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Research Experience in Molecular Biotechnology & Genomics
Summer 2010
Center for Integrated Animal Genomics
PGC1α over-expression rescues skeletal muscle from mdx mice
Connie M Santana¹,², Delphine Gardan-Salmon ¹, Katrin Hollinger¹, Joshua T Selsby¹,
Department of Animal Science, Iowa State University¹. Department of Biology, University of Puerto Rico, Rio Piedras Campus²
Abstract
Objective and Rationale
Duchenne muscular dystrophy (DMD) is the most severe
and common type of muscular dystrophy. Activation of
peroxisome proliferator-activated receptor γ coactivator 1α
(PGC-1α) is a possible therapeutic strategy for DMD
treatment due to its involvement in mitochondrial biogenesis
and regulation of utrophin expression, a dystrophy homolog.
Previous studies have indicated the positive effects of PGC1α gene transfer in preventing typical pathology and acute
injury in dystrophin-deficient muscles, however, the extent
to which this treatment is effective in rescuing dystrophindeficient skeletal muscle is unclear. In this experiment, mdx
mice, a mouse model of DMD, were allowed to age for three
weeks prior to being injected in the right hind limb with an
adeno-associate virus 6 (AAV6) driving expression of PGC1α and in the left hind limb with a null virus. At six weeks of
age, mice were euthanized and muscles removed. Overexpression of PGC-1α resulted in a reduction in total
damaged area. More specifically, PGC-1a over-expression
reduced areas of hypercontracted cells, immune cell
infiltration, and absent cells, all indicators of muscle injury.
Our results highlight the importance of PGC-1α in the rescue
of dystrophin deficient skeletal muscles, and present a
potential avenue for treating patients with DMD.
The objective of this study focuses on determining the extent
to which PGC-1α over-expression will rescue dystrophindeficient skeletal muscle from disease associated injury.
Introduction
Duchenne muscular dystrophy (DMD) is the most
prevalent type of muscular dystrophy, affecting one in every
3,500 boys [4].
DMD is caused by an aberration in the dystrophin gene,
which codes for the protein dystrophin [2].
Dystrophin is responsible for connecting the actin
cytoskeleton ultimately to the extracellular matrix [2].
The absence of dystrophin permits the entrance of
the sarcoplasm [2].
Ca2+
Methods
Histology: Upon being removed, muscles were placed in a
freezing media and frozen in liquid nitrogen. In accordance
with standard techniques, hematoxylin and eosin staining
was performed. These sections were utilized for
morphological assessments including determination of the
area of immune cell infiltration, hypercontracted cells, and
absent cells. Photoshop was used to reconstruct images of
whole muscles and OpenLab software was used to analyse
and quantify the area of the images.
Statistical analysis: Treated and control mice were
compared using paired T-tests, and the statistical significance
was set at p<0.05. * indicates significant differences between
these groups.
Muscle Mass
G
-P
G
m
dx
-P
Gastrocnemius
C
-1
a
dx
m

C
-1
dx
dx
In dystrophin-deficient muscle, we have previously
established that PGC-1a over-expression will prevent
pathologic muscle changes as well as muscle injury caused
by lengthening contractions, simulating normal activity [3].
*
1.0
0.5
-1
a
dx
C
dx
-P
G
m
m
dx
-P
G
C
-1
a
dx
m
-1

C
1.5
0.0
0
dx
-P
G
C
dx
-P
G
m
1
m
-1
a
dx
m
*
2.0
m
0
0
2
Percent Total Area
4
Percent Total Area
5
Percent Total Area
*
10
*
8
Hypercontracted cells
2.5
3
12
15
D
Absent Cells
Figure 2. Representative histological sections of dystrophin
deficient soleus muscle (10x). Dystrophin-deficient mice
were injected with an AAV driving PGC-1α (Right) or null
virus (Left) at three weeks of age. Three weeks following
injections we observed reduced total necrotic area in PGC-1
α expressing muscle when compared to null injected muscles
(A). More specifically, areas of immune cell infiltration (B;
Green arrows), absent cells (C; Black arrows), and
hypercontracted cells (D; Blue arrows) were also reduced in
PGC-1 overexpressing muscle compared to control limbs. *
indicates significant different from control (p<0.05).
Conclusions
These data suggest that 3 wks of PGC-1α over-expression in
mature dystrophin deficient mice:
 reduced areas of hypercontracted cells, immune cell
infiltration, and absent cells.
References
m
PGC-1α helps reduce the DMD pathology by increasing
the expression of oxidative proteins, and promoting utrophin
expression [2,3].
*
m
PGC-1α is a member of the transcription coactivators
family that regulates mitochondrial function and the
induction of slow gene expression, like utrophin.[1,3,4].
mg
Utrophin, a dystrophin related protein, has been shown to
function as a substitute for the missing dystrophin protein.
C
Together with previous data demonstrating disease
prevention, these data demonstrating disease rescue strongly
suggest that this pathway should be pursued as a potential
therapeutic intervention.
Ca2+
120
100
80
60
40
20
10
8
6
4
2
0
Immune Cell Infiltration
 was successful in rescuing dystrophin deficient skeletal
muscle.
Results
Increased
activates signaling cascades resulting in
mitochondrial dysfunction, oxidative stress, and proteolysis
leading to cell injury and death [2].
B
Total damaged area
20
Animal treatments: Three-week-old mdx mice were
injected in the right hind limb with an AAV6 driving
expression of PGC-1α (1x1011 gc) and in the left hind limb
with null AAV6. At six weeks of age the animals were
sacrificed and muscles removed. Muscles were stored for
histology or biochemistry, as appropriate.
to
A
dx
• Tissues are collected
• Inject virus into mice
• R leg – PGC-1α virus
• L leg – Null virus
Percent Total Area
• mdx mice were
obtained from our
colonies
mdx-PGC-1α
mdx
6 weeks
m
3 weeks
0 weeks
Soleus
Figure 1. Dystrophin-deficient mice were injected with virus
causing over-expression of PGC-1α at three weeks of age
and sacrificed at six weeks of age. PGC-1α over-expression
caused a reduction in the muscle mass of the gastrocnemius
but not in the soleus at time of sacrifice. * indicates p<0.05.
[1] Angus, L.M. et al. (2005) Calceneurin-NFAT signaling, together with GABP
amd peroxisome PGC-1α, drives utrophin gene expression at the neuromuscular
junction. Am J Physiol. Cell Physiol. Vol. 289, p.p. C908-C917
[2] Davies,K.E and Nowak,K.J. (2006). Molecular mechanisms of muscular
dystrophies: old and new players. Nature reviews. Molecular cell biology. pp.
762-773.
[3] Liang H. and Ward,WF.(2006) PGC-1alpha: a key regulator of energy
metabolism. Adv Physiol Educ.Vol. 30, p.p. 145-151.
[4]Miura,P. and Jasmin, BJ.(2006) . Utrophin upregulate for treating Duchenne
or Becker muscular dystrophy:how close are we?.Elsevier p.p. 122-129.
Acknowledgements
This work was partially supported by the National Science Foundation Research
Experience for Undergraduates and the Center of Integrated Animal Genomics. I
would like to thank Dr. Josh Selsby, Dr. Delphine Gardan, and Katrin Hollinger
for their guidance, support, and help during this project. I would also like to
thank Dr. Max Rothschild for the guidance given throughout the program.
Program supported by the National Science Foundation Research Experience for Undergraduates
DBI-0552371