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
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
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)
Laser induced lesions in isolated single fibers
Fluorescent dye FM 4-64
Diaz et al Cell Death and Disease (2010)
CHEMOTAXIS ASSAYMONOCYTES
CONDITIONED MEDIA
Skeletal muscle cultures
Myoblasts Myotubes
CONDITIONED MEDIA
Contains released factorsby skeletal muscle cells
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
TSP-1 EXPRESSION IN MOUSE MODELS
B.10 (Dysf +/+) A/J (Dysf-/-)
De Luna et al J. Neuropathol Exp Neurol 2010
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?
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