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Enhancing myoblast fusion for therapy of muscular dystrophies
Melissa WuThesis Defense
January 28, 2013
Overview
• Skeletal Muscle Structure
• Muscular Dystrophies
• Thesis1. Enhancing Fusion in
DMD2. Investigating Process of
Fusion
• Conclusions
Selections from the clinical works of Duchenne, ed. GV Poore, 1883.
Skeletal Muscle Structure
Hematoxylin and Eosin staining of muscle cross-
section
Myofiber nuclei
Satellite stem cell
Muscle myofiber
Skeletal muscle made up of bundles of
myofibers
Skeletal muscle drawing from MDA website
sarcolemma
Satellite Cells Regenerate Muscle
Satellite cell activation
Pax7Myf5MyoD
Differentiation and Fusion
MyoD and myogenin
Skeletal Muscle Dystrophies:Duchenne Muscular Dystrophy
• Affects 1:3300 boys
• Progressive muscle weakness
– Instability of sarcolemma
• High serum creatine kinase
– Muscle degeneration
– Satellite cells repair, but are exhausted
– Inflammation, fibrosis, adipogenesis
• Average lifespan ~30 years http://neuromuscular.wustl.edu/pathol/dmdpath.htm
Genetic Mutations in DAPC Proteins Result in Muscular Dystrophies
dystrophin
α β
sarcoglycans
γ dystroglycanYYl
YYl
YYl YYlF-actin
Limb Girdle Muscular Dystrophies 2C, 2D, 2E, 2F
Duchenne Muscular DystrophyBecker’s Muscular Dystrophy
Dystroglycanopathies:Muscle-Eye-Brain DiseasesFukuyama Congenital Muscular DystrophyWalker-Warburg SyndromeCongenital Muscular Dystrophy Type 1DLimb Girdle Muscular Dystrophy
δβ
αYYl
YYl
laminincollagen
Congenital Muscular DystrophiesUllrich Congenital Muscular DystrophyMerosin-deficient Congenital Muscular Dystrophy
sarcolemma
Treatment of Duchenne Muscular Dystrophy
Diagnosis and management of Duchenne muscular dystrophy, DMD Care Considerations Working Group, 2009
glucocorticoids VascularizationMembrane strengthMuscle growth
Therapeutics that address symptoms
Dystrophin replacement therapies
Gene therapy Read-through or exon-skipping therapies Cell transplantation
Enhancing fusion for therapy of DMD1. Enhance fusion of endogenous satellite cells to improve muscle2. Transplant cells to introduce dystrophin
Enhance endogenous fusion Transplant cells
• Can be used in different dystrophies• Can also be used in conjunction with transplanted cells
• Works in theory, but often injected cells die upon injection or do not fuse in • Need to understand better natural process of fusion
Hypothesis
Enhancing fusion can activate endogenous cells to repair damaged muscle or improve the efficacy of myoblast transplantation
1. Test carbamylated erythropoietin (C-EPO) as a therapeutic in mdx mice
2. Explore role of GPR56 in myoblast differentiation and fusion
Carbamylated Erythropoeitin Protects Tissues Without Overstimulating Blood Cells
Hematopoiesis Tissue Protection
Modified from slide created by Journal of Clinical Oncology
• Stimulates RBC production • Overstimulation leads to anemia
• Prevents apoptosis• Limits fibrosis • Stimulates satellite cell proliferation• Stimulates muscle fiber growth
EPO-R β-CR
XC-EPOEPO
Does administration of C-EPO improve dystrophic signs in mice with muscular dystrophy?
124
Week
Inject C-EPO 3x weekly intraperitoneally: 0, 50, 100 μg/kg
Assay for:Muscle growth (weight)
Changes in histologySarcolemmal integrity
mdx
C-EPO treatment does not affect weight of mdx mice
15
20
25
30
35
-1 1 3 5 7 9 11 13
Wei
gh
t (g
)
Time (Weeks)
0 ug/kg
50 ug/kg
100 ug/kg
0 μg/kg50 μg/kg100 μg/kg
4 w
eeks
C-EPO effects on mdx diaphragm0 μg/kg 50 μg/kg 100 μg/kg
50 m
12 w
eeks
C-EPO Induces a short-term increase in regeneration
4 weeks 12 weeks0
10
20
30
40
50
60
70
0 μg/kg
50 μg/kg
100 μg/kg
% fi
bers
with
cen
tral
ly lo
cate
d nu
clei
**
Myofiber size did not increase
4 weeks 12 weeks0
5
10
15
20
25
300 μg/kg50 μg/kg100 μg/kg
Aver
age
myo
fiber
dia
met
er (μ
m)
Treatment did not improve areas of fibrosis
4 weeks 12 weeks0
2
4
6
8
10
12
14
16
18
20
0 μg/kg
50 μg/kg
100 μg/kg
% in
ters
titial
are
a *
Serum Creatine Kinase levels did not improve
4 weeks 12 weeks0
500
1000
1500
2000
2500
3000
0 μg/kg50 μg/kg100 μg/kg
Seru
m C
reati
ne K
inas
e (U
/L) *
Conclusions
• C-EPO increased regeneration in the short-term (4 weeks)– Did not increase average muscle fiber size
• No effect on overall health of tissue– Did not reduce fibrosis– Did not improve muscle integrity
• Why no improvement?– Anemia noted in C-EPO treated mice, especially at 12 weeks– May have interfered with tissue-protective effects
• Overall, the effects of C-EPO treatment are modest
Studying Fusion Process
Enhancing fusion can activate endogenous cells to repair damaged muscle or improve the efficacy of myoblast transplantation
1. Test C-EPO as a therapeutic in mdx mice
2. Explore role of GPR56 in myoblast differentiation and fusion
NFATc1NFATc2
Myoblast differentiation
Mature myotubesCommittedmyocytes
Early myotubesProliferating myoblasts
Pax7Myf5
NFATc3SRF
MyoD
myogenin
FHL1Tran
scrip
tion
Fact
or A
ctivi
ty
Proliferating myoblasts
Late myotubes (>15 nuclei)
Early myotubes (2-5 nuclei)
Cerletti et al JCS 2006
Screen to find effectors of fusion
NH3 COO-
G-protein coupled receptor 56: Adhesion receptor
GbGg
Ga
q
12/13
l
ll
l
GPS
CD9 CD81
RhoA
Putatively partners with or activates proteins that have a role in myogenesis
SRE E2F NFAT
fusion
GPR56 causes “cobblestone brain” neuropathySome dystroglycanopathies are characterized by
a cobblestone brain phenotype
dystrophin
α β
γ
YYl
YYl
YYl YYlδ
β
αYY lYYl
Bilateral frontoparietal polymicrogyria (BFPP)
Piao et al, Science 2004.
BFPP
normal
Does GPR56 have a role in the muscle?
l
ll
l
?
GPR56 expression is upregulated during muscle cell fusion in vitro
D0 D1 D2 D3 D6
Rela
tive
mRN
A ex
pres
sion
D0 D1 D2 D3 D6 D0 D1 D2 D3 D6
GPR56
MyoD
myogenin
α/β tub
GPR56 is expressed in committed myoblasts
GPR56 GPR56 caveolin-1Nuclei
NFATc1NFATc2
GPR56 is expressed in early differentiation
Mature myotubesCommittedmyocytes
Early myotubesProliferating myoblasts
Pax7Myf5
NFATc3SRF
MyoD
myogenin
FHL1Tran
scrip
tion
Fact
or E
xpre
ssio
n
GPR56X
Knockout GPR56 myoblasts fuse less and have dysregulated effectors of differentiation
p < 0.05*p < 0.05
*p < 0.05
*
D2 D50
20
40
60
80
100Fusion Index
WT KO
Fusio
n In
dex
(%)
D2 D50
5
10
15
20
25
30Myotubes with >5 nuclei
WT KO>5
nuc
lei (
%)
GPR56 siRNA
0 1 2 3 4 5 60
0.5
1
1.5
2GPR56
uninfected
scrambled
shRNA 2
shRNA 3
Days in Differentiation Media
Rela
tive
mRN
A e
x-pr
essi
on
NH3 COO-GPS
D0 D1 D2 D3 D5
GPR56-silenced C2C12 cells fuse less and make smaller myotubes
unin-fected
scrambled shRNA2 shRNA305
101520253035
Fusio
n in
dex
(%)
*
uninfected scrambled shRNA2 shRNA3
p < 0.01
MHCDAPI
Fusion index
unin-fected
scrambled shRNA2 shRNA302468
101214161820
% tu
bes >
5 nu
clei
**
p < 0.01
p < 0.01
Myotubes >5 nuclei
Unlike in primary KO myoblasts, MyoD is not affected and myogenin is decreased
0 1 2 3 4 5 60
200400600800
10001200
myogeninuninfectedscrambledshRNA2shRNA3
Days in differentiation
Rela
tive
mRN
A e
xpre
ssio
n
Increased signs of proliferation seen in GPR56-silenced/KO myoblasts
0 2 4 6 8 10 120
200000
400000
600000
800000
1000000WTKO
Days in Proliferation
Num
ber o
f cel
ls (x
106) *
******
***
Pax7a/b tub
0 1 2 3 5 0 1 2 3 5 0 1 2 3 5 0 1 2 3 5
uninfected scrambled shRNA 2 shRNA 3
Cell proliferation in WT and KO myoblasts
In vitro studies show GPR56 promotes commitment and fusion
• GPR56 KO and GPR56 shRNA highlight two roles for GPR56– Promotion of MyoD maintenance for commitment
to differentiation and early fusion– Promotion of differentiation leading to fusion
• Loss of GPR56 results in less cells committing to differentiation and initiating early fusion
GPR56 knockout in vivo phenotype
WT KO
WT KO37
37.5
38
38.5
39
39.5
40
Average Myofiber Diameter
Myo
fiber
Dia
met
er
(µm
)
0.8 1 1.2 1.4 1.6 1.8 2 2.20
10203040506070
Serum Creatine KinaseSe
rum
Cre
atine
Kin
ase
(U/L
)
WT KO
*
p = 0.01
Loss of GPR56 does not alter muscle regeneration
0
1
2
3
4
5
6
0 5 10Rela
tive m
RN
A e
xpre
ssio
n
Days after Ctx injury
GPR56 mRNA expression
4 6 180
10
20
30
40
50WTKO
Days after Ctx injury
Myo
fiber
Dia
met
er (µ
m)
Myofiber diameter
WT KO
Regeneration accompanied by delayed expression of markers of differentiation
0 2 4 6 80.0
0.1
1.0
8.0
64.0Myf5
WT KO
*
*
0 2 4 6 80.0
0.1
1.0
8.0
64.0MyoD
**
0 2 4 6 80.256
2.56
25.6
256myogenin
*
0 2 4 6 80.16384
1.6384
16.384
163.84
1638.4
16384embryonic MHC
0 2 4 6 8
0.1
1
10NFATc2
0 2 4 6 80.16
1.6
16FHL1
*
NFATc2
Myf5MyoD
myogenin
FHL1Tran
scrip
tion
Fact
or E
xpre
ssio
n
* P < 0.05
l lll
Defining the GPR56 signaling pathway using luciferase assay
Ca2
+
Ca2
+
GbGg
Ga12/
13
PLC-b
NFAT
NFAT
• Gb/Gg signals to NFAT
GEFs
RHO
SRF
SRF
• Ga12/13 to Rho to SRF
• Transfect HEK293 with:– GPR56 or truncated
GPR56– Luciferase reporter gene
under various promoters– β-gal gene for
normalization
SRE NFAT-RE
GPR56 signals strongly to SRE, less strongly to NFAT-RE
* p < 0.05, *** p < 0.001
SRF and NFAT target transcripts in muscle cells
SRE
Gα
RhoA
Differentiation
SRF
SRF
FHL MyoD
NFAT
?
NFAT
MyoD, FHL1, NFATc2, and NFATc3 are decreased in knockout muscle
MyoD FHL1 NFATc1 NFATc2 NFATc3 NFATc40
0.5
1
1.5WT KO
Rela
tive
mRN
A e
xpre
ssio
n
* * **
NFAT targets include genes involved in myofiber specification
Myofiber growth and
Fiber type specification
NFATc3SRF
NFATc1
NFATc2
NFATc1
NFATc2
NFATc3SRF
NFATc4
Multi-nucleated Myotube/fiber
Commitment/differentiation
Fusion into early tubes
Proliferation
IIIA
IIB
Fiber type proportion is unchanged in KO mice compared to WT mice
MHC I MHC IIA MHC IIB
Laminin
WT KO WT KO
4 mo 9 mo
Summary
NFATc1NFATc2
Pax7Myf5
NFATc3SRF
MyoD
myogenin
FHL1Tran
scrip
tion
Fact
or E
xpre
ssio
n
GPR56
• Promotes commitment to differentiation and early fusion• Identified a cell surface molecule that can promote signaling to SRF and NFATc2
during differentiation• Loss is compensated by other factors• Does not have a role in mature muscle
Future implications for DMD therapy
• Fusion of endogenous muscle stem cells– Issues to consider:
• Duration of treatment• Target tissues• Delivery Method
– Perhaps better to use directly with transplanted cells
• Studying fusion for therapy– Identified a cell-surface molecule involved in promoting the
commitment to differentiation and early fusion of cells– Next step: overexpression further increase engraftment and
fusion of transplanted cells– Validation of the initial microarray to identify new players, can
study other candidates including other GPCRs
Thanks for your brains and laborC-EPO
Shire Therapeutics Arthur Tzianabos Gregory Robinson
Emanuela Gussoni Matt Mitchell
GPR56
Emanuela Gussoni Ariane Beauvais Chelsea Cherenfant
Michael Lawlor Hui Meng Alexandra Lerch Gaggl
Isabelle Draper Alan Kopin Jamie Doyle
Thanks for your brawn (muscle)
Great mentors and judges
Dissertation Advisory Committee
Dissertation Defense Committee
Thanks for keeping me sane!
Thanks for everything