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DYSTROPHINOPATHIES: Duchenne

Early age   Mild   Myopathic groups   Regeneration   Severe Later age Dystrophin Revertants General features

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DMD Muscle pathology: General features

Myopathy: Ongoing & Chronic Fiber sizes: Varied Small fibers: Round Hypercontracted (opaque) muscle fbers Sizes: Often large Necrosis & Regeneration of muscle fibers More early in disease course Patterns Scattered Myopathic grouping Muscle fiber pathology "Delta lesions" Internal architecture Internal nuclei Often coarse (Immature) Ring fibers Immaturity (2C fiber type): Many fibers Hypercontracted fibers Chronic changes: Other tissues Endomysial connective tissue: Increased More with: Later pathology Fat accumulation Locations: Endomysial & Perimysial Molecular Dystrophin staining Screening Antibodies: Target Epitopes N-terminus Dys-3: vs Exons 10 to 12 Rod domain 7G1: vs Exon 46 (Within deletion hotspot) 6A9: vs Exon 50 (Within deletion hotspot) Dys-1: vs Exons 26 to 30 C-terminus Dys-2: vs Exon 79 Less useful Few deletions in this region May be reduced in other myopathies Duchenne MD clinical phenotype: Staining patterns DMD Muscle Dystrophin: Absent staining for all epitopes Utrophin: Normal (Absent on muscle fibers) or Increased staining nNOS: Often absent with Exon 45-51 hotspot deletions Sarcoglycans: Often reduced in Duchenne MD Schwann cells: May stain for dystrophin with mutations proximal to Intron 55 Becker MD clinical phenotype: Staining patterns Overall reduced staining for all antibodies Relative reduction in intensity of staining for some dystrophin epitopes Complete loss of some dystrophin epitopes Dystrophin female carriers Non-necrotic, dystrophin-negative muscle fibers Other sarcolemma-related proteins Sarcoglycans: Reduced Aquaporin 4: Reduced Laminin-α2: Normal or Increased Utrophin: Increased β-Spectrin: Normal Calcium 1 Increased in myoplasm Pathomechanisms of Ca++ increase in DMD muscle fibers Enhanced Ca++ leak from sarcoplasmic reticulum (SR) Through oxidized Ryanodine receptor type 1 (RyR1) Ca++ release channels Hypernitrosylation of cysteine residues in RyR1 FKBP1A dissociation from RyR1 Destabilization of RyR1 channel closed state Increased RyR1-dependent SR Ca++ leak Uncontrolled production of reactive species Oxygen (ROS) Nitrogen (RNS) Reduced activity of SR/ER Ca++ ATPase(SERCA) (Ca++pump) Excessive extracellular Ca++ trans-sarcolemmal influx Micro-tears: Delta lesions Abnormal Ca++-permeable ion channels Enhanced store-operated Ca2+ entry (SOCE) Related molecules Stromal interaction molecule-1 (STIM1) Senses changes in luminal ER/SR Ca++ levels Orai1 Ca++ release-activated Ca++ channel in surface membrane SOCE trigger: Depletion of intracellular Ca++ stores

From: AG Engel Delta Lesions (Arrow) Description: Focal wedge-shaped lesions Locations: Subsarcolemmal regions of muscle fibers

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DMD
  Muscle fibers
    Size: Varied
    Shape: Small fibers round or polygonal
    Internal nuclei
    Hypercontracted
    Necrosis
  Endomysial connective tissue: Increased between muscle fibers
  Fat: Increased in endomysium & preimysium

H&E stain

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DMD
  Endomysial connective tissue: Increased between muscle fibers

VvG stain

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NADH stain

DMD: Immature muscle fibers
  Coarse internal architecture on NADH (Above)
  2C fibers: Intermediate staining on ATPase pH 4.3 (Below)

ATPase pH 4.3 stain

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Duchenne Muscular Dystrophy: Early Pathology

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H & E stain

Muscle fibers   Sizes: Varied; Small fibers are rounded or polygonal; Occasional hypertrophic fiber   Necrosis (Left): Fibers scattered & in small groups   Myopathic groups (Below): Clusters of small necrotic (Black arrow) & regenerating (White arrow) muscle fibers   Internal nuclei: Occasional Endomysial connective tissue: Normal to mildly increased Perimysium: Early replacement by fat

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Acid phosphatase stain Acid phosphatase + cells   Endomysium: Small Scattered cells   Necrotic muscle fibers: Clusters of phagocytic cells Ring fibers (Arrow)

Acid phosphatase stain Acid phosphatase + cells   Necrotic muscle fibers: Clusters of phagocytic cells

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DMD: Muscle Fiber Necrosis

Necrotic Fibers: Scattered; Varied stages

H&E stain

Gomori trichrome stain

Necrotic Fibers: Scattered; Replaced by cells

VvG stain

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Myopathic Grouping

Muscle fiber Stages   Necrosis   Regeneration     Early     Late

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Myopathic grouping: Necrotic muscle fibers, clustered

H&E stain

Necrosis, Early stage
  Necrotic muscle fibers: Pale stained
  Macrophages: Early invasion of muscle fibers

NADH stain Necrotic muscle fibers are pale on NADH stain

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Myopathic grouping: Clusters of very small regenerating muscle fibers & histiocytic cells

VvG stain

Myopathic groups: Clusters of small muscle fibers in similar, early, stage of regeneration or immaturity

VvG stain

Myopathic groups: Clusters of small muscle fibers in similar, early, stage of regeneration or immaturity

H & E stain

ATPase, pH 4.3

NADH

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Clusters of small cells   Many stain for Acid phosphatase Acid phosphatase

Acid phosphatase

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H & E stain

Myopathic Grouping: Late

H & E stain

Myopathic grouping: Intermediate-sized, Immature muscle fibers in clusters

H & E stain Immature fibers are small & basophilic

ATPase, pH 4.3 Immature fibers are: Small &   2C (Intermediate staining)

Alkaline phosphatase Immature small fibers have:   Cytoplasmic staining

NADH Immature fibers have coarse cytoplasmic staining

NADH

VvG Immature small fibers have coarse cytoplasmic staining

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Immature muscle fibers: Many

Staining properties of immature muscle fibers includes:   1. H & E (left): Basophilic fibers   2. Alkaline phosphatase positive (center)   3. 2C fibers: Intermediate staining on ATPase pH 4.3 (right)

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Duchenne Muscular Dystrophy: Severe at age 2 years

H&E stain

DMD
  Endomysial connective tissue: Increased
  Muscle fibers
    Necrosis & Regeneration
    Size: Varied

H&E stain

H&E stain

DMD
  Endomysial connective tissue: Increased
  Muscle fibers
    Necrosis & Regeneration
    Size: Varied

Gomori trichrome stain

Gomori trichrome stain

VvG stain

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DMD
  Many intermediate-stained muscle fibers

ATPase pH 4.3 stain

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Duchenne Muscular Dystrophy: Later Pathology (10 years)

Endomysial connective tissue Fat replacement Fiber types Internal architecture Necrotic fibers

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H&E stain

DMD: Later pathology
  Fiber size: Varied
  Endomysial connective tissue: Increased
  Fat replacement of muscle: Prominent

H&E stain

VvG stain

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Sudan stain

Replacement of Muscle fibers & Perimysial connective tissue by Fat (Dark black)

Sudan stain

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H&E stain Endomysial connective tissue: Increased Fiber size: Variable Small fibers: Rounded Large or hypercontracted muscle fibers: Scattered Necrotic fibers (Arrow): Scattered

	H&E stain Endomysial connective tissue   Increased between fibers

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Muscle fiber size: Varied
Small & Large fibers: May be are either type (I = Dark; II = Light)
Many type IIC fibers: Smaller size; Intermediate stain

ATPase pH 4.3 stain

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DMD late: Few necrotic muscle fibers

Acid phosphatase stain

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Muscle fiber internal architecture: Coarse

NADH stain

DMD: Ring Fibers

H&E stain

Gomori trichrome stain

NADH stain

DMD: Ring fibers

NADH stain

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Dystrophin staining

Normal	Duchenne Muscular Dystrophy:
Normal dystrophin staining around the rim of muscle fibers.	Absent dystrophin: Duchenne muscular dystrophy Left: No staining around the rim of any muscle fibers Right: No staining of most muscle fibers   One "revertant" fiber with dystrophin staining.   Revertant fibers reflect a somatic mutation allowing dystrophin expression

Normal: Dystrophin present near surface of muscle fibers Dys1 stain Duchenne MD: Dystrophin absent from surface of muscle fibers Dys1 stain

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Muscle from Duchenne MD male with large area of revertant muscle fibers

Muscle histology (Above): One fascicle (Arrow) has more normal muscle fibers & connective tissue amid otherwise myopathic muscle
Dystrophin staining (Below): Muscle fibers in preserved fascicle are revertants with dystrophin present around their rim
    Some revertant muscle fibers are present in myopathic regions as well.

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Western blot: Dystrophin from dystrophinopathies

from Novocastra Lane 1: Becker dystrophy; Dystrophin has reduced abundance but normal size. Lane 2: Becker dystrophy; Dystrophin has reduced size and abundance. Lane 3: Normal; Dystrophin has normal size and amount. Lane 4: Duchenne dystrophy; Almost no protein is present. Lane 5: Duchenne outlier; Dystrophin has severely reduced abundance.

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References
1. J Gen Physiol 2022;15:e202213081

8/2/2023