Download Fresh fibers 72 H rs culture Wild Type Mutant Cells per single

Role of PPARδ in satellite cells and muscle
development
A. Angione, S. Kuang
Department of Animal Sciences, Purdue University, West Lafayette, Indiana USA
•Satellite cells are muscle progenitor cells that are responsible for the
growth and maintenance of adult skeletal muscle1
WT
MUT
•PPARδ is a member of the peroxisome proliferation activated
receptor (PPAR) family nuclear receptors and has been shown to be
important in the maintenance of the slow fiber types2,3; however, the
function of Pparδ in satellite cells is still unknown.
1
0.8
0.6
0.4
0.2
0
WT
MUT
Wild Type
1.2
1
0.8
Ki67
Dapi
0.6
0.4
0.2
2.5
0
WT
MUT
Mutant
Pax7
Dapi
ki67 intensity ratio
*
2
1.5
1
0.5
0
WT
Fresh fibers
•Establish a mouse model for conditional mutation of PPARδ in the
myogenic progenitor cells
1.2
Pparγ
Mut
*p<0.05
percentage of fibers
•Fast muscle fibers (type 2; including 2a, 2b, and 2x) are mainly
glycolytic and much less resistant to fatigue2
*
Pparα
intensity
•Slow muscle fibers (type 1) use oxidative metabolism to produce
energy and are resistant to fatigue2
Pparδ
Relative mRNA levels
•Mature muscle cells (fibers) can be classified as either slow or fast
fibers based on differences in metabolism and rate of fatigue2
1.2
1
0.8
0.6
0.4
0.2
0
Mutant
*p<0.05
Relative mRNA levels
•Skeletal muscle is the most abundant tissue in the body and its
proper function is critical for motor performance and human health3.
Relative mRNA levels
Wild Type
14 day Regeneration after
injury
100%
80%
60%
40%
20%
0%
*
*
small
large
•Investigate the effect of Pparδ mutation on satellite cells
•Determine the role Pparδ in muscle differentiation and function
Mutant Pparδ
•A mouse model was created which has a conditional mutation of the
Pparδ gene restricted to the myogenic progenitor cells in the embryo and
later restricted to the muscle and brown fat lineage in the adult using
myf5-cre mediated recombination of loxP sites.
•Representative slow (Soleus; SOL) and fast (EDL) muscles were analyzed
for fiber type composition using myosin heavy chain isoform specific
monoclonal antibodies
•Single fibers were isolated from the EDL using collegenase B digestion
and the number of satellite cells per fiber determined
•Satellite cells were harvested from the hind limbs of wild type and
mutant littermates and cultured for proliferation and differentiation assay
•Glucose tolerance was tested to confirm the insulin sensitivity in wild
type and mutant littermates 8-9 months of age
Cells per single fiber
Number of cells per fiber
•Quantitative real-time (RT)-PCR was performed to confirm tissue-specific
knockdown of Pparδ.
Pax7
MyoD
Dapi
**
12
10
8
6
Percentage of cells per cluster
(SOL)
0.8
Percentage of cells
+ Myf5-Cre
72 Hrs culture
Wild Type Pparδ
Wild Type
**
4
2
0
EDL
Wild Type
0.6
0.4
0.2
0.0
*
*
•Pparδ expression has been reduced in the skeletal muscle, while the expression
of Pparα and Pparγ remain unchanged, suggesting that there is no compensation
from other PPAR isoforms.
•Fibers from slow and fast muscles have fewer satellite cells per fiber in the
mutant animals compared to wild type animals.
•Cultured fibers from the soleus had more differentiating satellite cells (MyoD+)
and fewer proliferating satellite cells (Pax7+/MyoD+)in the mutant animals
•Satellite cells from mutant animals showed less proliferation in vitro
•The expression of known Pparδ target genes was reduced in the skeletal muscle
of mutant animals
•Satellite cell mediated muscle regeneration after injury is delayed in mutant
animals
1. Kuang S, et al. Cell. 2007; 129(5):999-1010.
2. Wang YX, et al. PLoS Biol. 2004; 2(10):e294.
3. Schuler M, et al. Cell Metab. 2006; 4(5):407-14.
SOL
Mutant *p<0.05, **p<0.001
Mutant
This project is funded by grants from the USDA and MDA