Muscle Fiber Necrosis

MUSCLE FIBER NECROSIS

General features
Necrosis & Rhabdomyolysis
Stages
Early Features
Metabolic damage
Sarcolemmal membrane damage
C_5b-9 deposition
Macrophage activation
Fixed muscle, Paraffin sections
Later features
Phagocytosis & Regeneration
Myofiber collapse
Eosinophils
Distribution of Necrotic fibers
Clustered
Immune (Vascular)
Regional Ischemic Immune
NXP-2
Hereditary (Stretch-induced)
Myopathic grouping
Perifascicular: IMPP
Scattered
Necrosis: Types & Features
Muscle Fiber Patterns
Membrane damage
Delta lesions
Metabolic failure, Diffuse
Peripheral, Histiocyte-related
Dark

Etiologies
Differential Diagnosis
Necrosis
Necrosis, Immune
Immune
Antibody-related
HMGCR
Mi-2
NXP-2
SRP
BCIM
LHIM
Macrophage activation
RIIM
Phosphorylase deficiency
Toxicity
Alcohol
Statin

Segmental Necrosis of Muscle fibers: Varied stages

Necrosis: General features & Stages ¹

Cell & Metabolic Conparative features Morphology Dark Metabolic failure Membrane damage Large Histiocyte Phagocytosis	Necrosis, Early Stages: Muscle Fiber Patterns of Pathology
	Myofiber Necrosis Metabolic Muscle Fiber cytoplasm Pale stain: Diffuse Myonuclei: Lose staining	Myofiber Necrosis Sarcolemmal Membrane Δ Muscle Fiber Cytoplasm Pale stain: "C" or Δ Lesion Dark stain: Around C-region Myonuclei: Lose staining	Myofiber Necrosis Dark Necrosis Muscle Fiber Cytoplasm Dark stain: Diffuse Myonuclei: Lose staining	Myofiber Necrosis Histiocyte-Associated Neighboring histiocytes Around edge of fiber Muscle Fiber Cytoplasm Stain: Present
	Later Stages: Histiocyte Invasion & Phagocytosis of Muscle fibers			Later stage: Fiber Degeneration
	Myofiber Necrosis Cell invasion Stage: Intermediate Muscle Fiber Macrophage invasion	Myofiber Necrosis Phagocytic Stage: Late Muscle Fiber No cytoplasm remaining Replaced by histiocytes	Myofiber Necrosis Collapse Stage: Very Late Muscle Fiber Space: Collapsed Histiocytes Few remaining Many have migrated away	Myofiber Necrosis Primary Fiber Degeneration Stage: Late Muscle fiber Cytoplasm: Fragmented; Lost Histiocytes: Few or None

Necrosis: Cellular & Metabolic changes
- Cytoplasmic swelling (oncosis)
- Rupture of plasma membrane
- Cytoplasmic organelles: Swelling; Loss
- Chromatin: Condensation, moderate
- Absent: Apoptotic & Autophagic markers
- Necrosis in muscle
  - Associated with either
    - Metabolic failure: Ca⁺⁺ leak into cytoplasm
    - Break in, or loss of, muscle surface fiber membrane
  - Results in: Irreversible damage to whole, or segment of, muscle fiber
  - All organelles involved
- Associated metabolic alterations
  - No common biochemical denominator
  - Mitochondrial
    - Uncoupling
    - Production of reactive oxygen species (ROS)
    - Mitochondrial membrane permeabilization: Often controlled by cyclophilin D
    - Ca⁺⁺ overload
  - Lysosomal
    - ROS production by Fenton reactions
    - Membrane permeabilization
  - Nuclear
  - Lipid degradation
    - Activation of phospholipases, lipoxygenases & sphingomyelinases
  - Ca⁺⁺
    - Activation of non-caspase proteases: Calpains; Cathepsins

Necrotic muscle fiber morphology: Patterns during necrosis
- Necrosis due to: Metabolic failure
  - Myofiber changes: Sarcolemma & Cytoplasm
    - H&E & Gomori Trichrome
      - Cytoplasm staining: Pale
      - Intermyofibrillar network: Absent
      - Myonuclei: Absent or reduced staining
    - Necrotic muscle fibers have reduced or absent staining with
      - NADH
      - Mitochondrial (SDH)
      - Myosin ATPase
    - Necrotic muscle fibers: Molecular & cellular markers
      - Cytoplasm of necrotic muscle fibers: Stains for
        
        C_5b-9 Complement (Membrane attack complex (MAC))
        
        IgM
        
        Ca⁺⁺: Increased in necrotic muscle fibers
      - Around the rim of necrotic muscle fibers: Alkaline phosphatase
      - Muscle fiber phagocytosis
        
        Histiocytic (Phagocytic) cells stain for: Acid phosphatase; Esterase; HAM56; CD68; MHC Class I
    - Ultrastructure
      - Loss of plasma membrane
      - Myofibrils: Lysed or Lost
      - Mitochondria: Abnormal
      - Late: Granular or filamentous debris in fibers
    - Rare in intrafusil muscle fibers
- Necrosis due to: Membrane damage
  - Early damage pattern
    - Focal susarcolemmal area: Delta (Focal, subsarcolemmal) lesion: Pale stain
    - Other remaining cytoplasm: Dark stain
  - Disorders
    - Hereditary: Dystrophinopathy & Other sarcolemmal protein disorders
    - Toxic: Statin; Lysolecithin
- Necrosis due to: Ischemia
  - Early fiber damage pattern: "Dark" necrosis
    - Cytoplasm of involved muscle fibers: Diffusely dark on H&E & Gomori trichrome stains
  - Later fiber damage: 2 patterns
    - Pale fragmented, or No, cytoplasm staining + No, or few, associated histiocytes
    - Invasion, Phagocytosis & replacement of fibers by histiocytes
  - Disorders with "Dark" necrosis
    - RIIM
    - Rhabdomyolysis with
      - Phosphorylase deficiency
    - BACM
- Necrosis due to: Large Histiocyte-mediation
- Necrosis due to: Necroptosis (Regulated) ²
  - Programmed cell death
  - Stimuli
    - TNF-α : Pro-necrotic in muscle fibers
    - Death domain receptors: TNFR1 , Fas/CD95 , TRAIL-R
    - Toll-like receptors: TLR3 , TLR4
  - Pathway: Kinase RIP1 (RIPK1)
    - Molecules upregulated: RIPK1 , RIPK3 , MLKL
    - Execution pathway
      - RIP1–RIP3 necroptotic complex: Necrosome
      - Production of mitochondrial reactive oxygen species (ROS)
      - Depletion of cell energy under apoptotic-deficient conditions
      - MLKL increase
        
        Disrupts plasmalemma integrity
        Targets cellular organelles: Mitochondria & Endoplasmic reticulum,
        
        Releases: Damage-Associated Molecular Patterns (DAMPs) from ruptured cells
        
        DAMPs
        
        Molecules: HMGB1, Nuclear DNA, Mitochondrial DNA, ATP from necroptotic cells
        
        Effects: Induce tissue inflammation injury; Recruit immune cells
    - Caspase-independent
  - Regulation
    - Stimulated by: TNF-α; DUX4
    - Down-regulated: Muscle fiber differentiation; Adulthood
  - General effects
    - Inflammation: Induced
    - Cytokines, Pro-inflammatory
      - Activation
      - TNF-α, IL-1β, Interferons
    - DAMPs: Released
    - Tissue homeostasis
      - Satellite cells: Activation
      - During development, regeneration & cell damage
      - Inhibit tumor growth
      - Recruit cells to clear debris
  - Histologic marker: phospho-MLKL
Necrosis in muscle: Stages
- Early necrosis: Patterns
  - Cytoplasmic pallor: "Metabolic" pattern
  - Sectoral lesion & Hypercontraction
  - Subsarcolemmal lesion
- Phagocytosis (Clean-up) of necrotic muscle fibers
  - Early: Infiltration of muscle fibers by histiocytic cells
    - Proliferation or Migration of Phagocytic cells
    - Attraction of Phagocytic cells to necrotic muscle fiber
    - Phagocytosis of necrotic muscle fibers
    - Associated macrophages: M1 type
      - Classically activated inflammatory macrophages
      - Infiltrate at early time points after injury
      - Generate inflammatory cytokines
      - Sustain activation & proliferation of muscle stem cells
  - Late: "Collapse"
    - Migration of macrophages away from necrotic muscle fiber
    - "Collapse" of region with necrotic muscle fiber
- Cell remnant disposal
  - Related enzyme: MAP kinase phosphatase-1 (MKP-1; DUSP1)
- Cell proliferation
  - Muscle precursors
    - Function: Formation of regenerating muscle fibers
    - Origins
      - Satellite (Stem) cells
      - Vessel-associated muscle stem cells (Mesoangioblasts)
        
        Myogenic precursors
        
        Express pericyte markers
        
        Cross vessel walls
  - Connective tissue & Fat: Fibro/adipogenic progenitors (FAPs)
    - Sustain satellite cell-driven myogenesis
    - May differentiate into myoblasts, adipocytes or fibroblasts
  - M2 macrophages
  - Presence: Associated with tissue "Inflammation"
  - Late: Muscle healing
    - FAPs & Stem cells undergo apoptosis

Differential Diagnosis: Myofiber Necrosis in Immune Myopathies
Syndrome	Myofiber Necrosis	Myofibers MHC-1 expression	Pathology Other Tissues	Immune cells/ Location	Antibody or Marker (Serum)	IIM Clinical Syndromes
LHIM	Scattered Peripheral	Diffuse Most fibers	Anemia	Histiocytes/ Near myofibers	Ferritin (50%)	Necrotic Myopathy
IMPP	Near Perimysium	Fibers Near Perimysium	Perimysium Damage	Histiocytes/ Perimysium	tRNA synthetase (Jo-1)	DM/PM/ NM/ILD
SRP	Scattered	Immature fibers Scattered None on most fibers	Fibrosis Endomysium & Perimysium	No	Signal Recognition Particle 54 (SRP54)	Necrotic Myopathy
RIIM	Clustered in Border zones	Little or Diffuse	Vein damage	No	Tif1γ NXP-2	DM/PM/ Paraneoplastic
Checkpoint Inhibitors	Clustered	Regional: Around necrotic fibers	Mitochondrial Δ Cardiac; Eye	Histiocytes Clustered	Striation	Necrotic Myopathy
Active or Chronic Myopathy	Scattered or Near Perimysium	Immature fibers None on most fibers	Perimysium	No	HMGCR	Necrotic or Dystrophy-like Myopathy
BCIM	Scattered	Diffuse Most fibers	Endomysium C_5b-9 deposition	B-lymphocyte foci/ EGC (ELS)	ANA (Nucleolar) PM-Scl100	Polymyositis/ Other immune
Active Myopathy	Scattered	Diffuse Most fibers	Capillaries	No	U1snRNP	Necrotic Myopathy
Optimal stain for identifying Necrotic Muscle Fibers: C_5b-9 complement on fiber cytoplasm

Myopathies with Abundant Muscle Fiber Necrosis: Comparative Pathology

Myopathy
Type

Myopathy:
Examples

Necrotic Muscle Fibers: Features
Distribution in Muscle	Course	Fiber Cytoplasm Damage

Tissue Pathology:
Other

Hereditary

Sarcolemma
Disorders

Dystrophin
Dysferlin

Scattered
Myopathic Groups

Ongoing

Δ lesion, or
Diffuse

Endomysium
Increased

Rhabdomyolysis
Metabolic &
Membrane
Disorders

Phosphorylase
Malignant Hyperthermia
Membrane disorders

Scattered

Monophasic
± Ongoing

Diffuse

Toxic

Rhabdomyolysis

Statin toxicity, acute
Ethanol

Scattered

Monophasic

Δ lesion, or
Diffuse, Pale

Immune

IMPP

Jo-1 & Synthetase Ab
Multisystem Δ
Dermatomyopathy

Perifascicular
Predominant

Ongoing

Diffuse

Perimysium Damage

HMGCR
antibody

Myopathies:
Subacute or Chronic

Scattered &
Perifascicular

Ongoing

Diffuse

Perimysium Damage
Alkaline Phosphatase+

SRP antibody

SRP Myopathy,
Progressive, Severe

Scattered

Ongoing

Diffuse

Endomysial Proliferation
Perimysium Damage

LHIM

Myopathy, Subacute

Scattered

Ongoing

Peripheral

Histiocytes:
Surround Myofibers

NXP-2 & Tif1γ
antibodies

RIIM
Dermatomyopathy
Paraneoplastic: ↑ Age

Clustered: Vessel
Borderzone areas

Ongoing

Diffuse

Vessel: Veins & Capillaries
Connective tissue

Mi-2 antibody

Dermatomyopathy

Scattered &
Perifascicular

Ongoing

Diffuse

Focal

Toxic

Injections
Venoms

Clustered

Monophasic

Diffuse, or
Δ lesion

Ischemia

Vascular Disease

Clustered

Monophasic

Diffuse

Endomysium: Edema

Freeze

Clustered

Monophasic
Slow recovery

Diffuse

Satellite cells
Number reduced

Muscle Fiber Necrosis (Rhabdomyolysis episode): Morphology

Segmental necrosis: Scattered necrotic & hypercontracted muscle fibers

H & E Stain	← Normal muscle fiber
	← Necrotic muscle fiber: Pale; Little phagocytosis
	← Necrotic muscle fiber: Phagocytosis by macrophages
	← Hypercontracted necrotic muscle fiber
	← Perimysium

Segmental Necrosis (Arrow) in Muscle fiber

H & E Stain

Necrosis: Early; Metabolic failure
Pale muscle fibers with loss of cytoplasmic structures & staining

H&E stain
Necrotic muscle fibers: Pale; Little phagocytosis (Arrows)
Gomori trichrome stain	H&E Necrotic muscle fiber, Early Fiber is pale & enlarged. Internal nuclei may reflect invasion by macrophages.

Necrosis: Early; "Metabolic" pattern
Muscle fiber cytoplasm: Pale
Nuclear staining: Absent

Gomori trichrome stain

Necrotic muscle fibers: Internal architecture has lost NADH staining (Arrow)

NADH stain

Rhabdomyolysis
Some non-necrotic muscle fibers have increased lipid (Left)
Phagocytic cells have only minor staining of their cytoplasm (Right)

Sudan black stain

Necrosis: Early; Membrane damage, Statin toxicity
Varied internal structure: Hypercontracted material & Palor in different areas of single fibers

H&E stain

Membrane damage at several regions of individual fibers: Associated with clear regions in cytoplasm

H&E stain

H&E stain

"C"- or "Delta"-lesions
Membrane & Neighboring cytoplasm damage in a focal region of individual muscle fibers: Pale staining
Cytoplasm around focal lesion stains dark

H&E stain

NADH stain

"C"- or "Delta"-lesions
Cytoplasm around focal lesion stains pale

NADH stain

Congo red stain

Phagocytosis of Muscle fibers
Histiocytic cells invade & replace muscle fibers

H&E stain
Necrotic muscle fibers: Phagocytosis (Arrows)
Gomori trichrome stain

Necrotic muscle fiber
Largely replaced by cells
Some remaining debris

H&E stain

NADH stain

Phagocytic cells within pale necrotic muscle fibers
Phagocytic cells have
Mild NADH staining
Strong esterase staining
Necrotic muscle fibers: Cytoplasm has little, or no, staining

Esterase stain

Acid phosphatase stain
Necrosis: Varied Stages
Early: Pale stained muscle fibers
Later: Muscle fibers progressively replaced by acid phosphatase + cells (Right)

Acid phosphatasae stain

Esterase stain
Necrotic muscle fiber, Late
Muscle fiber is invaded and
replaced by phagocytes.

Esterase stain

Necrosis & Phagocytosis: Late

H&E stain

Phagocytic cells
Replace necrotic muscle fibers
Move out of necrotic muscle fiber areas

Acid phosphatasae stain

H&E stain

Collapsed, phagocytosed muscle fibers
Space occupied by necrotic muscle fibers "Collapses"
Some histiocytes remain

Acid phosphatase stain

MHC Class I stain
Very late stage necrosis: MHC Class I
Stains remaining cells phagocytsing collapsed muscle fibers
Stains capillaries (Normal)
No staining of most muscle fibers (Normal)

Alkaline phosphatase stain
Necrosis: Alkaline phosphatase stains around the rim of muscle fibers

Toluidine blue stain Necrotic muscle fiber (Longitudinal section) Pale Invaded by cells
Toluidine blue stain Necrotic muscle fiber (Cross-section) Pale Invaded by cells	Toluidine blue stain Necrotic muscle fiber Pale Invaded by cells

Toluidine blue stain
Cells in necrotic fiber

Toluidine blue stain
Non-necrotic, abnormal muscle fiber (Left): Dilated SR

Necrotic muscle fibers: Complement C5b-9 (MAC) deposition

Stain: C5b-9 components of complement (Membrane attack complex (MAC))
Necrosis: C_5b-9 deposition diffusely within muscle fiber cytoplasm

Rhabdomyolysis: PAS stain is pale in small & large muscle fibers

Necrosis: Ongoing

Necrosis: Early stage
Pale muscle fiber cytoplasm
Lipid droplets
Two other muscle fibers have large lipid droplets but are not pale & necrotic

H&E stain

H&E stain

Necrosis: Phagocytosis of varying degrees

H&E stain

Necrosis associated with Eosinophils

Unusual: Eosinophils may be a component of cells replacing necrotic muscle fibers
Reported with LGMD 2A (Calpain-3 mutations)

H&E stain

H&E stain

Gomori trichrome stain

H&E stain

H&E stain

H&E stain

Necrosis: Scattered Muscle fibers

H&E stain

Necrotic Muscle Fibers: Scattered
Ongoing: Muscle fibers in varied stages of necrosis & regeneration
Also see: Scattered necrosis, Severe

H&E stain

Necrotic Muscle Fibers: Scattered
Monophasic: Scattered muscle fibers in early stage of necrosis
Cytoplasm of Necrotic Muscle fibers stains diffusely for C_5b-9