1

WHAT HAPPENS WHEN YOU HAVE NO MUSCLESMuscular dystrophies

•Genetic disorders•Heterogeneous•Primarily affect the musculature•Tend to affect the connection between the sarcomeric structureand the basement membrane, surrounding the muscle cells

Muscular dystrophies, clinical features

Increased endomysial connective tissueVariable fiber sizeCentrally located nucleiInfiltrationHypercontracted muscle fibers

Which genes are involved??

• Mutations in more than 100 genes are likely to resultin myopathies

•~40 genes have so far been identified

•Transmembrane proteins•Extracellular matrix proteins•Membrane-associated proteins•Cytoplasmic proteases•Cytoplasmic proteins associated with sarcomeres•Protein modifying enzymes•Nuclear membrane proteins

LAMININ-α2α

α-DYSTROGLYCAN

β-DYSTROGLYCAN

ACTIN FILAMENT

DYSTROPHIN

γ-SARCOGLYCAN

α-SARCOGLYCAN

ββ SARCOGLYCANα

β β

α

ARMD, LGMD2D

CMD (dy/dy; dy)2J

ARMD, LGMD2E

ARMD, LGMD2C

DMD, BMD (mdx)

MUSCULARDYSTROPHIES:

C

N

modified according to : Campbell, 1995; Worton, 1995

α

γ

α DYSTROGLYCAN

β DYSTROGLYCAN

α SARCOGLYCAN

γ SARCOGLYCAN

BASEMENT MEMBRANE

Transmembrane receptors in skeletal muscle Dystrophin deficiency/Duchenne muscular dystrophy

• The most common form of muscular dystrophy in human.

• About 1 in 3000 boys is affected by the disease.

• Mutations are inherited or occur spontaneously due to the large size of the gene.

• The phenotype ranges from mild to severe.

Mild form (Becker muscular dystrophy) small in frame deletionsthe function of the protein is partially maintained. Severe form (Duchenne muscular dystrophy) complete absenceof the protein. Death post-puberty.

• The corresponding mouse model mdx, lacking dystrophin, develops histologically a muscular dystrophy but has a normal life span

2

Integrins The Integrin Super-Family

MTJMTJmyoblasts

primary myogenesis secondary myogenesis adult

NMJ

α5α5

α6 α6α7 α7

α7

α4

α4

αvαv

αv

α3

β1A β1D

β1D

α7β1 INTEGRIN

- two extracellular (X1, X2)two intracellular splice variants (A, B)

- specific laminin receptor

- mainly expressed in skeletal and cardiac musclebut also found in smooth muscle, neurons, melanocytes

- associates with β1D in skeletal and cardiac muscle

- deficiency of the α7 subunit leads to a muscular dystrophy in human and mice

Tendon

myofilaments

sarcolemma basement membrane

Tendon

myofilaments

3

Tendon

myofilaments

Tendon

Myotendinous junctions phenotype in integrin α7 null mice

WT

A7-/-

LAMININ-α2α

α-DYSTROGLYCAN

β-DYSTROGLYCAN

ACTIN FILAMENT

DYSTROPHIN

γ-SARCOGLYCAN

α-SARCOGLYCAN

ββ SARCOGLYCANα

β β

α

ARMD, LGMD2D

CMD (dy/dy; dy)2J

ARMD, LGMD2E

ARMD, LGMD2C

DMD, BMD (mdx)

MUSCULARDYSTROPHIES:

C

N

modified according to : Campbell, 1995; Worton, 1995

α

γ

MD INTEGRIN α7β1

α DYSTROGLYCAN

β DYSTROGLYCAN

α SARCOGLYCAN

γ SARCOGLYCAN

BASEMENT MEMBRANE

MUSCULARDYSTROPHIES:

Transmembrane receptors in skeletal muscle

WT

WT A7-/-

mdx

A7-/-A7-/-

WT

mdx

Mdx phenotype A7-/- phenotype

lateralstability

MTJstability

α-DYSTROGLYCAN

β-DYSTROGLYCAN

ACTIN FILAMENT

α

β β

α

C

N

modified according to : Campbell, 1995; Worton, 1995

α

γ

α DYSTROGLYCAN

β DYSTROGLYCAN

BASEMENT MEMBRANE

Transmembrane receptors in skeletal muscle

INTEGRIN α7β1

Integrin α7-/- mice

xmdx mice

(dystrophin-deficient)

4

WT α7-/- mdx mdx/α7-/-

Tibialisanterior

Peroneus

Flexorhallicuslongus

ExtensordigitorumLongus(EDL)

0 5 10 15 20 250

2

4

6

8

10

12

14 A7+/+; mdx, female, n=1 A7+/-; mdx; female, n=1 A7+/-; mdx; male, n=4 A7-/-; mdx; female, n=1

wei

ght (

g)

age (days)

Integrin α7/mdx double-mutant micegain less weight than controls

Cen

tral n

ucl

ei(%

)

EDL EDLPlantaris Plantaris

P10 P20

The number of fibers withcentrally located nuclei is low

WT mdx/α7-/- WT mdx mdx/α7-/-

P10

P20

EDL Plantaris

mdx

Nu

mb

er

of

mu

scle

fib

ers

EDL EDLPlantaris Plantaris

P10 P20

The number of muscle fibers is reduced

5

DKO

mdx

TUNEL + Lm α2 TUNEL + MHCdev H&E

Apoptosis is not prominent

adapted from Hawke and Garry, 2001

Satellite cell activation and function

adapted from Hawke and Garry, 2001

Satellite cell activation and renewal isimpaired in integrin α7/mdx double-mutant mice

DGC-/-

adapted from Hawke and Garry, 2001

Satellite cell activation and renewal isimpaired in integrin α7/mdx double-mutant mice

DGC-/-/α7-/-

WT α7-/- mdx mdx/α7-/-

Dystrophin

Integrin α7

Utrophin

α-Sarcoglycan

α-Dystroglycan

WT α7-/- mdx mdx/α7-/-

Laminin α2

Laminin α4

The laminin α2 chain is unaltered in double-mutant mice

6

α

β-DYSTROGLYCAN

ACTIN FILAMENT

α

β β

α

C

N

modified according to : Campbell, 1995; Worton, 1995

α

γ

α DYSTROGLYCAN

β DYSTROGLYCAN

BASEMENT MEMBRANE

Transmembrane receptors in skeletal muscle

INTEGRIN α7β1

ACTIN FILAMENT

α

β β

α

C

N

α

γ

α DYSTROGLYCAN

BASEMENT MEMBRANE

Transmembrane receptors in integrin α7/mdxdouble-mutant mice

UTROPHIN

The phenotype of Integrin α7/mdx double-mutant mice closely resemblesDuchenne muscular dystrophy

Could integrin α7 be a therapeutic target for DMD?