Transcript No Slide Title - Michigan State University

538.11 Increased Androgen Receptor Expression in Skeletal Muscle Fibers Leads to Muscle Pathology
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Jamie A. Johansen D. Ashley Monks S. Marc Breedlove
Cynthia L. Jordan
1Neuroscience Program, 2Department of Psychology
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Michigan State University; Department of Psychology University of Toronto at Mississauga
Introduction
• The spinal nucleus of the bulbocavernosus
(SNB) is a sexually dimorphic group of
motoneurons that innervate sexually dimorphic
perineal muscles, the bulbocavernosus (BC) and
levator ani (LA).
• These muscles are highly androgen responsive;
the size of LA muscle fibers is reduced by
castration and restored with testosterone treatment
in adulthood. In contrast, the extensor digitorum
longus (EDL) is not responsive to androgens,
showing no change in size after castration.
• These differences in androgen responsiveness
parallel a difference in androgen receptor (AR)
expression. LA muscle fibers contain much more
AR immunoreactivity than EDL muscle fibers.
• Question: Does AR expression within muscle
fibers account for differences in androgen
responsiveness?
• We generated transgenic mice that over-express
the rat AR selectively in muscle fibers using the
human skeletal actin (HSA) promoter to ask
whether EDL would now become androgen
responsive.
Methods
Results
Summary
AR immunoreactivity is selectively increased in
muscle fibers of transgenic (Tg) mice.
Testosterone (T) induces male-like pathology in muscles
from transgenic females as revealed by NADH stain.
• Unexpectedly, our mice develop an androgendependent pathology. Transgenic males exhibit
profound abnormal muscle pathology demonstrated
by atrophic and angular-shaped fibers, frequent
internal nuclei, occasional split fibers and ring
fibers.
Figure 1. Note that AR immunoreactivity is increased only in fibers and not
fibroblasts of muscles from transgenic males (right). Internal nuclei, an
indication of muscle pathology, also stain positively for AR (arrow).
Over-expression of wildtype AR in muscle fibers of
transgenic male mice leads to muscle pathology.
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• AR immunohistochemistry confirmed overexpression of AR specifically in muscle fibers, and
not in fibroblasts or Schwann cells.
• Testosterone treatment induced a male-like
pathology in transgenic females, including “ring”
fibers, internal nuclei, and small angular fibers.
Figure 4. NADH staining reveals that T treatment has marked effects on EDL
muscles from Tg females including alterations in the internal architecture of fibers,
and size and shape of the fibers. Note that ring fibers (arrows) are prevalent in the
T-treated Tg muscle, and absent in blank-treated Tg muscle. The somewhat darker
staining in T-treated female muscles also suggests that T induces a switch to a more
oxidative metabolism reminiscent of Tg male mice.
• A shift towards oxidative metabolism is seen in
transgenic males and testosterone treated transgenic
females.
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Significance
Figure 2. H&E staining of EDL muscles of wildtype (Wt) and transgenic male
mice indicate that transgenic muscles contain both abnormally small, angular
fibers (black arrow) and hypertrophied fibers (white arrow). Both fiber types
contain internal nuclei (stars).
T induces male-like pathology in Tg female
muscles as revealed by H&E stain.
• These results suggest that AR in muscle fibers
have an unexpected role in muscle health and
disease.
• Androgens may regulate myofibrillar organization
within muscle fibers.
Over-expression of wildtype AR leads to increased
oxidative metabolism as seen in NADH stain.
Acknowledgments
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• Construct: AR cDNA was subcloned from pCMVAR. cDNAs
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were ligated into the NotI site of pBSX-HSA.
• Histology: Muscles were dissected from transgenic and
wildtype males, placed in OCT filled cryomolds and flash frozen
in liquid nitrogen. Muscles were then cryostat sectioned at 10um,
and stained for Hematoxylin and Eosin (H&E), nicotinamide
adenine dinucleotide (NADH) and AR.
• Androgen Dependence: To look at androgen dependence,
transgenic females were ovariectomized and given either a
testosterone filled or blank Silastic capsule. Females were treated
for 8 weeks, and then muscles were harvested in a similar way.
Figure 3. EDL muscle from wildtype male mice stain moderately for NADH
(left). In contrast, the EDL from adult transgenic male mice show increased
NADH staining, indicating a switch to a more oxidative metabolism. “Ring”
fibers (arrow) are also evident in EDL transgenic muscles stained for NADH.
Figure 5. H&E staining in EDL muscles of wildtype and transgenic female mice.
Note that fibers in EDL muscles of blank-treated tg females are comparable in size
and shape to EDL fibers of both blank- and T-treated wt muscles. In contrast,
muscles of tg females exposed to T contain abnormal fibers that exhibit internal
nuclei (stars) and “ring” fibers (arrow).
Technical support was provided by Cindy Knaff,
Heather Malinowski, Diane Redenius, and Laura
Baum.
These studies were funded by operating grant NIH
NS-045195 (CLJ), a grant from the MSU
foundation (CJ, SMB) and a postdoctoral
fellowship (DAM) from the Canadian Institutes of
Health Research (CIHR).