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