NMJs.
• Mutations in LAMA2 gene that encodes laminin $\alpha2$ chain are responsible for MDC1A (congenital muscular dystrophy type1A) - 50% of all congenital muscular dystrophies.
• MDC1A is characterised by severe muscle weakness, hypotonia, joint contractures, dysmyelinating peripheral neuropathy, and brain defects.

Collagen IV

• Mechanical stability of the basement membrane is thought to be regulated by collagen IV network due to its unique structure and abundance.
• Protomer is a triple helix composed of three polypeptide chains (different combinations of $\alpha1$-6 chains).
• Protomers form a complex structured network.
• Connects with other components of the ECM.
• Interacts with the cell surface, mainly through $\beta1$ chain-containing integrins.
• Linked to various non-muscle diseases.
• Recently mutations in COL4A1 linked to Walker-Warburg Syndrome and muscle-eye-brain disease.

Assembly of Basement Membrane

• Laminin and type IV collagen self-assemble into covalently cross-linked networks.
• Nidogen and perlecan incorporate into the networks and provide stability to the matrix.
• The basement membrane matrix presents specific ligands that interact with cell-surface receptors, such as integrins and dystroglycans, and anchor cells to the BM.

Dystrophin

• Dystrophin functions as a shock absorber, protecting membranes from stress in contraction and relaxation.
• Structure and function:
  • 3685 amino acids, Molecular weight of 427kDa.
  • Dystrophin gene is on the X chromosome; the largest known human gene (>2500Kb; 90 times an average gene), composed of 79 exons.
  • Dystrophin links ECM with the contractile apparatus:
    • C-terminus binds to DGC at the sarcolemma.
    • N-terminus binds f-actin (actin cytoskeleton).
  • Structural role in stabilizing sarcolemma during contraction.
  • Transmits force laterally across sarcolemma to ECM.
  • DGC contains signaling proteins.
• The dystrophin protein has four functional domains:
  • The central rod domain (R) consists of 24 spectrin-like repeats arranged head-to-tail and interspersed by four flexible hinges (H).
  • The N-terminal (ABD) and the spectrin-like repeats bind to the cytoskeleton f-actin, but not to the actin of sarcomeric thin filaments.
  • The cysteine-rich domain (CR) binds to β-dystroglycan.
  • The C-terminal (CT) domain binds to α-dystrobrevin and syntrophin.

Duchenne Muscular Dystrophy (DMD)

• Out-of-frame mutation (deletion) leads to truncated and unstable protein; no dystrophin.
• Commonest - Duchenne muscular dystrophy.
• 1 in 3500 male births.
• Symptomatic <1 year - 5 years: late walking, toe walking, unable to run or jump, falls, stairs and climbing hard, Gowers’ manoeuvre, cramps.
• Loss of independent walking 6 -12 years.
• Progressive loss of muscle, contractures, scoliosis.
• 30% significant learning difficulties:
  • DMD: non-progressive learning difficulties, IQ shifted -1SD, 30% IQ<70, mean IQ 85 verbal IQ < performance IQ, speech delay very common.
• Death usually late teens/early 20s: respiratory or cardiac impairment.

Becker Muscular Dystrophy (BMD)

• In frame mutation (deletion) leads to partially functional protein; some dystrophin function.
• Usual onset 5-15yrs.
• Clinically similar to DMD.
• Milder with more variability.
• Slower progression.
• No contractures or scoliosis.
• Live into adulthood (normal lifespan possible).

Duchenne vs. Becker Muscular Dystrophies

• Normal: Bridges cytoskeleton to extracellular matrix.
• Becker: In-frame mutation, partly functional protein, bridge function maintained, milder phenotype.
• Duchenne: Out-of-frame mutation, prematurely truncated protein, bridge function lost, severe phenotype.

Histology of DMD Muscle

• Wide variation in myofibre size.
• Necrosis.
• Fibrosis and fat infiltration.
• Hypercontracted, split and whorled fibres.
• Internal nuclei and regenerating myofibres.
• Basophilic myofibres.

Absence of Dystrophin

• Major reduction of the entire DGC complex.
• Sarcolemma becomes fragile.
• Mechanical stress damage fibres.
• Influx of calcium through membrane leakage activates proteases that degrade myofibres and cause cell death.
• Roles of DGC in signaling pathways are impaired, inflammation, infiltration, fibrosis, exhaustion of satellite cells pool gradually impedes regeneration efficiency; gradual failure of regeneration leads to progressive lose of muscle function.
• Other possible pathological mechanism through absence of nNOS (muscle ischaemia).

Role of Satellite Cells in Normal Muscle

• Hypertrophy - during growth and in response to training.
• Maintenance - routine myonuclear turnover.
• Myofibre repair - after severe exercise or localised damage.
• Regeneration - in response to widespread damage.

DMD Therapy Approaches

1. Anti-inflammation/fibrosis drugs, exercise (e.g., hydrotherapy), corticosteroids etc.: slowing down the progression of the disease.
2. Dystrophin restoration approach:
   • Stop codon read-through (gentamycin, ataluren).
   • Exon skipping (restoring partial function; Duchenne into Becker).
3. Boosting alternative proteins (Utrophin) or blocking others (myostatin).
4. Gene Therapy:
   • Using mini/microdystrophin.
   • Viral gene therapy (lentivirus, AAV).
   • Non-viral DNA introduction.
5. Using CRISPR/Cas9-mediated genome editing to correct the dystrophin gene.
6. Cell transplants.

Dystroglycan

• α- and β-dystroglycan are encoded by a single polypeptide from one gene that undergoes posttranslational proteolytic cleavage.
• α-dystroglycan is an extensively glycosylated extracellular protein. It binds proteins with laminin-globular (LG) domains such as laminins with high affinity. The binding can be also influenced by the glycosylation of α-DG. The α-DG/laminin interaction is considered crucial for the stability of basement membranes.
• β-dystroglycan is a single pass transmembrane protein, binds directly to α-dystroglycan (N- terminus) and Dystrophin (C-terminus).
• In mouse animal model Dystroglycan is essential for Reichert's membrane in early embryonic stages in mouse leading to very early lethality of KO mouse.

Dystroglycanopathies

• Primary dystroglycanopathies: Amino acid changes in both α- and β-dystroglycan (missense/nonsense mutations) lead to impaired function and various muscle dystrophies.
• Secondary dystroglycanopathies: Abnormal glycosylation causes muscular dystrophies.
  • Abnormal glycosylation of α-dystroglycan affects the DGC In (muscle) and DGC-like (brain/neural).
  • Perturbs linkage of cell membrane to the ECM.
  • Various mutations in genes encoding to glycosyltransferases and other proteins involved in glycosylation give rise to muscular dystrophies (table).
  • Includes WWS(Walker Warburg Syndrome), Muscle-eye-brain disease, Fukuyama CMD, CMD1C, CMD1D, LGMD2L.

Walker-Warburg Syndrome (WWS)

• Mutations in protein O-mannosyltransferase (POMT1).
• Congenital onset.
• Absent psychomotor development.
• CNS: hydrocephalus type II lissencephaly (cobblestone), Cerebellar hypoplasia (vermis).
• Eye: anterior and posterior chamber defects.
• CK markedly elevated.
• Poor prognosis.

Muscle-Eye-Brain Disease

• Mutations in O-mannose β-1,2-N- acetylglucosaminyltransferase (POMGnt1).
• Neonatal onset.
• Severity of weakness variable.
• Mental retardation constant.
• Serum CK: grossly elevated.
• Eye: myopia, cataract, glaucoma.

Fukuyama CMD

• Mutation in the fukutin gene.
• Commonest dystrophy after DMD in Japan.
• Neonatal onset.
• Most patients sit but do not walk.
• Mental retardation invariable.
• Serum CK: grossly elevated.
• Death in the teens.

Sarcoglycan

• Sarcoglycans are single-pass transmembrane glycoproteins.
• Serve as anchorage for the peripheral DGC components.
• They display short intracellular tail and large extracellular glycosylated domain rich in cysteins, which are crucial for the assembly in a subcomplex. In striated muscle the subcomplex is made of α-, β-, γ- and δ-sarcoglycan.
• Interacts with DGC members as well as γ-filamin and probably integrins.

LGMD (Sarcoglycanopathies)

• Sarcoglycanopathies: a subgroup of autosomal recessive limb-girdle muscular dystrophies (LGMD 2C–F).
• Clinical phenotypes are very heterogeneous; in most cases-progressive proximal limb-girdle muscle weakness or wasting and elevated serum creatine kinase; in some cases cardiomyopathy, respiratory deficiency and retractions.
• Mutation in one of the SG genes has consequences on the stability of the entire SG subcomplex and the DGC (sometimes reduction of dystrophin level as well).

Integrins

• Large family of surface receptors bridge ECM and intracellular cytoskeleton and involved in signalling in both ways.
• Consist of two non-covalently-associated subunits, α and β.
• 18 α-types and 8 β-types make at least 24 known combinations (+spliced isoforms).
• $\alpha7\beta1$ integrin is the main muscle integrin (interacts mainly with laminin 211-merosin).
• Functional redundancy with the DGC (linking ECM and cytoskeleton in muscle).
• A few patients (ITGA7) and mouse models indicate involvement in maintaining MTJs and protection from stress-induced damage.
• Signaling through integrin $\alpha7\beta1$ is important to promote cell growth and myofiber survival.
• The integrin complex is involved in the initiation of skeletal muscle adaptation, in response to a mechanical stimulus in the form of contraction, exercise, or chronic loading.

The Nuclear Envelope

• Composed of a double membrane system interspersed with nuclear pore complexes, separating nuclear from cytoplasmic functions.
• The INM (inner nuclear membrane) juxtaposes the nuclear lamina, a meshwork-like structure mainly composed of the intermediate filament proteins nuclear lamins A, B and C, which in turn faces the nuclear interior.
• The ONM (outer nuclear membrane) is an extension of the ER (endoplasmic reticulum) network but also contains a distinct protein subpopulation.
• Between them lumen of 30-50nm.
• Nuclear pore complexes (NPCs)-large, macromolecular structures through which molecules are transported between the nucleus and the cytoplasm.
• Nuclear lamina-a fibrous-like structure in the nuclear side of the inner nuclear membrane, a mash of intermediate fibres, interacts with chromatin.

LMNA: One Gene, Many Diseases

• Mutations in LMNA can cause:
  • Myopathies.
  • CMT (Charcot-Marie-Tooth disease).
  • FPLD (Familial Partial Lipodystrophy).
  • HGPS (Hutchinson-Gilford Progeria Syndrome).
  • Mandibuloacral dysplasia (MAD).

Emery-Dreifuss Muscular Dystrophy (EDMD)

• Onset childhood (but neonatal to adult).
• Clinical phenotype similar to a mild form of BMD, i.e., muscle weakness, contractures, cardiac conduction defects.
• No loss of DGC components.
• Patients and carriers develop a unique cardiac conduction defect.
• Frequently die from sudden cardiac death, as do the carriers who can be asymptomatic!
• Two different genetic forms:
  • X-linked.
  • Autosomal dominant.
• Mutations:
  • Lamin A/C in rarer autosomal dominant EDMD.
  • Emerin in more common X-linked EDMD.
  • Dominant Missense mutations in nesprin-1 and nesprin-2 can also lead to EDMD.
• Nuclear lamina mimics the cytoskeleton forming a cage-like structure around the nucleus.
• Emerin-lamin A/C protein complex mimics DGC.
• Intermediate filament desmin connects nuclear envelope and sarcomeric actin and other cytoskeletal proteins together protects nucleus from mechanical stress.
• In EDMD: Increased nuclear fragility during normal mechanical stress associated with muscle function; whole cell mechanical vulnerability.
• Aberrant nuclear architecture remodeling throughout the cell cycle and DNA damage repair.
• Incorrect activation/localization of nuclear factors and cofactors at specific chromatin sites; cell proliferation and gene expression could also be affected, producing some of the EDMD phenotype.