Mechanisms of muscle membrane repair: The dysferlin model

Eduard GallardoLaboratori de Neurologia ExperimentalInstitut de Recerca Hospital Sant Pau

2nd MuscleTech Network WorkshopSeptember 27–29, 2010Barcelona

Group objectivesOur main focus of interest is a muscular dystrophy caused by mutations in the gene DYSF (dysferlin).

Specific aims:• To study possible functions of the protein (membrane

repair, cell fusion, muscle differentiation, regeneration, inflammation...)

• To elucidate which proteins interact with dysferlin• Therapeutic approaches ( minidysferlins, cell therapies).• To develop new diagnostic tools and search for

biomarkers of the disease.

Caveolin-3

Telethonin

Filamin

C

DysferlinCalpain-3

Nebulin

Tropomodulin Titin

Myotilin

TRIM32

Integrins

complex

SEPN1

α-actinin

Z-disk

Collagen

IV

Dystrophinα

Laminin a-2

α-dystroglican

β-dystroglican

β

α

α γδ β

β1Distrobrevin

SintrophinsNO sintase

nNOS

γα

Sarcoglycans

complex

Sarcospan

Fukutin

Actin TropomyosinMyosin

Lamin

A/C Emerin

Z-disk I A M A I

Sarcomere

Desmin

Desmin

Por I. de Andrés

Nucleus

Sarcoplasm

Sarcolemma

DYSF + DYSF -

IMMUNOHISTOCHEMISTRY

Dysf +/+

Dysf -/-

Poor differentiation, fusion defect

De Luna et al J Biol Chem 2006

MEMBRANE REPAIR MODEL

Glover et al Traffic 2007

In vitro studies

250--160--105--75--50--

58--

CD14- CD14+CD14+ CD14-

Dysferlin 237 kDa

β-actin 42 kDa

Peri

pher

al b

lood

Skel

etal

mus

cle

7.5 --

4.5 --

2.0 --

kb

Diagnostic tool minimally invasive

Dysferlin is also expressed in peripheral blood monocytes

Ho et al Ann Neurol 2002

In vivo studies

Phenotypic

description

of

mouse

adult

mesoangioblasts

+ TGF‐β

Smooth

muscle

SMA

+ BMP

Bone

Alkaline

Phosphatase

Adipogenicmedium

Oil red

Low

serum

medium

Fat

Myosin

Skeletal

muscle

Diaz et al Cell Death and Disease (2010)

IM+CTX IM

IA+CTX IA Results at 1 month

Recovery

of

dysferlin expression

after

mesoangioblasts

trasnplantation

of

A/J SCID mice

Laser induced lesions in isolated single fibers

Fluorescent dye FM 4-64

Diaz et al Cell Death and Disease (2010)

Inflammation in dysferlin myopathy

MHC-I Macrophag. CD8

H&E Macrophag. CD8 Neurology 2001

MHC-I

CHEMOTAXIS ASSAYMONOCYTES

CONDITIONED MEDIA

Skeletal muscle cultures

Myoblasts Myotubes

CONDITIONED MEDIA

Contains released factorsby skeletal muscle cells

Results•

Chemotaxis

assay

De Luna et al J. Neuropathol Exp Neurol 2010

Results

•

Thrombospondin-1 (TSP-1) (3.2 fold

increase)

Microarray

analysis

comparing

dysf+/+vs

dysf

-/-

human myotubes (PROGENIKA)

Human genome

U133 Plus 2.0 array

De Luna et al J. Neuropathol Exp Neurol 2010

ELISA

TSP-1 EXPRESSION “in vitro”

De Luna et al J. Neuropathol Exp Neurol 2010

TSP-1 EXPRESSION IN MOUSE MODELS

B.10 (Dysf +/+) A/J (Dysf-/-)

De Luna et al J. Neuropathol Exp Neurol 2010

TSP-1 EXPRESSION “in vivo”Macrophages TSP-1 Merged

DYSF-/-

FSHD

IBM

HOT TOPICS IN THE AREA

• Phenotypic variability.How come some patients start developing the disease much later than others (from congenital forms to very late onset forms) although all of them present null or very low levels of dysferlin expression?

2009

HOT TOPICS IN THE AREA

• Patients with a single mutation (this is a recessive disease???)

HOT TOPICS

• Increased exercise accelerates the onset of the disease (clinical observation)?.However, recent works using mouse models, suggest that mild exercise may be benefitial for the patients. Concentric (good)versus eccentric (bad)

KEY QUESTIONS

• What are the functions of dysferlin?Membrane repairMuscle differentiation

• What is the origin of muscle inflammation in dysferlin myopathy?Abnormal upregulation of TSP-1 in muscleAbnormal function of monocytes due to the lack of dysferlin

From translational research to translational medicine

Question 1:Insights into how could it be done to translate the day to dayscientific discoveries into practical applications, clinical level,or patient's “bedside” to improve human health.

• Development of diagnostic tools (dysferlin expression in monocytes)• Development of biomarkers of the disease :

1. Use of dysferlin expression in peripheral blood to asses the efficacy of drugs instead of repeated muscle biopsies2. Levels of TSP-1 in serum as a specific marker of the disease (instead of CKs) or as a marker of disease progression…..

Question 2: Impact the research on muscle and tendon could have onhealth and society, both socially and economically. • Use of cell therapy to treat muscle diseases• Discovery of drugs that may improve muscle membrane resealing both in

patients with muscular dystrophies and in injured muscle in healthy people.

• Unitat Neuromuscular HSCSP, BarcelonaDra. ILLADr. RojasDr. DiazDra. IturriagaDra. HankiewiczDra Martinez

• Lab Neurologia ExperimentalDra. De LunaB. FlixJ. AraqueE. OrtizX. Suárez

• Hospital St Rafaele, MilanDr. Cossu

• Lab. Morfología Celular. Unidad Mixta. CIPF-UVEG

Dr García-VerdugoMario Soriano

• University of MassachusettsDr. Brown