ANDROGEN TOXICITY ON NEUROMUSCULAR PHYSIOLOGY IN A NOVEL MODEL
OF SBMA
By
Kentaro Oki

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
Neuroscience – DOCTOR OF PHILOSOPHY
2013

ABSTRACT
ANDROGEN TOXICITY ON NEUROMUSCULAR PHYSIOLOGY IN A NOVEL MODEL
OF SBMA
By
Kentaro Oki
Spinal bulbar muscular atrophy (SBMA) is a progressive motor disease that appears only in men
around mid-life and results in limb weakness, dysphagia (swallowing difficulties), and dysarthria
(speech difficulties). The disease is believed to be neurogenic, originating from motoneuron
dysfunction and its slow progressive death. Thus, most of the studies characterizing the disease
in mice have focused on motoneuron as the site of disease although there is some clinical
evidence suggesting skeletal muscle may be an important site of disease. SBMA is caused by a
mutation that leads to an expansion of CAG repeats coding for glutamine in the androgen
receptor (AR) gene, and the male-specific phenotype is believed to be androgen-dependent as
females carrying the mutation have little to no symptoms. The male-specific disease phenotype
has been replicated in mouse models expressing similar mutations and can be improved with
castration, reinforcing that the disease is androgen-dependent. Furthermore, female mice in
these models are asymptomatic and only exhibit disease symptoms with androgen treatment.
Although a CAG expansion in the AR gene is thought to underlie the disease, a similar
phenotype is observed in a transgenic (Tg) mouse line engineered to express a rat AR cDNA with
a wild type (WT) number of glutamine residues (22) at very high levels exclusively in skeletal
muscle fibers. Although alteration of gene and protein expression is exclusive to the skeletal
muscles, mice from this myogenic (141) model exhibit a phenotype similar to the other CAGexpanded mouse models of SBMA. Tg 141 female mice that exhibit an androgen-dependent loss
of motor function after 3-5 days of testosterone (T) treatment exhibit skeletal muscle

dysfunction, recorded by electrically stimulating isolated preparations of the extensor digitorum
longus (EDL) and the soleus (SOL), prototypical fast- and slow-twitch muscles, respectively. T
treatment in female 141 Tg mice over 3-5 days was enough to induce a precipitous decrease in
force production in both muscles and alterations to kinetics during contractions in the EDL. To
confirm that skeletal muscles could be a primary site of disease during SBMA, male mice of the
same 141 model, as well as two other SBMA mouse models were examined. The other models
were one expressing the full-length human AR with 97 CAG repeats (97Q model) and another
expressing 113 CAG repeats in the first exon of the AR gene. Muscle dysfunction in the other
models would further support myogenic contributions as being critical to the SBMA motor
phenotype. Motor dysfunction was recorded in all mouse models, and male mice from the 141
and 97Q models exhibited dysfunction in the EDL and SOL. All muscles exhibited some deficit
during force production, and contraction kinetics were altered in the EDL of Tg 141 males.
These results indicate that severe muscle dysfunction can underlie the phenotype during SBMA,
and androgens can act on the skeletal muscles to induce motor weakness. Furthermore, skeletal
muscles may be an important target for therapeutics that could ameliorate disease symptoms.

ACKNOWLEDGMENTS

Thank you to Dr. Cynthia Jordan, my graduate advisor, who gave direction to my
research and has given so much time and effort to guiding my writing. I hope that I can have a
similar ability to guide so many different projects in the future. Thank you to Dr. Bob Wiseman
who advised me on how to conduct studies on skeletal muscles. Without your collaboration and
expertise, this work would not have been possible. Thank you to Dr. Marc Breedlove whose
questions and insight at each step of the planning, benchwork, analysis, and writing helped us fill
in missing details. Thank you to Dr. Cheryl Sisk and Dr. Peter Cobbett who comprised the rest
of my committee and provided valuable guidance throughout my research. Thank you to Dr.
Greg Demas who guided my first research project and whose enthusiasm for research influenced
me to pursue a career in science.
Thank you to the other members of the Jordan and Breedlove laboratories, past and
present, particularly Diane Redenius, Kate Mills, Kelsey Korabik, Jessica Poort, Mike Kemp,
Jamie Johansen, Chieh Chen, and Kayla Renier as well as past and present members of the
Wiseman laboratory, Jeff Ambrose, Natalie Pizzimenti, and Jon Kaspar for technical assistance
and scientific discussions. Thank you to the individuals in ULAR who have cared for the mice
used in this research. Thank you to Matt LaTourette and Chris Hunley for collaborating and
guiding data analysis. Thank you to Irene Li who guided me in making figure illustrations in
Adobe Photoshop. All of you have helped me in my research and allowed me to work smoothly
in two laboratories on campus.
I am thankful for my friends at Michigan State University who were involved with the
Navigators and the Ballroom Dance Team. You have all been amazing friends who have helped

iv

me outside of academics. Thank you to Mulyanto Poort, Rehan Baqri, and Amit Shah for your
collegiality in a professional environment as well as your personal friendship outside of our
respective laboratories.
I am forever thankful to my parents and my brother, Tai, for their support,
encouragement, and counsel throughout my life and all of my education. You have shaped me to
be the person I am today and have stood by me through all things. You always encouraged me to
strive towards my goals, especially through struggles, and I could not have done this without
you. Thank you for everything. Finally, I am grateful to Robin Coan for her support and grace.
You have made my life a much happier one, and I have truly enjoyed some of the best moments
of my life with you. Your constant support was also critical in the latter stages of this work.
This work was supported by R01 NS045195 (CLJ) and DK095210 (RWW).

v

TABLE OF CONTENTS

LIST OF TABLES …………………………………………………………………………….viii
LIST OF FIGURES …………………………………………………………………………….ix
LIST OF ABBREVIATIONS …...…………………………………………………………….xv
INTRODUCTION ...………………………………………………………………………….....1
Motor Function ..……………………………………………………………………………...1
Neuromuscular Diseases ...……………………………………………………………………3
Spinal and Bulbar Muscular Atrophy .......……………………………………………………8
Experiments to determine disease origin ...………………………………………………….11
Anatomy and Physiology of Skeletal Muscle ...……………………………………………..13
Overview .....…………………………………………………………………………………16
CHAPTER 1: Androgen receptors in muscle fibers induce rapid loss of force but not mass:
Implications for SBMA .………………………………………………………………………..18
Abstract ….…………………………………………………………………………………..19
Introduction .…………………………………………………………………………………19
Methods .……………………………………………………………………………………..21
Animals and hormone treatment .………………………………………………………..21
Motor function tests ...…………………………………………………………………...22
In vitro studies to assess muscle mechanics ...…………………………………………..23
Histology ...………………………………………………………………………………26
Androgen receptor protein expression ..…………………………………………………26
Protein gels ...…………………………………………………………………………….27
Testosterone levels ...…………………………………………………………………….27
Data analysis and statistics ...…………………………………………………………….27
Results…..……………………………………………………………………………………28
Testosterone (T) treatment impairs motor function without loss of muscle mass ...…….28
Skeletal muscles from T-treated female mice show striking androgen-dependent loss of
force .......………………………………………………………………………………...29
Five Day Treatment …………………………………………………………………29
Tetanic Force ……………………………………………………………………29
Muscle histology and protein content …………………………………………...30
Skeletal muscles from T-treated Tg females exhibit androgen-dependent changes in
twitch force and kinetics ...………………………………………………………………31
Twitch Force ………………………………………………………………………...31
Contraction Kinetics ………………………………………………………………...32
Tetanic force production is reduced in the SOL but not EDL of Tg mice after three days
of T treatment ...………………………………………………………………………….32
Only the EDL from motor-impaired mice show deficits in fatigue resistance ...………..33
Discussion ...…………………………………………………………………………………34

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CHAPTER 2: Muscle dysfunction may underlie androgen-dependent motor dysfunction in
mouse models of SBMA ...…………………...…………………………………………………40
Abstract ……………………………………………………………………………………...41
Introduction ...………………………………………………………………………………..41
Methods ...……………………………………………………………………………………44
Animals and treatment ...………………………………………………………………...44
97Q males …………………………………………………………………………….....44
141 males ………………………………………………………………………………..45
KI males ...……………………………………………………………………………….46
Motor function tests ..……………………………………………………………………46
In vitro studies to assess muscle mechanics …………………………………………….47
Histology ………………………………………………………………………………...50
Testosterone levels ………………………………………………………………………50
Data analysis and statistics ………………………………………………………………50
Results ……………………………………………………………………………………….51
Motor dysfunction correlates with muscle dysfunction in 97Q Tg mice ………………..51
Motor dysfunction correlates with muscle dysfunction in myogenic Tg males ………...53
Histology ..……………………………………………………………………………….55
97Q Model …………………………………………………………………………..55
141 myogenic model ………………………………………………………………...56
Knock-in model …………………………………………………………………………56
Discussion …………………………………………………………………………………...56
GENERAL DISCUSSION …………………………………………………………………….65
General Findings ...…………………………………………………………………………..65
Overview of findings ………………………………………………………………………..65
Mechanisms of Muscle Dysfunction ………………………………………………………..69
Calcium Mechanisms ………………………………………………………………………..70
Trophic Factors ……………………………………………………………………………...74
Metabolic Mechanisms ……………………………………………………………………...75
Future Directions ……………………………………………………………………………76
Conclusions ………………………………………………………………………………….77
APPENDIX ……………………………………………………………………………………..78
REFERENCES ………………………………………………………………………………..105

vii

LIST OF TABLES

Table 1
Biometric data for female 141 mice. T, testosterone; mg, milligrams; nmol/L,
nanomolar per liter; N/g, newtons/gram. Values are means + standard error of means. * P<0.05.
……………………………………………………………………………………………………83

Table 2
Body weight, muscle mass and plasma testosterone (T) levels at end stage.
Motor-impaired Tg male mice in each SBMA model show significantly decreased body weights
compared to their respective WT controls. All but the soleus (SOL) muscle in 97Q males had
significantly less mass. Circulating T levels were comparable between Tg and WT males within
each line but notably higher for Tg and WT males in the 97Q model because these animals were
castrated and given exogenous T starting at puberty. *P<0.05 difference between transgenic
mice and their respective WT controls, values = mean + standard error of mean ………………90

Table 3
Kinetics analysis of individual twitch traces for motor-impaired Tg males and
their WT controls. Analyses revealed few significant effects of disease on twitch kinetic; only
rise time (time to peak force) in the EDL muscle of 141 Tg males was significantly prolonged
compared to WT controls. Note however a consistent trend toward slower twitch kinetics in
diseased muscles of both models. *P<0.05, values = mean + standard error of mean …………91

Table 4
Muscle morphology for male mouse muscles. Total number of muscle fibers and
percent of total number of fibers containing centralized (cent) nuclei for EDL and SOL of the
two Tg SBMA models (141 myogenic and 97Q). Only the EDL from myogenic males had
significantly fewer fibers than WT controls. Fiber number was unaffected in both the EDL and
SOL of 97Q Tg males and also in the SOL of 141 Tg males. The EDL of 97Q males and the
SOL of 141 males contain a higher percentage of fibers with centralized nuclei, indicative of an
abnormal muscle state. Note that these two muscles also showed the severest loss in force (Figs
2, 4). n=5/group in all except 141 EDL (n=3) *P<0.05, values = mean + standard error of mean.
1
2
values from (Johansen et al., 2011); unpublished data (Johansen and Jordan). ………………92

viii

LIST OF FIGURES

Figure 1
For interpretation of the references to color in this and all other figures, the reader
is referred to the electronic version of this dissertation. The whole skeletal muscle is comprised
of multiple fascicles with capillaries between fascicles providing a blood supply. Fascicles are
then comprised of multiple myofibers. Myofibers are large, multinucleated individual cells
of a skeletal muscle and are comprised of myofibrils. ……………………………………….78

Figure 2
Large multinucleated myofibers contain the mitochondria on the muscle periphery
to aid in muscle energetic. Myofibrils comprise the myofiber and are, in turn, comprised of
myofilaments. Along myofibrils are sarcomeres, the most basic unit of a skeletal muscle that
allows for a contraction. Upon electrical stimulation, multiple sarcomeres shorten within a
muscle resulting in whole muscle shortening to produce a contraction. ………………………..79

Figure 3
The myofilaments that comprise myofibrils and sarcomeres are shown when the
overlying sarcoplasmic reticulum (SR) is removed from the myofibril. Myosin and actin form an
interdigitating pattern throughout the myofibril to allow contractions. Myosin are anchored at
the M line while actin is anchored by Z disks. Regions of the SR closer to the T tubules are
termed the terminal cisternae. T-tubules that run on either side of the interdigitating region
contain openings that form a triad with the terminal cisternae. These regions are where the
electrical stimulus initiates muscle contractions. ………………………………………………..80

Figure 4
0) The first image at the top right lists the key proteins involved during a skeletal
muscle contraction. A T-tubule is in contact with the sarcoplasmic reticulum (SR) containing
calcium (Ca) ions that will initiate contraction. Ryanodine receptor 1 (RyR1) is closed, and
sarcoendoplasmic reticulum Ca ATPase (SERCA) is inactive while Ca is within the SR. On the
actin filament, tropomyosin covers the binding site preventing myosin heads from binding to
actin. 1) With a voltage change, T-tubules conduct the electrical depolarization to initiate the
opening of RyR1, releasing Ca from the SR. Upon Ca release from the SR, it binds to the Casensitive subunit of the troponin complex. This also initiates SERCA function, but the large
release of Ca into the cytosol is greater than the rate at which Ca is replaced into the SR. 2) Ca
binding of troponin induces a conformational change in troponin removes the troponintropomyosin complex away from the actin filament, exposing the actin binding sites. 3) A
myosin head to binds to the actin binding site, and 4) ATP is hydrolyzed, resulting in ADP and
inorganic phosphate. Once phosphate is released, the myosin head turns. 5) With the release of
ADP, the myosin head is turned even further, moving the actin filament, shortening the
sarcomere, and resulting in a power stroke to initiate skeletal muscle contraction. This
shortening in the sarcomeres produces the contractile force generated by muscles. In cases
where ATP is depleted, the myosin head remains in this locked position on the actin filament. 6)
As long as ATP is available, another ATP binds to the myosin head. 7) This releases the myosin
head from the actin filament, returning the myosin head to its original position (same as step 2).

ix

8) Once the skeletal muscle returns to resting potential, RyR1 closes, and SERCA, its function
initiated when Ca entered the cytosol, is able to return Ca back to the SR against the Ca
concentration gradient (dashed arrow). Consequently, TnC no longer binds Ca, and the troponintropomyosin complex returns to its resting state where tropomyosin covers the myosin binding
sites on actin. 0) With Ca return to the SR, the entire region is ready for another contraction. ..81

Figure 5
Motor function declines rapidly in testosterone (T)-treated adult female transgenic
(Tg) mice that express a wild-type androgen receptor (AR) transgene only in skeletal muscle
fibers. Baseline performance at Day 0 before hormone treatment did not differ across treatment
groups. T treatment induced a rapid decline in grip strength (A), hang time (B), and body weight
(C) only in Tg females. By Day 5, motor performance on both tests was decreased significantly
for T-treated Tg (T Tg) females compared to baseline or compared to the other groups, including
asymptomatic females that were blank-treated wild-type (B WT), testosterone-treated wild-type
(T WT), and blank-treated transgenic (B Tg). Each asymptomatic group maintained pretreatment performance levels on both the grip strength and hang tests across the 5 days of
treatment, indicating that only the combination of AR transgene expression in muscle fibers and
male levels of androgens instigates motor dysfunction. Values are means + standard errors of
means based on N = 5-6 mice/muscle and group. *P<0.05, T Tg versus B Tg or T WT. ……..84

Figure 6
Tetanic force produced by both EDL and SOL of motor-impaired female mice is
reduced significantly compared to tetanic force produced by corresponding muscles of
asymptomatic controls after 5 days of testosterone (T) treatment. Representative traces for T
WT muscles and T-treated transgenic (T Tg) muscles are shown (A, B). Tetanic force produced
by the EDL of T Tgs is about half of control females (C), whereas tetanic force produced by the
SOL of T Tg females is about a quarter of T WT controls (D). Tetanic force produced by
muscles of control-treated Tg females (B/Tg) is equivalent to that of WTs, indicating that AR
transgene expression per se does not impair muscle strength. Comparable force deficits are
observed at the beginning and end of experiments (indicated as first and last tetanus) for both
EDL and SOL of T Tg mice, suggesting that force deficit is present in vivo and is not due to a
more rapid deterioration of these muscles in vitro. Force (in Newtons-N) was normalized to
muscle wet weight (grams-g). Values in histogram are means + standard errors of means, N = 56 mice/group. *P<0.001, T Tg versus B Tg or T WT. …………………………………………85

Figure 7
Neither histopathology nor expression of the AR transgene explains the severe
deficit in SOL force production of T-treated Tg mice. Cross sections of SOL muscle (scale bar =
500 μm) from Tg females after 5 days of T treatment stained with H&E exhibit no signs of
abnormal morphology (e.g. fiber atrophy/hypertrophy, centralized nuclei, A), nor alterations in
fiber number (B) compared to the SOL from other treatment groups (N = 4-5 mice/group). Bars
represent means + standard errors of means. AR immunoblot (C) shows AR expression levels in
the EDL and SOL from asymptomatic (B Tg) versus symptomatic (T Tg) mice. We find no
consistent differences in AR expression (~110 kD) between EDL and SOL muscles, indicating
that a difference in AR protein content between the EDL and SOL is not likely to underlie the
relatively greater force deficit in the SOL compared to the EDL in T-treated Tg mice. Actin

x

(~40 kD) loading control shows no consistent differences in protein loading between the EDL
and SOL of Tg mice. …………………………………………………………………………….86

Figure 8
Twitch kinetics are slowed only in the EDL of motor-impaired Tg mice after 5
days of T treatment. Representative twitches from the fast-twitch EDL and the slow-twitch SOL
(A, B) reveal decreased peak twitch force for both muscles of T-treated Tg mice (see Table 1 for
mean values) but altered twitch kinetics only for the EDL. Quantitative analysis of individual
twitches (C – F) confirms a significant prolongation in the time it takes to reach peak twitch
force (C) and to relax (E) in EDL, but neither parameter is significantly altered by T in the SOL
muscle of Tg mice (D, F). Plotted values are means + standard errors of means, with N = 5-6
mice/group. *P<0.001 T Tg versus B Tg or T WT. ……………………………………………87

Figure 9
The SOL but not the EDL of motor-impaired mice shows the same deficit in peak
tetanic force after 3 days of T treatment. Tetanic force in the slow-twitch SOL from T Tg mice
was significantly decreased after 3 days of T treatment (A), comparable to the magnitude of the
effect of 5 days of treatment (Fig 6). A subtle, but not significant difference was seen in EDL
from the same motor-impaired mice (T Tg versus T WT mice, p = 0.07). However, peak twitch
force in both muscles was significantly reduced in T Tg mice compared to T WT mice (B).
Twitch kinetics were largely unaffected in T-treated Tg mice after 3 days of treatment (C, D)
except that relaxation time was slowed significantly in EDL, consonant with the effect of T on
this parameter after 5 days of treatment (Fig 8). N = 5-7/group. *P<0.05. ……………………88

Figure 10
The EDL but not the SOL from motor-impaired Tg mice showed compromised
resistance to fatigue after 5, but not 3, days of T treatment. Fatigue resistance was measured as
time in seconds from onset of tetanizing stimulation to when the muscles decreased in force by
50% of maximal tetanic force. The EDL from Tg mice after 5 days of T treatment fatigued
significantly faster, dropping to half maximal force several seconds sooner than the EDL from
either T-treated WT or blank-treated Tg mice (A). Interestingly, the EDL from blank-treated Tg
mice showed a greater resistance to fatigue than the EDL from blank-treated WT (B Tg versus B
WT), indicating a beneficial effect of the transgene itself on resistance to fatigue in the EDL.
Five days of T treatment had no effect on fatigue resistance in SOL (B), and there was no effect
of T on fatigue resistance after 3 days of treatment in either muscle from Tg mice (C and D). 5day EDL: N = 5-6 mice/group; 5-day SOL, N = 5-6 mice/group; 3-day EDL, N = 6 mice/group;
3-day SOL, N = 4-7 mice/group. *P<0.05. …………………………………………………….89

Figure 11
Transgenic (Tg) males in both the 97Q and 141 ‘myogenic’ models of SBMA
show the expected impairment in motor function. Mean number of rearings in an open field,
taken as an indirect measure of hindlimb strength, was decreased significantly in Tg males of
both the 97Q (A) and myogenic 141 (D) models of SBMA compared to their respective WT
controls. Similar significant deficits were also found in mean frontpaw grip strength (B, E) and
hang times (C, F). These measures indicate that Tg males in both models show the expected

xi

SBMA phenotype. *p < 0.05 compared to WT controls. Error bars represent standard error of
the mean, with n = 6-7 mice/group. ……………………………………………………………..93

Figure 12
Both the fast twitch extensor digitorum longus (EDL) and slow twitch soleus
(SOL) muscles from motor-impaired 97Q Tg males show deficits in contractile force. Raw
twitch and tetanic force (A,D, and G) were significantly reduced, with the one exception that raw
tetanic force was unaffected in the SOL (J). However, twitch and tetanic forces once normalized
to muscle mass were significantly decreased in both muscles, including tetanic force produced by
the SOL (B, E, H, and K), indicating primary defects in contractile function of muscles of
diseased 97Q Tg males that are independent of their mass. The force deficit is also readily
apparent in examples of individual traces of EDL (C) and SOL (I) twitches. Although somewhat
reduced, the twitch/tetanus (Tw/Tet) ratio in the diseased EDL was not significantly different
than WT (F). In contrast, the Tw/Tet ratio in the SOL of diseased males was significantly
reduced (L), suggesting defects in calcium handling mechanisms. Such defects in muscle
function may contribute to if not underlie defects in motor function. *p < 0.05 compared to WT
controls. Error bars represent the standard error of the mean, with n = 4-7 mice/group. ………95

Figure 13
Both the EDL and SOL from motor-impaired 97Q Tg males fatigue more rapidly.
The fatigue profile for both the EDL and SOL indicates that both muscles in Tg males produce
less force per gram muscle at the start of stimulation (A, C) followed by a drop in force for all
muscles indicating muscle fatigue during tetanizing stimulation. While the Tg EDL loses less of
its original force than the WT EDL over the stimulation period (A), the Tg EDL appears to
fatigue faster, losing 50% of its original force (time to half of maximal force loss) significantly
sooner than WT EDL (B). The Tg SOL also appears to have a faster time course of fatigue than
WT SOL, losing 50% of its original force significantly sooner than WT SOL (D). *p < 0.05
compared to WT controls. Plotted values are means + standard error of the mean, with n = 4-7
mice/group. ……………………………………………………………………………………...97

Figure 14
Both the fast twitch EDL and slow twitch SOL muscles from motor-impaired
myogenic (141) Tg males show deficits in contractile force. Raw and normalized twitch and
tetanic force were significantly reduced in both the EDL and SOL of motor-impaired males (A,
B, D, G, H, J and K) with the exception of normalized tetanic force in Tg EDL (E). On the other
hand, the deficit in raw tetanic force in the EDL probably is due to the deficit in fiber number in
this muscle (Table 4). Samples of twitch traces reveal such deficits in twitch force (C, I). The
Tw/Tet ratio is also significantly reduced in both the Tg EDL (F) and SOL (L), also suggesting
that that calcium mobilization from intracellular stores may be perturbed. Muscles of myogenic
141 males show similar defects in contractile properties as 97Q males, although disease affects
force most in the SOL of 141 males while affecting most the EDL in 97Q males. Nonetheless,
these results suggest that motor dysfunction likely involves primary defects in muscle function.
*p < 0.05 compared to WT controls. Error bars represent the standard error of the mean, with n
= 4-7 mice/group. ………………………………………………………………………………..98

xii

Figure 15
Only the SOL from motor-impaired 141 Tg males fatigues more rapidly. The
fatigue profile indicates that the Tg EDL shows increased resistance to fatigue, losing less total
force than WT EDL overall (A). However, both Tg and WT EDL appeared to fatigue at about
the same rate, reaching a 50% drop at about the same time (B). The apparent increase in
oxidative metabolism in the diseased EDL (Johansen et al., 2011; Monks et al., 2007) may help
stave off some of the later losses in force exhibited by WT EDL. The SOL of diseased 141 Tg
males on the other hand fatigues rapidly, losing 50% of its starting force in less than 9.8+0.9 s
compared to 42.1+2.4 s in WT SOL. The marked increase in fatigue susceptibility of the SOL
muscle in 141 Tg males also likely contributes to motor dysfunction in this model. *p < 0.05
compared to WT controls. Plotted values are means + standard error of the mean, with n = 4-6
mice/group. …………………………………………………………………………………….100

Figure 16
Muscles from diseased and healthy mice remain stable in vitro during the course
of the experiment. Viability of each skeletal muscle was monitored with tetanus stimulations
throughout an experiment. Force produced by tetani at the beginning of the experiment (open
bar) was comparable to force produced at the end (filled bar). These data suggest that the force
deficits found in vitro in diseased muscles likely reflect a genuine force deficit in vivo, rather
than a defect that emerges in vitro. In vitro measurements were taken during a 2-3 hour window.
Plotted values are mean + standard error of the mean. ………………………………………...101

Figure 17
Representative photomicrographs of cross sections of muscle used for fiber
counts. The EDL from the myogenic model was previously characterized (Johansen et al., 2011)
and therefore not included. EDL muscle fibers in diseased 97Q Tg males are markedly
heterogeneous in size, with some fibers notably smaller than the rest (arrow). EDL fibers from
97Q males also contain centralized nuclei (arrow). Since mass of the EDL from diseased 97Q
males is reduced by half (Table 2) without loss of fibers (Table 4), it is likely that atrophy of
individual fibers underlies the deficit in EDL mass. Note however that decreases in muscle mass
contribute little to the deficits in twitch and tetanic force (Fig 2A, B, D and E) since a large
proportion of the deficit persists once force measures are normalized to muscle mass. No major
histological changes were observed in cross section of SOL from either 97Q males (middle
panels) or myogenic (141) males (bottom panels) except for centralized nuclei which were more
frequent in the SOL of 141 Tg males compared to WT SOL. Despite the lack of striking
histopathology, SOL function is impaired in both models, but particularly in the 141 myogenic
males, which show a three-fold loss of normalized force (Fig 14H, K). These data indicate that
impairments in muscle function need not be accompanied by marked histopathology. Scale bar
= 50 µm. ………………………………………………………………………………………..102

Figure 18
KI males with mild motor deficits show negligible deficits in muscle function.
Measurements of forepaw grip strength and hang times are significantly decreased in KI males
relative to WT controls (A, B), showing the expected impaired motor function in KI males.
While force measures in the KI EDL suggest small deficits in twitch and tetanic force consistent
with the mild motor phenotype in this model, these apparent deficits are not significant (C – H).
Likewise, we found no significant deficits in twitch and tetanic forces produced by the SOL from

xiii

KI males (I - N). These data raise the possibility that motor deficits in KI males may reflect
synaptic defects upstream to the skeletal muscles. *p < 0.05 compared to WT controls. Plotted
values are mean + standard error of the mean, with n = 5-7 mice/group. ……………………..103

xiv

LIST OF ABBREVIATIONS
ACh – acetylcholine
AChR – acetylcholine receptor
ALS – amyotrophic lateral sclerosis
AR – androgen receptor
B – blank
Ca – calcium
CK – creatine kinase
DHPR – dihydropyridine receptor
DM – myotonic dystrophy
DMD – Duchenne’s muscular dystrophy
EDL – extensor digitorum longus
EMG – electromyography
GDNF – glial cell line-derived neurotrophic factor
H&E – hematoxylin and eosin
HSA – human skeletal α-actin
IGF-1 – insulin-like growth factor 1
KI – knock-in
N – newtons
PV – parvalbumin
RyR1 – ryanodine receptor
SBMA – spinal and bulbar muscular atrophy
SERCA – sarcoendoplasmic reticulum ATPase

xv

SMA – spinal muscular atrophy
SMN – survival motor neuron
SOD1 – superoxide dismutase 1
SOL – soleus
SR – sarcoplasmic reticulum
T – testosterone
Tg – transgenic
TnC – troponin C
TnI – troponin I
TnT – troponin T
VEGF – vascular endothelial growth factor
WT – wild type

xvi

INTRODUCTION
The overall aim of the thesis is to determine whether skeletal muscle dysfunction has a role
in the onset and/or progression of motor deficits in a neuromuscular disease known as spinal and
bulbar muscular atrophy (SBMA). All experiments were conducted using mice as the model
organism. This chapter briefly introduces motor function as a result of motor unit activity. It
then presents how alterations to elements of the motor unit result in motor dysfunction, with a
particular emphasis on SBMA, a male-specific motoneuron disease. It will discuss how skeletal
muscle dysfunction might contribute to the symptoms of this disease based on case studies in
humans and animal models of SBMA. Finally, a description of the mechanisms involved in
skeletal muscle function is given to help readers understand the measurements that were used to
assess muscle function in my studies.

Motor Function
Motor function is critical for survival in vertebrates and most invertebrates. Muscle
contractions initiated by excitation-contraction coupling, i.e. when a presynaptic electrical
stimulus is converted to a postsynaptic mechanical response (Sandow, 1952), generates the
necessary in vivo force to perform locomotion and many other functions. Basic motor function
in animals is generated by the motor unit, comprised of an α-motoneuron and the muscle fibers
that it innervates. When a single motoneuron is excited above threshold, all the muscle fibers it
innervates are stimulated to contract. This stimulus response follows an “all or none” pattern of
activation where any stimulus below threshold will not induce a response, and any stimulus at or
above threshold results in a complete response by the motor unit (Sherrington, 1929).

1

Different unit types are classified according to differences in size (i.e., the number of target
muscle fibers a motoneuron innervates). Motor units and α-motoneurons vary in size where
smaller motoneurons innervate fewer muscle fibers, and larger motoneurons innervate more
muscle fibers. Smaller motoneurons and motor units are more easily recruited to contract and
perform small tasks. As the need for greater muscle force arises, the larger motor units are
recruited to accommodate the task. This phenomenon occurs as the smaller motoneurons have
higher input resistance, resulting in a large change in potential with smaller input (rheobase)
currents and a greater probability of being activated. Conversely, larger motoneurons have
greater surface area and lower input resistance requiring larger rheobase currents to induce
changes in potential, resulting in a lower probability of being activated. This model for motor
unit recruitment has been called Henneman’s size principle (Clamann and Henneman, 1976;
Henneman, 1957; Henneman and Olson, 1965). Once motor units have been recruited, larger
units with larger axons tend to have faster conduction velocities due to decreased resistance
within the axon, and smaller motor units with smaller axons have slower conduction velocities
due to increased resistance within an axon (Bawa et al., 1984). Order of motor unit recruitment
based on size can also predict the metabolic properties of the skeletal muscles they innervate as
being fast-twitch or slow-twitch (Zengel et al., 1985). Smaller motoneurons tend to innervate
muscles that have higher fatigue resistance and can be tonically activated due to the greater
levels of mitochondria and vascularization and the dependence on aerobic mechanisms in these
muscles. Conversely, larger skeletal muscles innervated by larger motor units produce more
force, but fatigue more easily and are only activated for shorter periods due to lower levels of
mitochondria and vascularization but being dependent on anaerobic mechanisms (Burke et al.,
1973; Henneman and Olson, 1965; Hennig and Lomo, 1985).

2

Proper motor function allows the execution for vital functions such as movement and
respiration. However, dysfunction in α-motoneurons, junctions, or skeletal muscles can result in
difficulties with vital functions, and such cases of motor dysfunctions, termed neuromuscular
diseases, can be fatal.

Neuromuscular Diseases
Neuromuscular diseases are a group of conditions that affect the ability of an organism to
move or coordinate movements. Although other diseases like Parkinson’s and Huntington’s
diseases also exhibit a motor component, their underlying symptoms originate in brain regions,
and the current review specifically focuses on diseases originating in the motor unit components.
Neuromuscular diseases are characterized by motor performance deficits and are separated based
on where the agents of disease are presumed to act. Neuromuscular diseases include myopathies
(e.g. muscular dystrophies, mitochondrial myopathies), diseases directly affecting the junction
(e.g. Lambert-Eaton myasthenic syndrome, myasthenia gravis), and diseases in which
pathogenesis is presumed to originate in the motoneurons (e.g. amyotrophic lateral sclerosis –
ALS, spinal muscular atrophy – SMA). Depending on the condition, symptoms may be apparent
anywhere from infancy (seen in some cases of SMA) to the middle-later stages of life (seen in
cases of ALS). The discussion will focus on two primary myopathies, Duchenne muscular
dystrophy and myotonic dystrophy, and two motoneuron diseases, ALS and SMA.
Myopathies are conditions for which the agents of disease originate in skeletal muscles.
They can be categorized based on how they present in patients and are divided as primary or
acquired myopathies. Primary myopathies typically have an underlying genetic cause, whereas
acquired myopathies result from an insult to the body, such as inflammation. The focus will be

3

on primary myopathies and their etiology. The most common myopathies are muscular
dystrophies, conditions marked by progressive skeletal muscle degeneration and regeneration
that result in weakness. Duchenne’s muscular dystrophy (DMD) is the most frequently
occurring dystrophy with an occurrence of 1 in every 3300 live births (Deconinck and Dan,
2007). DMD occurs due to a mutation in the DMD gene on the X chromosome (Lindenbaum et
al., 1979). The gene codes for dystrophin, a structural protein found in the sarcolemma of
skeletal muscles (Hoffman et al., 1987; Koenig et al., 1988; Zubrzycka-Gaarn et al., 1988).
Males and females can carry the gene, but females rarely exhibit disease symptoms due to the Xlinked recessive pattern of inheritance (Morton and Chung, 1959). Although the precise function
of dystrophin is unknown, it is thought to stabilize the sarcolemma against the force imposed by
muscle activity. In affected individuals, the absence of dystrophin removes the anchor between
muscle cytoskeleton and the extracellular matrix, disrupting the costameric lattice (Ervasti and
Campbell, 1993). This leads to muscle membrane fragility usually confirmed using orange
Procion or Evans Blue (Matsuda et al., 1995), which enter only muscle fibers that lack intact
plasma membranes. It is postulated that in DMD, multiple pathophysiological processes are
activated due to the membrane fragility, and this may alter intracellular calcium (Ca) handling as
increased Ca content has been recorded in skeletal muscle fibers from DMD individuals
(Bertorini et al., 1982; Emery and Burt, 1980). Symptoms present in DMD patients between 3
and 5 years of age and are characterized by a delayed onset in walking, toe walking, or waddling.
Serum creatine kinase is elevated 50-100 times normal levels in patients, indicative of damaged
fibers (McMillan et al., 2011). Fatty replacement and fibrosis (pseudohypertrophy) manifest in
calf muscles (Schreiber et al., 1987). Generally, loss of ambulation occurs between 7 and 12
years of age and death by the second decade (Eagle et al., 2002).

4

Another primary myopathy is myotonic dystrophy, the most common form of muscular
dystrophy in adults with a minimum incidence of 1 in 8000. Myotonic dystrophy is classified
into two types (DM1 and DM2), and both conditions have an autosomal-dominant inheritance
pattern (Harper, 1989). DM1 (also referred to as Steinert’s Disease in the literature) is the more
common form of myotonic dystrophy that results from an expansion of the codon CTG in the
untranslated 3’ region of the myotonic dystrophy protein kinase (DMPK) gene (Harley et al.,
1992). The CTG expansion in DM1 individuals ranges from 50 to >1000 repeats, and
individuals screened for the disease are classified into three subcategories based on the number
of repeats. DM1 patients are also classified on a spectrum based on symptoms and time of onset
during the lifetime: congenital, childhood, adult, and late adult (Harper and Dyken, 1972). The
variable age of onset is seen not just across affected families but also within a family and is
correlated with the number of CTG repeats. Analysis of genetic markers also reveals that the
repeat expansion can increase in size in successive generations, starting with a mild phenotype in
early generations but resulting in a more severe and earlier onset of the disease in the later
generations due to the lengthened expansion (Brook et al., 1992). Patients generally exhibit
muscle weakness, and similar to DMD, have elevated levels of serum creatine kinase in the
blood (Heatwole et al., 2006). Electromyographic recordings indicate delayed muscle relaxation
and hyperexcitability in myofibers, and spontaneous activity resulting in myotonia (Romeo,
2012). The underlying mechanism for the abnormal muscle activity is thought to be aberrant
splicing of a chloride (Cl) channel (ClC1) due to the CTG expansion. This leads to decreased Cl
channel expression in the muscle membrane, decreased Cl conductance, and increased input
resistance. Thus, muscles respond to a lower stimulus intensity, resulting in hyperexcitability
and myotonia (Mankodi et al., 2002). Associated histopathology in human muscle biopsies

5

include centralized nuclei within muscle fibers, generally atrophic slow-twitch (type I) muscle
fibers , and muscle fibers with ring-shaped myofibrils (Heidenhain, 1918 reviewed in (Romeo,
2012)).
Neuromuscular diseases also include conditions where motor function is characterized by
motoneuron dysfunction and degeneration, such as ALS and SMA. Unlike primary myopathies,
these conditions are classified as motoneuron diseases with muscle dysfunction and atrophy
presumed to be secondary to the defects in motoneurons. ALS is a neurodegenerative disease
marked by loss of motor coordination, muscle atrophy, and degeneration of upper and lower
motoneurons. Symptoms include difficulties with movement, speech, and breathing that worsens
progressively until the terminal stage. Onset generally peaks around the sixth decade of life, and
life expectancy of a patient after diagnosis is generally 2-5 years though it varies from case to
case (Gubbay et al., 1985; Haverkamp et al., 1995; Ringel et al., 1993). The first identified
causative gene for ALS is a mutation in the superoxide dismutase 1 (SOD1) gene (Rosen et al.,
1993). Cases with an underlying genetic defect are termed “familial” and account for roughly
2% of all ALS cases. Since the discovery of the mutant allele, much of the research on ALS has
relied on mouse models expressing a mutated SOD1 allele, which codes for a metalloenzyme
that protects cells from oxidative stress by catalyzing the dismutation of toxic superoxide to
oxygen and hydrogen peroxide (McCord et al., 1971). Although the specific mutation in the
SOD1 gene differs across models, the ALS mouse models generally exhibit a similar and
predictable pattern of disease. Mice in these models generally exhibit paralysis in one or more
limbs starting at around 3-6 months of age, lose body mass, eventually exhibit total immobility,
and die shortly afterwards. Spinal cords from affected mice indicate motoneuron pathology or

6

loss resulting may underlie this phenotype (Bruijn et al., 1997; Gurney et al., 1994; Ripps et al.,
1995; Wong et al., 1995).
SMA is a neuromuscular condition characterized by α-motoneuron degeneration in the spinal
cord, resulting in proximal muscle weakness and paralysis. It is estimated to occur for 1 in every
6,000-10,000 live births (D'Amico et al., 2011). SMA is classified by 1) the underlying genetics
and pattern of inheritance and 2) the age of onset and phenotype progression and severity. The
causative gene was found to be the survival motor neuron (SMN) gene (Lefebvre et al., 1995)
and is present in multiple copies in the genome (Burglen et al., 1996). Type 0 is designated for
prenatally diagnosed individuals who rarely survive beyond 6 months of age. Type I is
designated where age of onset is between 0-6 months of age. Type I patients are never able to sit
unsupported and generally do not live beyond 2 years of age. Type II patients are able to sit
unsupported but are never able to walk. These individuals generally experience proximal
weakness in the legs more than the arms and generally live into their 30’s but have shortened life
expectancies. Type III patients are able to walk but experience weakness later in life and may
require a wheelchair. In spite of the symptoms, individuals with type III SMA generally have a
normal life expectancy (Dubowitz, 1995; Markowitz et al., 2012). The severity and spectrum of
SMA result from the nature of SMN1 deletion. SMA type I is generally caused by a homozygous
deletion of the SMN1 allele, whereas SMA types II and III result mostly from conversion of
SMN1 to SMN2 (Campbell et al., 1997). Type II is due to a conversion event at one allele and a
hemizygous deletion in the other allele while type III can be due to homozygous conversion of
SMN1 to SMN2 (Hahnen et al., 1996; van der Steege et al., 1996). Sometimes referred to as Xlinked SMA, spinal and bulbar muscular atrophy (SBMA) is a condition in which recent

7

evidence suggests that motor dysfunction may originate in the skeletal muscles. Thus, the
dividing line between primary myopathies and motoneuron diseases is becoming blurred.

Spinal and Bulbar Muscular Atrophy
Spinal and bulbar muscular atrophy (SBMA) is a so-called polyglutamine as well as a
motoneuron disease for which proper motor coordination is disrupted. SBMA is also known as
Kennedy Disease for the researcher who characterized the disease (Kennedy et al., 1968).
Unlike other forms of SMA, a mutation in the androgen receptor on the X-chromosome has been
linked to SBMA. The mutated gene is passed on in an X-linked inheritance pattern where
affected males have the mutation expressed in all somatic cells. In females, if the mutation is on
one X chromosome, the mutation is expressed in roughly half of all somatic cells due to random
X inactivation (Lyon, 1961, 1962). The mutation, itself, is due to an expansion of three
nucleotides, similar to what is observed in myotonic dystrophy. A region of CAG codons, which
code for glutamine in the N-terminus of AR, is expanded in SBMA (La Spada et al., 1991). All
individuals have this expanded CAG region. However, whereas unaffected males have 10-37
repetitions, individuals affected by SBMA have 40-85 repeats. Thus, in addition to being classed
as a type of SMA, the underlying genetic mutation has led to SBMA being classified as a
polyglutamine disease, a group of neurodegenerative conditions which include Huntington’s
disease, dentatorubropallidoluysian atrophy, and spinocerebellar ataxia types 1, 2, 3, 6, 7, and 17
(Ross, 2002). Although all the conditions are progressively neurodegenerative and grouped
together, the symptoms and localization of neurodegeneration differ across conditions. SBMA is
unique among polyglutamine disorders because the function of the affected protein is well
characterized (Fischbeck et al., 1999).

8

Although males and females can express the disease allele, SBMA is a male-specific disorder
where affected individuals generally show symptoms sometime between their third to fifth
decades of life (Rhodes et al., 2009). Recent evidence suggests that the reason only males are
affected is because SBMA symptoms depend on male levels of androgen (specifically
testosterone – T) rather than random X inactivation. T administration to an SBMA sufferer
resulted in motor performance decline. Subsequent cessation of T administration allowed motor
function to return to baseline (Kinirons and Rouleau, 2008). Additionally, females (one with two
mutated X chromosomes) are unaffected or exhibit only minor symptoms, presumably because
of a lack of testosterone (Schmidt et al., 2002).
AR is a ligand-activated transcription factor. Androgen receptors bind their ligand
(testosterone or the metabolite dihydrotestosterone) to exert their effects, and the ligand-receptor
binding results in receptor dimerization and translocation from the cytoplasm into the nucleus
(Wong et al., 1993). Once ligand-receptor complexes are formed, co-activators are recruited to
aid in binding DNA and increase protein translation (MacLean et al., 1997). Although
testosterone normally promotes growth of skeletal muscles, the AR mutation linked to SBMA
leads to dysfunctional AR signaling when bound to T and results in loss of motor function in
men with SBMA. In addition to motor deficits, which are thought to arise through a gain of new
and toxic functions of the mutant AR, individuals with SBMA also show a partial loss of normal
AR signaling. Signs of partial androgen-insensitivity include gynecomastia (development of
male breasts) and reduced virility of patients with decreased sperm count and testicular atrophy
(Arbizu et al., 1983). Of note, XY individuals with a total loss of AR function because of a
different mutation in the AR gene are feminized but report no difficulties with motor function,

9

arguing that a loss of normal AR function plays little to no role in the motor dysfunction caused
by SBMA.
Deficits in motor coordination are seen in patients as they have limb muscle weakness,
difficulties climbing stairs, and may require a cane or a walker for mobility. Tests have
concluded there are delays in motor conduction velocities to limb muscles in SBMA patients
compared to control patients (Suzuki et al., 2008). Results from postmortem analyses show that
SBMA causes a loss of spinal motoneurons (Kennedy et al., 1968; Sobue et al., 1989). In
addition, muscles are also clearly affected in SBMA but whether the effects of muscle are via a
loss of innervations (neurogenic) or are due to the action of a mutant AR directly in muscle is not
clear. Electromyographies (EMGs) have revealed fasciculations (Kennedy et al., 1968), and
biopsies have revealed muscle atrophy, pathology typically attributed to a neurogenic origin but
also split myofibers which is typically attributed to a myogenic origin, in patients suffering from
SBMA (Arbizu et al., 1983). Additionally, consistently finding elevated creatine kinase levels in
SBMA patients implicates primary myopathic mechanisms in SBMA (Chahin and Sorenson,
2009). Thus, the question of whether primary muscle involvement in SBMA should be assessed
in considering the etiology of SBMA and potential targets for therapy.
In addition to the clinical data, Drosophila and murine models have also been crucial to
better understanding the neurodegenerative properties of SBMA. Drosophila research has
examined the survival of photoreceptor neurons when mutant AR is expressed in the eye.
Results from one study indicate that receptors with expanded CAG regions but confined to the
cytosol do not induce degeneration of photoreceptor neurons. However, a chimeric protein fused
to the nuclear localization signal of AR was enough to induce neuron degeneration in the eyes,
indicating that translocation of the AR to the nucleus regulates the androgen dependence of

10

SBMA (Takeyama et al., 2002). Additionally, the SBMA phenotype requires a functional AF-2.
AF-2 is a region on AR activated by ligand binding and recruits co-activators in order to initiate
androgen signaling (Nedelsky et al., 2010). This suggests some of the native functions of WT
AR are still present during SBMA onset and/or progression. Thus, Drosophila research has
advanced our understanding of the mechanisms associated with AR toxicity to cause neuronal
death. Murine models have also added to our understanding of SBMA. Five mouse models of
SBMA have been engineered by broadly expressing AR containing the CAG repeat expansions
(Chevalier-Larsen et al., 2004; Katsuno et al., 2002; McManamny et al., 2002; Sopher et al.,
2004; Yu et al., 2006). Three of the five models (named the 112Q, 97Q, and KI models) have
been confirmed to recapitulate the androgen-dependence of the disease observed in humans
(Chevalier-Larsen et al., 2004; Katsuno et al., 2002; Monks et al., 2008; Yu et al., 2006). Males
are symptomatic, and motor conditions can improve with castration. Conversely, females are
asymptomatic until treated with T. More recently, another mouse model was engineered where
transgenic (Tg) mice overexpress wild type rat androgen receptors (WT AR) specifically in
skeletal muscles. Contrary to expectations, Tg mice exhibit androgen-dependent motor
dysfunction similar to what is seen in the other five models; T treatment in females induces
disease, and castration ameliorates disease in males (Monks et al., 2007). As the altered genetic
expression is confined to the skeletal muscle fibers, data from this model suggest skeletal
muscles may be affected primarily, with the disease then spreading to the motoneurons
secondarily. Motoneuron function has been shown to be compromised in retrograde axonal
trafficking studies in this novel SBMA model (Kemp et al., 2011).

Experiments to determine disease origin

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While previous results have suggested the importance of skeletal muscle in SBMA (Monks et
al., 2007; Palazzolo et al., 2009), whether motoneuron diseases have a myogenic origin is an area
of active research in other conditions, such as ALS and SMA. Thus, while findings relevant to
skeletal muscle (dys)function during SBMA will be useful in better characterizing SBMA
specifically, that research may also serve to inform mechanisms involved in other so called
motoneuron diseases such as ALS and SMA. In several animal models of neurodegenerative
diseases, the broad expression of genetic mutations does not allow determination of specific preor postsynaptic site(s) of action for disease onset and progression. The lack of a confirmed site
of action has led to studies trying to determine the exact origin of the disease by selectively
expressing or suppressing the disease-causing genes in either motoneurons or skeletal muscles
(Cifuentes-Diaz et al., 2001; Dobrowolny et al., 2005; Johansen et al., 2011; Kieran et al., 2005;
Monks et al., 2007; Palazzolo et al., 2009; Perlson et al., 2009; Wong and Martin, 2010; Yu et
al., 2006).
A feature reported in one SBMA mouse model is the loss of motoneurons and nuclear
inclusions in some of the motoneuron soma (Thomas et al., 2006). However, other data from
another model suggest nuclear inclusions and neuronal dysfunction can occur in the absence of
cell death (Adachi et al., 2001). This is also seen in ALS models where overexpressing mutant
SOD1 specifically in motoneurons does not result in motoneuron pathology or an ALS
phenotype (Lino et al., 2002). Moreover, another study using an ALS model where uncoupling
protein 1 was specifically overexpressed in skeletal muscles has suggested distal defects in the
neuromuscular junction may be caused by early pathology of the skeletal muscle, leading to
dysfunction in the motoneuron (Dupuis et al., 2009). Thus, early myogenic factors could
contribute to the later disease effects in motoneurons in conditions often thought to be due to a

12

primary effect directly on motoneurons. One possibility is the loss of neurotrophic factors in
muscles that retrogradely promote the viability and function of motoneurons.
Typically, muscle function has been assessed by one of two ways: 1) stimulating the nerve
and measuring force production in skeletal muscles, or 2) isolating and field-stimulating skeletal
muscles directly with electrodes to induce contractions. While most labs have utilized indirect
muscle stimulation via the nerve to trigger muscle contractions in ALS models and SBMA
patients (Hegedus et al., 2008; Kieran et al., 2005; Suzuki et al., 2008), this approach does not
allow one to determine whether muscle weakness is due defects intrinsic to the muscle or due to
defects upstream of the muscle. Thus, the approach I took was to examine muscle function
directly in isolation from the nervous system (the latter of the two methods previously
mentioned). To better understand skeletal muscle measurements, a description of skeletal
muscle architecture and function is necessary.

Anatomy and Physiology of Skeletal Muscle
Skeletal muscles are large organs that comprise about 40% of total body mass in humans.
They are organized in descending orders of complexity as a single skeletal muscle is divided into
fascicles, bundles within a muscle that contain myocytes or myofibers surrounded by a
connective tissue layer known as fascia. The myofibers in fascicles are large multinucleated
cells that are the primary components of muscles (Figure 1). Myofibers come in a diverse array
of types and several fiber types are usually found within one muscle. Fiber type composition of
a muscle determines its contractile speed and the amount of force that it can produce. Myofibers
are comprised of myofibrils, which contain repeat structures in series known as sarcomeres,
which form the basic contractile unit within a single fiber. Inside the sarcomere are

13

myofilaments and the organelles necessary for contraction, such as the sarcoplasmic reticulum
(SR), T-tubules and mitochondria (Figure 2). The myofilaments are comprised of thick
filaments (myosin) and thin filaments (actin) (Figure 3). Actin contains the binding site where
myosin heads attach and initiate skeletal muscle contractions. Additionally, troponins are
located near the actin filament. Troponin is comprised of troponin C (TnC), troponin I (TnI),
and troponin T (TnT) which play regulatory roles in muscle contraction (Szent-Gyorgyi, 2004).
Voluntary in vivo contractions are initiated at the motor endplate, where a presynaptic signal
is transmitted to the muscle via release of acetylcholine (ACh) from the nerve terminal. Upon
binding to its receptors (AChR) postsynaptically, sodium rushes into the muscle to induce a
depolarization via the T-tubule. When skeletal muscles are isolated, electrode(s) replace the
input from the nervous system and the voltage change in the electrode(s) induce(s) a
depolarization within the muscle. T-tubules are invaginations of the sarcolemma that surround
myofibrils and are in close contact with the SR. The regions where T-tubules contact the SR are
called the terminal cisternae, and the association of the T-tubule to two terminal cisternae form a
triad (Figure 3; reviewed in (Al-Qusairi and Laporte, 2011)). Within a skeletal muscle fiber,
there are thousands of triads, and this arrangement enables myocytes to overcome the spatial
limits of using calcium (Ca) as a second messenger. T-tubules conduct the electrical
depolarization to initiate conformational changes in the dihydropyridine receptor (DHPR), a
voltage-gated Ca channel. The DHPR is mechanically coupled to the skeletal muscle ryanodine
receptor (RyR1), which opens and releases Ca from the sarcoplasmic reticulum (SR) (Figure 41). It is this change in intracellular Ca concentration that critically mediates muscle contraction.
Upon its release from the SR, Ca binds to TnC, the Ca-sensitive subunit of the troponin complex.
TnC binding Ca induces a conformational change in the troponin complex which removes

14

troponin I, the inhibitory subunit of the troponin complexes, from the actin filament (Figure 4-2;
(Ebashi and Endo, 1968)). As TnI moves away from the actin binding sites, tropomyosin is
pulled away, allowing myosin heads to bind to actin filaments with TnT linking the other
troponins to tropomyosin (Figure 4-3).
Once the myosin heads bind to the binding sites on actin, ATP is hydrolyzed, resulting in
ADP and inorganic phosphate. Phosphate is released, turning the myosin head (Figure 4-4).
With the release of ADP, the myosin head is turned even further, moving the actin filament,
shortening the sarcomere, and resulting in a power stroke to initiate skeletal muscle contraction
(Figure 4-5; (Eisenberg and Hill, 1985)). This results in shortening in the sarcomeres and
produces the contractile force generated by muscles (Huxley and Niedergerke, 1954). As long as
Ca ions and ATP are available, another ATP will bind to the myosin head to release it from the
actin filament and return the myosin head to its original position for another power stroke to
occur, and the cycle can repeat (Figure 4-6&7). In cases where ATP is depleted, the myosin
head remains in the locked position on the actin filament (Figure 4-5). Once the membrane
potential returns to resting potential, the DHPR-RyR1 complex closes, and sarcoendoplasmic
reticulum ATPases (SERCAs) actively return Ca ions to the SR (Figure 4-8). Consequently,
TnC no longer binds Ca, and the troponin-tropomyosin complex returns to its resting state where
tropomyosin covers the myosin binding sites on actin (Figure 4-0).
Force output of the skeletal muscle can vary based on fiber properties of skeletal muscles: I,
IIA, IIB, IIX, and hybrids (Pette and Staron, 2000). Skeletal muscles containing primarily type
IIB/X fast fatigable glycolytic fibers generate higher levels of force over a faster time course.
The fast-twitch muscles fatigue over a relatively short time course due to low vascularization and
mitochondrial content. In contrast, skeletal muscles with primarily types IIA fast fatigue-

15

resistant or I slow fatigue-resistant fibers generate lower levels of force over a slower time
course. Slow-twitch muscles also fatigue over a relatively long time course due to greater
vascularization and mitochondria content (Burke et al., 1971). In addition to differences in force
production and fatigability, fast-twitch and slow-twitch muscles exhibit different kinetic
properties. Fast-twitch muscles, so named due to the relatively shorter amount of time to
contract, have faster acting ryanodine receptors (RYR1) and sarcoendoplasmic reticulum
ATPases (SERCA) to release and restore Ca ions from/to the SR, respectively. Additionally, in
smaller mammals, fast-twitch muscles contain a Ca-buffering protein, parvalbumin (PV), which
shortens the contraction duration (Heizmann et al., 1982). In contrast, slow-twitch muscles have
longer contraction times due to slower RYR1 and SERCA subtypes and lack PV (Brandl et al.,
1987; Heizmann et al., 1982; Imagawa et al., 1989). Defects in the function of one or more such
proteins in skeletal muscles could lead to dysfunction, often observed as motor weakness,
without changes in kinetics of muscle contraction.

Overview
Like SBMA in humans, several of the animal models show a male-specific phenotype that
depends on male levels of androgens. Male mice present a phenotype, and symptoms can be
ameliorated by castration (Chevalier-Larsen et al., 2004; Katsuno et al., 2002). In some models,
T can also be given to female Tg mice, and disease can be rapidly induced (Katsuno et al., 2002).
Thus, females of SBMA models are critical to understanding the early symptoms and pathology
of the disease, whereas males are useful in understanding pathology and weaknesses due to a
chronic disease phenotype. For this reason, females and males were tested, and these
experiments form the two research chapters to the thesis. Originally, it was uncertain if muscle

16

weakness would be measured in SBMA mice, and only female mice overexpressing a WT AR
transgene only in skeletal muscle fibers were tested and forms the first chapter of the thesis.
Once muscle weakness was confirmed in these females, males from the same model were also
tested. However, as the expectation of observing muscle weakness was confirmed, the scope of
the study was expanded to also include males from two other SBMA models. The so-called
“97Q” model is a Tg mouse model in which a full length AR transgene containing 97Qs is
broadly expressed in mice. The KI model expresses a humanized AR gene in mice. Exon 1 of
mouse AR gene was replaced with exon one of the human AR gene containing 113 CAG repeats.
This mutant AR is expressed in all cells that normally express AR because its expression is being
driven by native AR promoters. This study indicates that muscle weakness is also likely
involved in the disease phenotype of male SBMA mice and forms the second chapter of the
thesis.

17

CHAPTER 1: Androgen receptors in muscle fibers induce rapid loss of force but not mass:
Implications for SBMA
Oki, K., Wiseman, R.W., Breedlove, S.M., Jordan, C.L. (2013) “Androgen receptors in muscle
fibers induce rapid loss of force but not mass: Implications for SBMA.” Muscle & Nerve
47(6):823-34.

18

Abstract
Introduction: Testosterone (T) induces motor dysfunction in transgenic (Tg) mice that
overexpress wild type androgen receptor (AR) in skeletal muscles. Since many genes implicated
in motor neuron disease are expressed in skeletal muscle, mutant proteins may act in muscle to
cause dysfunction in motor neuron disease. Methods: We examined contractile properties of the
extensor digitorum longus (EDL) and soleus (SOL) muscles in vitro after 5 and 3 days of T
treatment in motor-impaired Tg female mice. Results: Both muscles showed deficits in tetanic
force after 5 days of T treatment, without losses in muscle mass, protein content, or fiber
number. By 3 days of T treatment, only SOL showed a deficit in tetanic force comparable to that
at 5 days of treatment. In both treatments, EDL showed slowed twitch kinetics, whereas SOL
showed deficits in the twitch/tetanus ratio. Conclusions: These results suggest calcium handling
mechanisms in muscle fibers are defective in motor-impaired mice.

Introduction
The long-held assumption that motor dysfunction caused by motoneuron disease is the direct
result of motoneuron loss has been challenged recently (Dupuis et al., 2009; Gould et al., 2006).
Furthermore, whether motor impairments associated with motoneuron disease are due to defects
that originate in the motoneurons, muscles, or both has also come under scrutiny recently.
Because genes linked to motoneuron disease are expressed widely, typically in both
motoneurons and muscles, deficits in motor performance associated with motoneuron disease
could arise from dysfunction in the motoneurons, skeletal muscles, or both. Recent data from
muscle-specific mouse models of motoneuron disease suggest that skeletal muscle may indeed
be a primary site of action for disease genes, and dysfunction in skeletal muscles may contribute,

19

if not underlie, motor dysfunction (Braun et al., 1995; Cifuentes-Diaz et al., 2001; Di Giorgio et
al., 2007; Dupuis et al., 2009; Monks et al., 2007; Nagai et al., 2007; Wong and Martin, 2010).
The transgenic (Tg) model used here recapitulates a polyglutamine disease called spinal
bulbar muscular atrophy (SBMA) (Monks et al., 2007), a late-onset motoneuron disease that
affects only men and is linked to a CAG repeat mutation in the androgen receptor (AR) gene.
While not interfering with masculine development and function, this CAG expansion mutation in
AR causes men with SBMA to develop progressive loss of strength that greatly impairs their
motor function (Fischbeck, 1997). This muscle-specific or “myogenic” Tg model was
engineered by inserting a transgene into mice that overexpresses wild type (WT) rat androgen
receptors (AR) selectively in skeletal muscle fibers using the human skeletal α actin promoter
(Monks et al., 2007). Like SBMA in humans and other mouse models of this disease, motor
dysfunction in such myogenic Tg mice is triggered by male levels of androgens and can be
relieved by lowering circulating androgen levels (Banno et al., 2009; Chevalier-Larsen et al.,
2004; Katsuno et al., 2002; Kinirons and Rouleau, 2008; Monks et al., 2007; Yang et al., 2007;
Yu et al., 2006). While Tg females in this model also express the AR transgene at comparably
high levels, they are asymptomatic throughout life. However, when provided with male-typical
levels of testosterone, the motor function of Tg females deteriorates rapidly. Motor defects
develop in only a few days, comparable to those seen in Tg males, including shortened stride
length and deficits in grip strength (Johansen et al., 2009; Monks et al., 2007). Significantly, Tg
male mice with a mutation in endogenous AR, which eliminates endogenous AR function, also
show the same androgen-dependent loss of motor function (Johansen et al., 2011). This
demonstrates that testosterone activates functional transgenic AR to impair motor function.
While Tg males show several markers of motoneuron disease, including atrophic and angulated

20

muscle fibers and deficits in the number of axons in ventral roots, acutely diseased Tg females
do not (Johansen et al., 2009; Monks et al., 2007). Thus, AR acting directly in muscles can
produce profound deficits in motor function without the expected histopathology in muscle,
and/or loss of motoneurons, a dissociation that is also evident in other mouse models of SBMA
(Chevalier-Larsen et al., 2004; Katsuno et al., 2002; Yu et al., 2006).
To explore whether muscle dysfunction might underlie motor dysfunction in myogenic mice,
we directly examined the contractile properties of muscles from diseased and healthy female
mice. We chose to study females over males, because unlike Tg males, who are chronically
impaired, the disease state can be “turned on” by exposure to androgens in Tg females. Thus,
given that the time of disease onset is known, acutely-impaired Tg females offer the potential to
distinguish proximal pathogenic mechanisms that underlie motor dysfunction from secondary
effects of disease. Muscle mechanics of the fast-twitch extensor digitorum longus (EDL) and
slow-twitch soleus (SOL) were examined in vitro from adult Tg females and age-matched WT
controls following 5 and 3 days of testosterone treatment. We observed a rapid loss of intrinsic
force in both the EDL and SOL that occurs independent of muscle mass in this model.

Methods
Animals and hormone treatment. Transgenic (Tg) mice were generated and genotyped using
PCR as previously described (Monks et al., 2007). Tg animals from the 141 founding line
express a WT rat AR cDNA under the control of the human skeletal α actin promoter. Tg
females were mated to C57/B6J mice for several generations to produce progeny for this study.
Water and food were provided ad libitum. All animal procedures were approved and performed
in compliance with the Michigan State University Institutional Animal Care and Use Committee,

21

in accordance with the standards in the NIH Guide for the Care and Use of Laboratory Animals.
Adult (90-120 days) Tg and age-matched wild type (WT) females were deeply anesthetized with
isoflurane, and Silastic capsules (1.57 mm i.d. and 3.18 mm o.d., effective release length of 6
mm) containing either crystalline testosterone (T) (~0.15 mg per capsule) or nothing (blank)
were implanted subcutaneously just caudal to the scapula, as previously described (Johansen et
al., 2009). Incisions were closed with 9 mm surgical staples. All animals received the analgesic
ketoprofen (5 mg/kg, sc) immediately following surgery. In the first experiment, mice were
treated for 5 days before muscle function was examined. Treatment groups were as follows:
blank-treated wild type (B WT), testosterone-treated wildtype (T WT), blank-treated transgenic
(B Tg), and testosterone-treated transgenic (T Tg) with n=5–6 mice/group. Separate cohorts of
mice were used to examine the EDL and the SOL after 5 days of treatment. In a second
experiment, contractile properties of the EDL and SOL harvested from the same set of mice were
examined after 3 days of T treatment, comparing muscles from T WT to T Tg mice, n=5-6
mice/group.

Motor function tests. Motor function was assessed using the grip strength and hang tests 1 hour
before surgery to establish baseline performance (Day 0) and on each day following surgery
(Days 1-3 or 1-5) until the time of sacrifice. Forelimb grip strength was assessed using a grip
strength meter (Columbus Instruments) oriented in the horizontal plane (Chevalier-Larsen et al.,
2004; Johansen et al., 2009; Yu et al., 2006). Mice were held by the tail and lowered to the
apparatus, allowed to grasp the metal grid with their forelimbs only, and were pulled by the tail
vertically at a 90° angle away from the grid until their grip was released. Force (g) applied to
the bar at the moment of release was recorded as grip strength. Seven consecutive measurements
22

were done within a single 2-minute session for each subject with the lowest and highest grip
values excluded, and the mean of the remaining 5 values recorded as the grip strength for that
animal and session. The hang test was conducted by placing mice on a wire grid that was then
turned upside-down 40 cm above a counter. Latency to fall (up to 120 seconds) from the wire
grid was measured (Monks et al., 2007; Sopher et al., 2004).

In vitro studies to assess muscle mechanics. To determine whether impairments in skeletal
muscle function might underlie motor dysfunction, muscle contractile characteristics of the EDL
and SOL were examined in vitro in motor-impaired Tg females and controls after 3 or 5 days of
hormone treatment. These hindlimb muscles were selected, because the EDL and SOL are
prototypical fast and slow twitch muscles, respectively, with considerable background
information available on their normal contractile properties (Crow and Kushmerick, 1982;
Kushmerick et al., 1992). Moreover, we were interested in determining whether the toxic effects
of an activated AR in this model system might depend on fiber type, as observed in other
diseases (Atkin et al., 2005; Hegedus et al., 2008).
Mice were anesthetized deeply with a single intraperitoneal injection of sodium pentobarbital
in saline solution (0.1 mg/g body weight, Abbott Laboratories, North Chicago, IL, USA). We
chose sodium pentobarbital as opposed to ketamine/xylazine as our anesthetic, which has little to
no residual effect on the contractile properties of muscles assessed in vitro, yielding estimates
that best fit those reported in the literature (Lapointe and Cote, 1999). Once the muscles were
exposed, 5-0 silk sutures were tied to the proximal and distal tendons in situ, and muscle resting
length was recorded. Muscles were then excised by cutting above the suture knot on the
proximal tendon and below the suture knot distally. Excised muscles were incubated in an 8 mL

23

organ bath containing oxygenated Ringer solution (117.1 mM NaCl, 4.6 mM KCl, 25.3 mM
NaHCO 3 , 2.5 mM CaCl 2 , 1.2 mM MgSO 4; pH 7.4; equilibrated with 95% O 2 /5% CO 2 ) (Dentel
et al., 2005; Wiseman et al., 1996). The temperature of the bath was monitored continuously
using a K-type thermocouple (Omega Engineering, Stamford, CT) adjacent to the muscle and
maintained at 25±0.2° C by circulating water through a glass-jacketed organ bath (Radnoti Glass
Technology, Monrovia, CA). Isolated muscles were fixed at their approximate resting length by
tying 1 end of the muscle ligature to a glass hook and the other to an isometric force transducer
(Astro-Med, West Warwick, RI) fitted with a positioning micrometer. Muscles were initially set
to their in vivo resting length by using the suture distances between the proximal and distal
tendons. The optimal length (Lo) for each muscle was determined precisely using the lengthtension relation. Electrical stimulation was delivered via 2 platinum plate electrodes (3-4 mm
apart, 25 mm long) parallel to the long axis of the muscle using a Grass S88 Stimulator (Grass
Instruments, Quincy, MA). Force output was digitized using an analog-to-digital converter
(ADC) (model AT MIO16E; National Instruments, Austin, TX) controlled via commercially
available software (LabScribe/NI; iWorx, Dover, NH) operating at a sampling rate of 1 KHz (1
ms time resolution). Muscles were stimulated to produce successive twitches (1 ms duration, 1
Hz) at 10-60 volts (V) to determine the supramaximal stimulation voltage (voltage at which
maximal twitch force was elicited X 1.35) used subsequently during the experiment (generally
30-40 V). Although the voltage used to directly stimulate the SOL muscle after 5 days of
treatment was slightly higher for diseased mice (T Tg) compared to the other groups, there was
no statistical difference in stimulation voltages between treatments and genotypes for the SOL
and EDL muscles at either the 5- or 3-day time points. After muscle excision, mice were
euthanized.
24

Muscle function was assessed based on directly evoked twitches and tetani. Stimulation
parameters used to evoke muscle twitches were 1 ms duration pulses delivered at rates ranging
from 0.2 Hz to 120Hz at supramaximal voltage to generate the full range of function from single
twitch to fused tetani. These data were used to generate the force-frequency relation for each
preparation. Muscle tetani were evoked by stimulating the muscle directly with 1 ms duration
pulses at fusion frequency, defined operationally as the frequency at which ripples in the plateau
phase of the tetanic force are < 3% of the peak force (Wiseman et al., 1996). The fusion
frequency ranged from 80-100 Hz for the EDL and 30-40 Hz for the SOL. Because the kinetics
of force development are slower in the SOL muscles, the length of the stimulus train to induce
the plateau phase of a tetanus also differed between muscle fiber types. Tetanic contractions
were elicited by delivering a 500 ms train to EDL and a 1 s train to the SOL. In all instances
both the twitch and tetanic peak forces were normalized to the tendon-free muscle mass.
Resistance to fatigue was assessed by stimulating the EDL and SOL with repeated tetanic stimuli
(1 train/s; 330 ms duration, ~80-100 Hz in EDL and 670 ms duration, 30-40 Hz in SOL) until
force decreased to approximately 10% of initial force. This was determined experimentally to be
90 s for wild type EDL and 135 s for wild type SOL from start of the fatigue stimulation, and
this parameter was used for all treatment groups. Fatigue resistance was measured by
determining the duration in seconds it took for maximal tetanic force to drop to half its starting
value. Following the experiment, muscles were removed rapidly from the apparatus, dissected
free from tendons, blotted of excess media, and weighed to estimate mass and then stored at 80°C.
In a separate series of experiments with other mice (B WT mice and T Tg, 90-120 days old;
n=2-4/group), D-tubocurarine was added to bath (100 μM, Sigma-Aldrich) to test whether the

25

motor nerve contributed to the force produced (Cairns et al., 2007). Force production was not
affected by the addition of curare, indicating that field applied electrodes were not relying on
stimulation of neuromuscular junctions to elicit muscle contractions (data not shown).

Histology. In a separate cohort of age-matched WT and Tg females (90 -120 days of age) treated
for 5 days with either T or blank capsules, the SOL was dissected, placed in OCT compound
(Sakura Japan) in cryomolds (Cryomolds) and snap frozen in liquid nitrogen (n = 5 mice/group).
SOL were sectioned on a Leica cryostat at 10 μm, thaw-mounted onto gelatin-coated slides, and
stained with hematoxylin and eosin (H&E) for fiber counting, as previously described for the
EDL (Johansen et al., 2009).

Androgen receptor protein expression. Upon completion of force recordings, we assessed total
protein content and AR expression in muscles, using previously described methods (Johansen et
al., 2007). In brief, the EDL and SOL were homogenized in lysis buffer [14 mM NaCl, 0.268
mM KCl, 0.147 mM KH 2 PO 4 , 0.802 mM Na 2 HPO 4 1% Nonidet P-40 (v/v), 0.5% sodium
deoxycholate (wt/v), 1% sodium dodecyl sulfate (wt/v), 10% protease inhibitor (v/v; SigmaAldrich P2714)] using a Bio-Gen PRO 200 homogenizer (PRO Scientific). Samples were
centrifuged at 12,000x at room temperature for 5 minutes. Supernatant from each sample was
isolated to determine protein concentration using a Pierce BCA protein assay (Thermo Scientific)
and a spectrophotometer (Beckman DU 530). Ten µg of protein was loaded per lane on an 8%
Tris-Glycine gel (Invitrogen), run at 125 V for 2 hours, transferred to a nitrocellulose membrane
at 45 V for 2 hours, and probed using a rabbit polyclonal antiserum directed at AR (N-20, Santa
Cruz Biotechnology; Santa Cruz, CA, USA; dilution 1:500; 0.4 μg/mL). This was followed by
26

incubation of the membrane in anti-rabbit goat antiserum labeled with horseradish peroxidase
(HRP; Santa Cruz, sc-2004; 0.4 μg/mL) and detected by Luminol (Santa Cruz Biotechnology).
Films were exposed and developed using an X-OMAT 1000A Processor (Kodak). After probing
for AR, nitrocellulose membranes were stripped and re-probed for actin using goat polyclonal
anti-actin antiserum directly conjugated to HRP (I-19 Santa Cruz; 0.4 μg/mL).

Protein gels. Muscle homogenates were prepared as described previously and loaded into 12%
SDS gels. Gels were run until the dye front had reached the bottom of the gel (~2 hours at 125
V). Protein bands were visualized by incubating in Coomassie Brilliant Blue R250 (Thermo
Scientific Pierce) and destaining (40% methanol, 10% acetic acid v/v in distilled water).

Testosterone levels. Blood was collected via transcardial puncture from deeply anesthetized
mice used for studies on muscle mechanics and held on ice for 30 minutes before centrifugation
at 3000 rpm for 20 minutes at 8° C (Sorvall Biofuge Fresco, Thermo Scientific, Asheville, NC).
After centrifugation of blood samples, plasma was collected and held at -80° C until it was
assayed for circulating testosterone levels as described previously (Zuloaga et al., 2008).

Data analysis and statistics. Analysis of mechanical transients was performed using a custom
algorithm (Jayaraman et al., 2006) in a MatLab programming environment (Mathworks, Natick,
MA). In brief, raw digital signals were passed through a low-pass filter to smooth the data and
then analyzed for force output and tension-time integrals. Kinetic parameters for the rise time
(time to peak force) and relaxation time (time of return to baseline) were also measured in
individual muscle twitches as described previously (Jayaraman et al., 2006). Statistical analysis
27

of data after 5 days of treatment was performed using two-way analysis of variance with
genotype and hormone treatment as independent variables (SPSS Inc., Chicago, IL). Group
differences were considered significant at P<0.05, and post hoc comparisons were done using the
Tukey post-hoc test. Independent group t-tests were used to evaluate significant differences
between muscles from Tg versus WT mice after 3 days of T treatment.

Results
Testosterone (T) treatment impairs motor function without loss of muscle mass
At Day 0, before testosterone treatment, comparisons across treatment groups indicated
similar body weights (data not shown) and comparable motor function based on both the hang
and grip strength tests (Fig 5A, B). Replicating previous findings (Johansen et al., 2009; Monks
et al., 2007), T induced a rapid and progressive decline in motor performance only in Tg mice.
None of the 3 control groups (B WT, T WT, B Tg) showed any change in motor function.
Although T also induced decreases in the body weight of Tg females (Figure 5C), losses in body
weight lagged significantly behind the loss of muscle strength, as seen previously (Johansen et
al., 2009). For example, a significant deficit in hang time was detected by day 2 of T treatment
in Tg females (P < 0.001, compared to T WT or B Tg), with performance on this test reaching 0
s by Day 3, whereas a significant loss in body weight did not emerge until 3 days of T treatment
(P < 0.05 at Days 3-5 in T Tg females versus asymptomatic controls, T WT, or B Tg). These
data indicate that a rapid loss of motor function occurs only when T activates transgenic AR in
skeletal muscle fibers and that neither the AR transgene nor T alone affect motor performance,
replicating previous results (Johansen et al., 2009; Monks et al., 2007).

28

Although T induced marked losses in both body weight and muscle strength based on the
hang and grip strength tests in Tg females, 5 days of T treatment did not induce any apparent
decrease in muscle mass. Specifically, neither the EDL nor SOL muscle of Tg females showed a
significant decrease in mass following T treatment (Table 1).
As expected, circulating T levels were greater in females given T capsules (T WT, T Tg)
than those given blanks (B WT, B Tg; Table 1, P<0.05). However, T capsules of the same size
and randomly selected from the same stock produced greater levels of circulating T titers in Tg
females than WT females, but both averages were within the physiological range of gonadally
intact WT male mice. We suspect that this elevated level of T in Tg mice is due to their reduced
body weight by 5 days of T treatment, as we have obtained similar findings in rats that differ in
body weight (Johnson et al., 2012).

Skeletal muscles from T-treated Tg female mice show striking androgen-dependent loss of
force
Five-Day Treatment
Tetanic Force. Tetanic force measurements from WT or Tg mice with normal motor
function indicate that the EDL and SOL exhibit the expected characteristics of fast and slow
muscle, respectively, with the fast-twitch EDL requiring less time but higher stimulation
frequencies than the slow-twitch SOL to attain maximal force production (data not shown). Also
as expected, the EDL produces nearly twice as much force per gram of muscle wet weight as the
SOL (Fig 6A-D). While these same fiber-type specific differences were also seen in the EDL
and SOL muscles of motor-impaired, T-treated mice, both muscles showed striking deficits in
maximal tetanic force normalized to muscle mass (Fig 6). T induced a significant decrease in

29

maximal tetanic force only in Tg females, resulting in a significant interaction of genotype with
treatment and significant main effects of both genotype and treatment (P < 0.001 for both). Both
the EDL and SOL muscles of T-treated Tg females showed significantly less tetanic force
compared to either T WT or B Tg females (P < 0.001 for both). T also induced a greater
proportional deficit in tetanic force in the SOL than in the EDL of motor-impaired mice. While
tetanic force for the fast-twitch EDL from T-treated Tg mice was approximately half that
produced by the EDL from control mice, tetanic force for the slow-twitch SOL was decreased
even further in T-treated Tg mice, to about a quarter of that produced by controls. All muscles
tested had comparable force production at the end of the experiment as at the beginning (Fig 6C,
D), indicating that there was little deterioration of the muscles in vitro, and suggesting that the
force deficits observed in vitro reflect primary muscle dysfunction in vivo.
Muscle histology and protein content. The SOL was chosen for detailed histological
analysis, because we had previously established that the EDL from motor-impaired Tg mice
treated with T for 9 days shows no notable histopathology in H&E stained cross sections
(Johansen et al., 2009). Likewise, we found no signs of histopathology in the SOL of motorimpaired, T-treated Tg mice. For example, muscle fibers in the SOL from T-treated Tg mice
appear homogeneous in size (Fig 7A), without evidence of atrophy. While centralized nuclei
were occasionally observed, they were no more frequent in the impaired SOL than in control
SOL (data not shown). Moreover, we found no differences in the number of SOL muscle fibers
from motor-impaired versus control mice (Fig 7B).
Because loss of contractile proteins might not be reflected in overall muscle weight, we also
measured the total amount of protein in the EDL and SOL muscles of motor-impaired and
control mice but we found no evidence for a net loss in total protein in either the EDL or SOL of

30

motor-impaired Tg mice compared to control Tg and WT mice with normal motor function
(Table 1). Moreover, muscle protein gels revealed no consistent differences in specific
contractile proteins in either muscle (data not shown).
We also addressed whether the SOL expresses AR at a higher level than the EDL in Tg
females, since this could potentially explain the more severe loss of force in the SOL than the
EDL following T-treatment (Fig 6). To assess this possibility, AR expression was examined in
Western blots of muscle homogenates from Tg mice. As expected, AR expression was higher in
both the EDL and SOL of Tg mice compared to the AR-enriched bulbocavernosus/levator ani
(BC/LA) muscles of WT mice (Monks et al., 2007), but there were no consistent differences in
AR expression between the two muscles (Fig 7C) that might account for the relatively greater
loss of tetanic force induced by T in the SOL compared to the EDL from Tg mice (Fig 6).

Skeletal muscles from T-treated Tg females exhibit androgen-dependent changes in twitch
force and kinetics
Twitch Force. Peak force of individual twitches (normalized to muscle weight) produced by the
EDL and SOL from T-treated Tg females were decreased to about 60% and 7%, respectively, of
peak twitch forces produced by these same muscles from blank-treated Tg mice (Table 1; Fig
8A, B). Although the twitch to tetanus ratio did not differ across treatment groups in the EDL
(Table 1; P > 0.05), SOL from T-treated Tg females had a significantly lower twitch to tetanus
ratio than muscles from Tg and WT control mice (Table 1; P < 0.001), due to T treatment
reducing SOL peak twitch force more than peak tetanic force in Tg mice.
Contraction Kinetics. Analysis of twitch kinetics revealed a slower rise time to peak force
and a slower relaxation time to baseline in EDL from motor-impaired T-treated Tg mice

31

compared to the EDL from control-treated Tg or WT mice (Fig 8C, E; p < 0.001 for both rise
and relaxation in T Tg versus T WT or B Tg). Delays in both the rise and relaxation times
suggest the kinetics of calcium release from, and uptake to, the sarcoplasmic reticulum or
binding to troponin C of the thin filament are altered in the EDL of T-treated Tg mice. The
observed left-ward shift in the force-frequency curves in T-treated EDL also supports this view
(data not shown). However, neither the times to peak force nor relaxation time of the twitch
were altered significantly by T in the SOL of Tg mice (Fig 8D, F).

Tetanic force production is reduced in the SOL but not EDL of Tg mice after three days of
T treatment
Deficits in motor function are observed as early as 2 days after the initiation of treatment. Ttreated Tg mice were unable to perform the hang test by Day 3, raising the question of whether
changes in muscle function are also evident this early. Thus, we measured muscle mechanics in
a separate cohort of Tg and WT females given T for 3 days.
We found the same time course of motor dysfunction after 3 days of T treatment as seen in
the previous cohort (data not shown). In vitro measures of contractile properties after 3 days of
T treatment revealed that both peak tetanic and twitch forces were reduced significantly in the
slow-twitch SOL of Tg mice compared to the SOL of WT mice (P < 0.05, Fig 5A, B) but that
only peak twitch force was significantly decreased in the EDL from the same Tg mice at this
timepoint (P < 0.05, Fig 5B). Maximum tetanic force produced by the EDL in Tg mice was
slightly but not significantly less than in WT controls (P=0.07, Fig 5A). Only the SOL muscle
of Tg mice showed a significant deficit in the twitch-to-tetanus ratio after 3 days of T exposure
(P < 0.01, Table 1), comparable to the effects of T after 5 days of treatment. Twitch kinetics for

32

the SOL of Tg mice were unaffected by T after 3 days of treatment, as expected. However, for
the EDL of Tg mice, in which both speed to contract and relax were prolonged after 5 days of T,
only relaxation was significantly slowed after 3 days of T treatment (P < 0.05, Fig 5D), with no
effect of T on rise time to peak force (Fig 5C). In sum, the same pattern of deficits was seen in
the SOL after both 3 and 5 days of T treatment whereas only some deficits, namely peak twitch
force and relaxation time, were seen in the EDL after both 5 and 3 days of T treatment,
suggesting that deficits in the SOL generally emerge sooner than in the EDL.

Only the EDL from motor-impaired mice show deficits in fatigue resistance
The only effects on fatigue resistance were seen in the EDL after 5 days of T treatment. The
EDL from Tg mice treated with T for 5 days fatigued significantly faster, dropping to half
maximal tetanic force sooner than the EDL from either blank-treated Tgs or T-treated WTs (Fig
10A, P < 0.01). Interestingly, the EDL from blank-treated Tg mice showed a greater resistance
to fatigue than the EDL from blank-treated WT mice (P < 0.05), indicating that the transgene
alone enhanced fatigue resistance in the EDL (Fig 10A). We found no effect of T on fatigue
resistance in the SOL from motor-impaired Tg mice after either 3 or 5 days of T treatment (Fig
10 B, D) nor did we find an effect of T on fatigue resistances in the EDL of Tg mice after 3 days
of treatment (Fig 10D). That deficits in both maximal tetanic force and fatigue resistance in the
EDL emerge later than other defects in this muscle suggests that the same mechanism, perhaps
involving mitochondrial dysfunction, may underlie both.

Discussion

33

This study shows that male levels of androgens not only trigger a pronounced and rapid loss
of motor function in female Tg mice, but they also trigger a concomitant loss of contractile
function in skeletal muscle in such mice; maximal force production of both the fast twitch EDL
and slow twitch SOL plummeted to about half that of WT controls. This marked loss in
contractile strength develops within a few days, in step with the emergence of motor dysfunction,
and occurs without detectable losses in muscle mass or protein. Given that the AR transgene is
expressed only in skeletal muscle fibers in the Tg mice (Monks et al., 2007), our data show that
androgens can act directly on muscle fibers to reduce their contractile strength. Because ARs are
normally expressed in skeletal muscle (Johansen et al., 2007; Monks et al., 2004; Sinha-Hikim et
al., 2004), mutant AR may act directly in muscle, independent of the motoneurons, to cause
muscle weakness in SBMA. Importantly, the deleterious effects of transgenic AR on both motor
and muscle function were evident only when Tg mice were exposed to male-like levels of
androgens, in line with the androgen-dependence of SBMA in humans.
We have confirmed that the loss in force production reflects neither a loss in muscle mass nor
a disproportionate decay in muscle function in vitro. While force production of muscles from all
four treatment groups declined somewhat during the course of the experiment, this decline was
no greater in muscles from T-treated Tg mice than in muscles from control mice (Fig 6),
indicating that the reduced force exhibited by the EDL and SOL muscles of motor-impaired mice
cannot be attributed to a more rapid demise of muscle function in vitro. Moreover, while muscle
mass generally correlates with the amount of force produced, the robust loss of maximal tetanic
force to less than half that of normal was not accompanied by reduced muscle mass in motorimpaired mice. This was surprising, particularly because the mice lose nearly 20% of their body
weight during the 5-day treatment period. This weight loss likely reflects the increased

34

metabolic rate and loss of fat stores triggered by androgen activation of transgenic AR in muscle,
which is known to occur in such myogenic rodent models (Fernando et al., 2010). Indeed, at the
end of the 5-day treatment, fat pads in T-treated Tg females are negligible in size (unpublished
observation). Measures of twitch and tetanic force were normalized to the wet weight of each
muscle, which takes into account any change in mass that might contribute to changes in force.
We also saw no change in muscle mass per se, based either on their weight or total protein
content (Table 1). Neither did we see any apparent histological changes. The impaired SOL
contained a normal number of fibers, and those fibers appeared intact, with no signs of atrophy
ordinarily associated with deficits in muscle strength (Fig 7). Our results in the SOL also agree
well with prior results from the EDL of similarly treated mice (Johansen et al., 2009). Given that
myosin and actin make up most of the bulk in muscle fibers, these data suggest that AR perturbs
the contractile capacity of skeletal muscles in motor-impaired mice, not by inducing protein loss,
but by perturbing the function of critical proteins that mediate muscle contraction either directly
or through modulation of calcium handling properties.
Contractile strength was more severely and more rapidly affected in the SOL than in the EDL
in T-treated Tgs; it declined by about 75% of force after only 3 days of androgen exposure, a
time when effects on the EDL are negligible (Fig 5). This finding raised the question of whether
transgenic AR is expressed at appreciably higher levels in the SOL than in the EDL. However,
AR Western blots revealed no consistent difference in AR content between the EDL and SOL of
Tg mice. It is also noteworthy that the EDL of Tg mice showed other androgen-dependent
defects in contractile properties that the Tg SOL did not. These results also indicate that
differences in AR expression cannot readily explain the more severe effects of T on force in the
SOL.

35

While deficits in contractile force favored the SOL, the kinetics of force development
showed the opposite pattern. Only the EDL of Tg mice affected by T showed significant slowing
in both the rise and relaxation times compared to control-treated mice (Fig 8). We also found
that fatigue resistance was reduced in the EDL, but not in the SOL, after 5 days of T treatment in
Tg mice, with no effects on fatigability at 3 days (Fig 10). Interestingly, other investigators who
have studied the same model report that the number and size of mitochondria in the EDL
increases significantly after a week of T exposure (Musa et al., 2011), suggestive of
mitochondrial dysfunction. While the EDL normally derives much of its ATP from glycolytic
metabolism, the EDL from female Tgs after 9 days of T has shifted to a more oxidative
metabolism based on NADH staining (Johansen et al., 2009). This apparent shift toward
oxidative metabolism in the face of possibly impaired mitochondrial function could explain why
fatigue resistance is reduced in the EDL after 5 days of T. In sum, these findings suggest that
there are other inherent differences between these muscles, perhaps related to their fast- versus
slow-twitch fiber type composition (Crow and Kushmerick, 1982), that predispose them to
respond differentially to the toxic effects of an activated AR.
The overall profile of defects in both the EDL and SOL of motor-impaired mice suggest that
mechanisms controlling intracellular calcium fluxes in muscle fibers may be perturbed. For
example, the prolonged rise time to peak twitch force in the EDL of T-treated Tg mice suggests
that the function of the ryanodine receptor (RyR1) may be impaired. This receptor controls the
release of intracellular calcium from the sarcoplasmic reticulum to trigger muscle contraction.
Likewise, the decreased twitch to tetanus ratio seen in SOL after both 3 and 5 days of T
treatment, as well as the loss of tetanic force, suggests inefficient or impaired release of calcium
from the sarcoplasmic reticulum controlled by the RyR1. Moreover, the prolonged relaxation

36

times seen at both 3 and 5 days of T treatment in the EDL of Tg mice suggest that the
sarcoplasmic reticulum calcium ATPases (SERCA), which pump calcium back into the
sarcoplasmic reticulum to allow muscles to relax, may also be impaired (Berchtold et al., 2000).
While it is possible that other mechanisms involved in the initiation of muscle contraction
may be impaired, such as troponin C which binds calcium to trigger contraction, compelling
evidence suggests that post-translational modifications to the RyR1 may have a role in the
contractile defects we find. Recent studies of the mechanisms behind a loss of contractile
strength in both aging and dystrophic muscles indicates that the RyR1 becomes modified by
reactive oxygen and nitrogen species, causing the receptor to become leaky and leading to a
deficit in muscle force (Andersson et al., 2011; Bellinger et al., 2009; Bellinger et al., 2008).
Moreover, mitochondria likely play a key role in these maladaptive changes in the RyR1. In our
model, loss of muscle function is associated with both an increase in mitochondrial number and
activity of complexes I, II, and IV in the respiratory chain of mitochondria (Musa et al., 2011).
These mitochondrial complexes are the primary source of reactive nitrogen and oxygen.
Paradoxically, the production of ATP may at the same time be decreasing, as suggested by the
reduced resistance to fatigue in the EDL and a deficit in citric synthase activity found in the fast
twitch anterior tibialis muscle of these same T-treated Tg mice (unpublished observation).
Together, these data are consistent with the idea that excessive amounts of reactive oxygen and
nitrogen species produced by dysfunctional mitochondria in muscles of T-treated Tg mice may
modify the RyR1, impairing its function. Such modifications of the RyR1 could explain many, if
not all, the defects in muscle contraction that we observe. It is also noteworthy that such changes
in the RyR1 can occur quite rapidly. A bout of intense exercise has been shown to induce such

37

changes in the RyR1 in less than 24 hours (Bellinger et al., 2008), consistent with the rapid
decline in muscle force that we observe, particularly in the slow twitch SOL.
These data from our Tg mouse model may be relevant to human disease, particularly SBMA,
an androgen-dependent motoneuron disease that affects only men (Fischbeck, 1997; Kinirons
and Rouleau, 2008; La Spada et al., 1991; Schmidt et al., 2002). Several different mouse models
of SBMA have been developed that broadly express a full length human AR harboring the
disease-causing CAG repeat expansion. Such SBMA models show androgen-dependent loss of
motor function (Chevalier-Larsen et al., 2004; Katsuno et al., 2002; Yu et al., 2006). However,
because the mutated AR is expressed in both skeletal muscle and motoneuron in these various
models, whether mutant AR acts in 1 or both of these cell types or in some other cell population
is unknown. While androgen-dependent motor dysfunction in this model is triggered by WT AR
acting exclusively in muscle fibers, such mice show a disease phenotype remarkably similar to
that of other SBMA mouse models, indicating that AR may act in muscle to directly cause
muscle weakness. Consistent with this view, humans with SBMA show elevated levels of serum
creatine kinase (CK), indicative of skeletal muscle damage (Chahin and Sorenson, 2009). Such
elevations are observed in patients with primary myopathies, such as muscular dystrophy but not
in patients who have primary neuropathies (Pearce et al., 1964). Evidence from a knock-in (KI)
mouse model of SBMA also suggests that primary myopathy is caused by muscle AR (Yu et al.,
2006). Significantly, muscles from KI males show signs of pathology before motoneurons.
Affected muscles in those males also have decreased expression of the ClCN1 chloride channel,
and show abnormal spontaneous activity, like muscles affected by myotonic dystrophy, a
primary myopathy. Finally, given that WT AR and the polyglutamine-expanded AR can exert
toxicity through common mechanisms (Nedelsky et al., 2010), it is possible that the pathogenesis

38

of SBMA originates through different pathways of which mutant AR is only one such route. This
is not unlike other neurodegenerative diseases, where known genetic mutations account for only
a minority of the cases. Recognizing that an apparently normal AR protein can cause disease by
a failure of mechanisms which ordinarily limit its inherent toxic potential could explain why
some individuals diagnosed with SBMA due to muscle weakness and reduced androgen
sensitivity, nonetheless lack the CAG expansion in their AR gene (Mariotti et al., 2000). In
short, SBMA could be caused by different pathogenic events that perturb normal AR function in
comparable ways, triggering the same disease. Because the AR transgene is expressed
exclusively in muscle fibers of our myogenic model but nonetheless leads to expression of an
SBMA phenotype, these findings are consistent with the possibility that AR acts directly in
muscle fibers to cause or substantively contribute to SBMA pathogenesis.

39

CHAPTER 2: Muscle dysfunction may underlie androgen-dependent motor dysfunction in
mouse models of SBMA
Oki, K., Katsuno, M., Adachi, H., Sobue, G., Wiseman, R.W., Breedlove, S.M., Jordan, C.L
(submitted). “Muscle dysfunction may underlie androgen-dependent motor dysfunction in
mouse models of SBMA.”

40

Abstract
Spinal and bulbar muscular atrophy (SBMA) is a late-onset motoneuron disease associated
with the progressive loss of muscle strength. This disease is linked to a CAG/polyglutamine
(polyQ) repeat expansion in the androgen receptor (AR) gene. Current views about SBMA
suggest that cell dysfunction rather than cell death triggers motor dysfunction and that muscle
dysfunction per se may underlie or contribute to the loss of motor function in this disease. Using
a myogenic transgenic (Tg) mouse model in which androgen exposure can acutely induce a
motor phenotype recapitulating SBMA in female Tg mice, both fast and slow twitch muscles
from such mice show a precipitous drop in specific force that is independent of muscle mass. In
the current study, we set out to determine whether muscle contractile function was similarly
perturbed in chronically diseased males of three different SBMA mouse models: a Tg model that
broadly expresses a full length human AR with 97 CAGs (97Q), a knock-in (KI) model that
expresses a ‘humanized’ AR containing a disease-causing CAG expansion, and a Tg myogenic
model that overexpresses wildtype AR only in skeletal muscle fibers. Significant deficits were
found in muscle contractile strength independent of muscle mass, twitch/tetanic ratio and in the
fatigue resistance of muscles, although the pattern of results was not uniform across models.
These results raise the possibility that primary muscle dysfunction may be a key underlying
feature of SBMA, and implicates skeletal muscle as a prime target for SBMA therapeutics.

Introduction
Whether functional deficits in motoneurons or muscles or both underlie motor dysfunction in
motoneuron disease is not clear. Current evidence on amyotrophic lateral sclerosis (ALS), for
example, indicates that dysfunction likely exists in both presynaptic and postsynaptic elements of

41

the neuromuscular system (Dekkers et al., 2004; Diaz-Amarilla et al., 2011; Dobrowolny et al.,
2008a; Dobrowolny et al., 2008b; Gifondorwa et al., 2007; Kaspar et al., 2003; Li et al., 2007;
Miller et al., 2006). Thus, functional impairments in one or both parts of the system may
underlie the loss of motor function in motoneuron disease.
Spinal and bulbar muscular atrophy (SBMA), another motoneuron disease, is distinct from
ALS in its strict male bias and apparent dependence on male levels of androgens. SBMA is
linked to a CAG expansion mutation in exon one of the androgen receptor (AR) gene (Fischbeck
et al., 1991; La Spada et al., 1991). This polyglutamine (polyQ) expansion in AR triggers a
slowly progressing neurodegenerative phenotype that typically emerges in middle aged men.
Female carriers of the mutated AR allele are largely unaffected (Soraru et al., 2008), even in
those rare cases where females have two copies of the disease allele (Schmidt et al., 2002).
Symptoms of SBMA include difficulties in ambulation due to limb weakness, and problems with
speech and swallowing due to weakness in bulbar and facial muscles (Soraru et al., 2008).
Findings from studies on SBMA mouse models indicate that the loss of motor function depends
critically on male levels of androgens, with castration rescuing males from disease (ChevalierLarsen et al., 2004; Katsuno et al., 2002; Yu et al., 2006) and testosterone (T) inducing disease in
otherwise asymptomatic females (Katsuno et al., 2002; Monks et al., 2007). Because T
reversibly exacerbates SBMA symptoms in a male patient (Kinirons and Rouleau, 2008), it is
likely that T also plays a role in promoting SBMA in humans.
Data from animal models of SBMA suggest that cell dysfunction is a critical mediator of
motor dysfunction with cell death being a rather late, end-stage event (Chevalier-Larsen et al.,
2004; Johansen et al., 2009; Yu et al., 2006). Perturbations in axonal transport may be one
hallmark feature of cellular dysfunction in neurodegenerative disease (Chevalier-Larsen and

42

Holzbaur, 2006). For example, retrograde axonal transport in motoneurons of affected males is
impaired in three different mouse models of SBMA, an impairment which emerges early and is
androgen-dependent (Katsuno et al., 2006; Kemp et al., 2011). Surprisingly, while muscles of
SBMA patients exhibit marked histopathology, little is known about the effects of disease on
muscle function.
Whether muscles are dysfunctional in SBMA has recently been addressed using a musclespecific transgenic (Tg) mouse model of SBMA. This so-called “myogenic” model shows an
androgen-dependent loss in motor function (Monks et al., 2007), in step with other SBMA
mouse models. Exposing asymptomatic females to male levels of androgens triggers a
precipitous loss in motor function. Using an in vitro approach, muscles from impaired female
mice were found to produce 50 – 75% less contractile force after five days of treatment (Oki et
al., 2013). On the other hand, muscle mass did not decrease over the five days of T treatment,
suggesting that the loss of contractile force reflects changes in function rather than net losses of
contractile machinery.
These data beg the question of whether comparable deficits exist in muscles of diseased
SBMA male mice. Thus, we used the same in vitro approach of stimulating isolated muscles
attached to a strain gauge to examine the contractile properties of skeletal muscles in affected
male mice of three different SBMA models, a myogenic Tg model in which rat wildtype (WT)
AR is expressed only in skeletal muscle fibers, the ‘97Q’ Tg model in which a full length 97
polyQ AR is globally expressed, and a KI model in which a humanized polyQ-expanded mouse
allele of AR is broadly expressed. Our results show that muscle force is compromised
independent of muscle atrophy in two of the three models, suggesting that primary impairments
in the contractile properties of skeletal muscles may contribute to motor dysfunction in SBMA.

43

Methods
Animals and treatment. The contractile properties of the fast twitch extensor digitorum longus
(EDL) and the slow twitch soleus (SOL) muscles of affected male mice from three different
models of SBMA were examined. Myogenic’ transgenic (Tg) mice express under the control of
the human skeletal α-actin (HSA) promoter a wildtype AR allele only in skeletal muscle fibers
(Monks et al., 2007). Mice of the 97Q Tg model globally express under the control of a CMV
promoter/chicken β-actin enhancer a full length human AR transgene harboring 97 CAGs
(Katsuno et al., 2002). KI mice express a humanized CAG-expanded mouse AR allele under the
control of native AR promoters (Yu et al., 2006). Heterozygous female carriers in each model
were mated to C57/B6J male mice for several generations to produce progeny for this study.
Animals were group housed and had access to water and food ad libitum. All animal procedures
were approved and performed in compliance with the Michigan State University Institutional
Animal Care and Use Committee, in accordance with the standards in the NIH Guide for the
Care and Use of Laboratory Animals.
97Q males. Because disease onset is highly variable in transgenic males of the 97Q model,
we attempted to synchronize disease onset and progression by exposing castrated juvenile males
to adult levels of testosterone (T). Age-matched (30-32 days old) WT and Tg male mice (n=5–6
mice/group) were deeply anesthetized with isoflurane, castrated, and implanted subcutaneously
just caudal to the scapula with Silastic capsules (1.57 mm i.d. and 3.18 mm o.d., effective release
length of 6 mm) containing crystalline T (~0.15 mg per capsule), as previously described
(Johansen et al., 2009). Incisions were closed with 9 mm surgical staples. All animals received
ketoprofen analgesic (5 mg/kg, sc) immediately following surgery. One T capsule was used

44

throughout the duration of the study for each mouse and treatment was continued until Tg mice
developed motor impairments.
Motor function was assessed using the grip strength and hang tests as described below one
hour before surgery to establish baseline performance (Day 0) and twice weekly following
surgery up to the time of sacrifice when end-stage motor function was reached. End-stage motor
function was defined as hang times < 24 s or 20% of the latency to fall in WT mice and grip
strengths around 16 g force, roughly half of WT control values. On average, this level of
dysfunction was reached by around 50 days of age and only in Tg mice. Mice were sacrificed at
this point, and muscles were removed under a surgical plane of anesthesia for in vitro muscle
testing as previously described (Oki et al., 2013).
141 males. Normally, most males in the myogenic model carrying the HSA-AR transgene of
the 141 line die on the day of birth (Monks et al., 2007). However, such Tg males can be
rescued from perinatal death by blocking the effects of prenatal T with the AR antagonist,
flutamide (Johansen et al., 2011; Monks et al., 2007). Thus, timed pregnant Tg dams were given
daily sc injections of flutamide (5 mg flutamide dissolved in 100 µl in propylene glycol) on
gestational days 14-18. Flutamide-rescued Tg males show the same androgen-dependent disease
phenotype as Tg male mice that survived without the aid of prenatal flutamide (Johansen et al.,
2011). The motor function of Tg and age-matched WT male mice (90-135 days old, n=5-6
mice/group) was assessed based on grip strength and hang test. Since Tg males in this model are
chronically impaired, motor function was assessed three times: three days before harvest, the
day before harvest, and on the day of harvest.
KI males. The length of CAG repeat in the AR gene of the KI model tends to contract across
generations, necessitating sequencing the first exon to determine the length of the repeat and

45

thus, potential for toxicity. KI males used in this study had a repeat length ranging from 84-93.
Motor function of KI male mice and yoked WT controls (n=5-7/group) was measured starting at
30 days of age and tested twice weekly using the grip strength and hang tests until a > 30%
decline was seen on one or both tests in KI males, at which point muscles were harvested from
the KI male and its yoked WT control for in vitro examination.

Motor function tests. In the 97Q and 141 lines, forelimb grip strength was assessed using a grip
strength meter (Columbus Instruments) oriented in the horizontal plane (Chevalier-Larsen et al.,
2004; Johansen et al., 2009; Katsuno et al., 2002). Mice were held by their tails and lowered to
the apparatus, allowed to grasp the metal grid with their forelimbs only, and were pulled by their
tail vertically at a 90° angle away from the grid until their grip was released. Force (g) applied
to the bar at the moment of release was recorded as grip strength. Six consecutive measurements
were done within a single two minute session for each subject with the lowest and highest grip
values excluded, and the mean of the remaining four values recorded as the grip strength for that
animal and session. In the KI line, consistent with previously used methods (Kemp et al., 2011;
Yu et al., 2006), a triangle gauge was used to measure grip strength as deficits in these mice are
more easily detected with the triangle. Out of five values, the lowest and highest was dropped
and the middle three values were used to estimate average grip strength for that mouse on that
day. We saw no evidence of a consistent drop in grip strength over the five bouts of this test.
The hang test was conducted by placing mice on a wire grid that was then turned upsidedown 40 cm above a counter. Latency to fall (up to 120 seconds) from the wire grid was
measured (Monks et al., 2007; Sopher et al., 2004).

46

The number of rears (i.e. number of vertical movements) was also assessed in an open field
(16 × 16 inch Plexiglas chamber) for five minutes using the Versamax activity monitor
(AccuScan Instruments, Columbus, Ohio, USA). The chamber was cleaned with 70% ethanol
between tests, and data were analyzed using the Versamax software (Johansen et al., 2009).
Rearing behavior is an indirect readout of hindlimb muscle strength and was conducted in the
97Q and 141 models.

In vitro studies to assess muscle mechanics. To determine whether impairments in skeletal
muscle function might underlie motor dysfunction, muscle contractile characteristics of the fasttwitch EDL and the slow-twitch SOL were examined in vitro in motor-impaired Tg/KI males and
their respective WT controls using freshly excised preparations. These hindlimb muscles were
selected because the EDL and SOL are the prototypical fast- and slow-twitch muscles,
respectively, with considerable background information available on their normal contractile
properties (Crow and Kushmerick, 1982; Kushmerick et al., 1992). Moreover, the toxic effects
of an activated AR in SBMA might depend on fiber type, as we have previously seen in diseased
females of the myogenic model (Oki et al., 2013) and as observed in other diseases (Atkin et al.,
2005; Hegedus et al., 2008).
Mice were deeply anesthetized with a single intraperitoneal injection of sodium pentobarbital
in sterile saline (0.1 mg/g body weight, Abbott Laboratories, North Chicago, IL, USA). Once
muscles were exposed, 5-0 silk suture was tied to the proximal and distal tendons in situ and
their resting length approximated by recording the intersuture distance prior to excision.
Muscles were then removed by cutting above the suture knot on the proximal tendon and below
the suture knot distally. After muscles were excised, mice were euthanized. Excised muscles

47

were incubated in an 8 mL organ bath containing oxygenated Ringer’s (117.1 mM NaCl, 4.6 mM
KCl, 25.3 mM NaHCO 3 , 2.5 mM CaCl2 , 1.2 mM MgSO 4; pH 7.4; equilibrated with 95%
O 2 /5% CO 2 ) (Dentel et al., 2005; Oki et al., 2013). Temperature of the bath was continuously
monitored using a K-type thermocouple (Omega Engineering, Stamford, CT) adjacent to the
muscle and maintained at 25±0.2°C by circulating water through a glass-jacketed organ bath
(Radnoti Glass Technology, Monrovia, CA). Isolated muscles were fixed by tying one end of the
muscle ligature to a glass hook and the other to an isometric force transducer (Astro-Med, West
Warwick, RI) fitted with a positioning micrometer. Muscles were then set to their in situ resting
length using the length-tension relation.
Electrical stimulation was delivered via two platinum plate electrodes (3-4 mm apart, 25 mm
long) parallel to the long axis of the muscle using a Grass S88 Stimulator (Grass Instruments,
Quincy, MA). Force was recorded using an analog-to-digital converter (model AT MIO16E;
National Instruments, Austin, TX) controlled via commercially available software
(LabScribe/NI; iWorx, Dover, NH). Muscles were stimulated with successive twitches (1 ms
duration, 1 Hz) at 10-60 volts (V) to determine the supramaximal voltage (generally 30-40 V).
Muscle function was assessed based on directly evoked twitches and tetani. Stimulation
parameters used to evoke muscle twitches were 1 ms duration pulses delivered at a rate of 0.2 Hz
for 3 seconds at supramaximal voltage. Muscle tetani were evoked by stimulating the muscle
directly with 1 ms pulses at the fusion frequency which was operationally defined as the
frequency at which ripples in force on the tetanic plateau are < 3% of the total peak force. The
fusion frequency ranged from 80-100 Hz for the EDL and 30-40 Hz for the SOL in WT animals.
Length of the stimulus train to induce tetanus also differed between muscle fiber types, with a

48

500 ms train and 1 s train used for EDL and SOL, respectively. Absolute force was recorded in
grams and converted to Newtons (N) using the conversion
-3

1 gram of force = 9.80665 X 10 N.
This force value was then normalized to the grams tendon-free muscle mass (N/g). While
muscle atrophy could contribute to motor dysfunction via a loss in strength, normalizing force to
mass reveals changes in force that are independent of mass. Contraction kinetics were also
measured using the trace of individual twitches. Time to peak force in the twitch (rise time) and
time to return to baseline (relaxation time) were measured in all muscles using customized
software (details below).
Resistance to fatigue was assessed in the EDL by stimulating with tetani at ~80-100 Hz, each
train lasting 330 ms, repeated every 1 second (Burke et al., 1971). This was done until the final
tetanus was ~10% of initial force, determined to be 90 s in EDL. A similar protocol was used for
the SOL. Each SOL was stimulated with tetani at ~30-50 Hz repeated every 1 second.
However, due to a higher percentage of slow-twitch, fatigue resistant fibers being present, the
SOL fatigues much slower than the EDL, and the protocol was adjusted to induce fatigue (final
tetanus decreased to ~10% of initial) in a shorter window of time. The train duration was
increased to 670 ms in duration, and the tetani were repeated for 135 s in the SOL. Fatigue
profile was determined by generating a curve that calculated the amount of force relative to
muscle mass produced at each stimulus pulse. Following the experiment, muscles were rapidly
removed from the apparatus, dissected free from tendons, blotted of excess media, weighed to
estimate mass, and then stored at -80° C.

49

Histology. The contralateral SOL was dissected from the same mice of the 141 line that were
used for the mechanical studies, placed in OCT compound (Sakura Japan) in cryomolds
(Cryomolds) and snap frozen in liquid nitrogen (n = 5 mice/group). Unlike the EDL, the SOL
had not been previously examined in this model for histopathology (Johansen et al., 2009). The
SOL were sectioned on a Leica cryostat at 10 μm, thaw-mounted onto gel-subbed slides, and
stained with hematoxylin and eosin (H&E). Both the EDL and SOL from 97Q males and WT
controls were prepared in a similar fashion for histological analyses. Because we did not find
any significant deficits in the contractile properties of either the EDL or SOL of KI males, we did
not conduct similar morphological analyses of muscles in this model.

Testosterone levels. Blood was collected via a transcardial puncture from deeply anesthetized
mice used for studies on muscle mechanics, and held on ice for 30 minutes before centrifuging at
3000 rpm for 20 minutes at 8° C (Sorvall Biofuge Fresco, Thermo Scientific, Asheville, NC).
After centrifugation of blood samples, plasma was collected and held at -80° C until assayed for
circulating testosterone levels as previously described (Zuloaga et al., 2008).

Data analysis and statistics. Analysis of mechanical transients was performed using a custom
algorithm created for the MatLab programming environment (Mathworks, Natick, MA). In brief,
raw digital signals were passed through a low-pass filter to smooth the data and then analyzed for
force output and tension-time integrals. Kinetic parameters for the rise time (time to peak force)
and relaxation time (time of return to baseline) were also measured in individual muscle twitches
as previously described (Jayaraman et al., 2006). Briefly, a point for peak force was determined
in the digitally acquired twitch traces. The ascending portion of the twitch trace up to the peak
50

force was defined as the rise time, and the descending portion of the trace from the peak force
back to baseline was defined as the relaxation time. Independent groups T-tests were used to
evaluate significant differences between muscles from affected Tg or KI males versus their WT
controls.

Results
Motor dysfunction correlates with muscle dysfunction in 97Q Tg mice.
Motor tests confirmed that castrated 97Q Tg males treated with T show the same androgendependent motor dysfunction as previously reported for gonadally intact 97Q Tg males (Katsuno
et al., 2002). All three indices of motor function—number of rears in an open field (Fig 11A),
frontpaw grip strength (Fig 11B), and hang times (Fig 11C) indicate that the motor function of
97Q Tg males was significantly impaired compared to WT controls (p-values < 0.002). Serum T
levels were comparable between Tg mice and their respective controls, suggesting these
functional changes were not due to differences in circulating T (Table 2). 97Q Tg and WT
males did have higher T levels than gonadally intact males of either the 141 myogenic or KI
models (data not shown for KI model) as expected because of the exogenous T treatment.
Relatively severe motor deficits emerged in Tg males by 50 days of age (age range: 40–57
days). At the time of testing, all symptomatic Tg mice had lower body weights than their WT
controls (Table 2), although deficits in body weight lagged behind deficits in motor function by
~3 days.
Both the isolated EDL and SOL from 97Q males showed significant deficits in force that
were evident for both raw and normalized force measures (Fig 12). With one exception (raw
tetanic force in SOL, Fig 12J), raw twitch and tetanic force were significantly decreased in

51

muscles of Tg males compared to WT males (p-values < 0.007 for raw EDL twitch and tetanus
and raw SOL twitch). However, once force measures for individual muscles were normalized to
their mass, all force measures showed significant and robust deficits in both EDL and SOL
muscles of Tg males (p-values < 0.002 for all measurements), indicating a loss of force in both
fast and slow twitch muscles in diseased 97Q males that occurred independent of the losses in
muscle mass. Notably, the EDL from Tg males exhibited a greater loss in both normalized
twitch and tetanic force than did the Tg SOL, producing less than 20% of the normal WT force
whereas the Tg SOL exhibited more than 50% of WT force. These data are consistent with the
idea that a loss of motor function may be due to a loss in contractile force of muscles, some of
which reflects muscle atrophy, but much of which seems to occur independent of losses in
muscle mass. We found no evidence of a drop in force production from the first to the last
recording of peak tetanic tension (Fig 16) prior to stimulating the muscle to induce fatigue. This
apparent stability of muscle performance in vitro strongly argues that the force deficits reflect
genuine deficits in vivo.
Although force was more affected in the Tg EDL, only the Tg SOL showed significant
changes in the twitch/tetanus ratio (Fig 12L), with significant decreases in this ratio compared to
WT (p < 0.02). The diseased EDL did however show a similar trend (Fig 12F) raising the
possibility that with a larger sample size, the decreased twitch/tetanus ratio may be significant.
These data suggest impairments in calcium handling mechanisms in the SOL and possibly the
EDL of motor-impaired 97Q Tg males.
Twitch traces (Fig 12C, I) were subjected to a kinetics analysis to assess whether time to
peak force (rise time) and time to baseline (relaxation time) was also affected by disease.

52

Although both contraction and relaxation times were consistently longer in Tg muscles compared
to WT controls, such differences fell short of significance (Table 3).
Initial fatigue analysis examining the percentage of force lost over the entire stimulation
period suggested the EDL from 97Q Tg males were less fatigable than WT controls (Fig 13A),
that is, losing less force over a given period of time (45% force loss in Tg vs. 92% force loss in
WT). We also determined the time it took for muscles to lose 50% of their original force (i.e.,
time to half maximal force loss). Based on this measure, the EDL from Tg males fatigued faster,
reaching a 50% drop in force twice as fast as WT EDL (Tg versus WT: 14.3+3.0 s versus
25.8+0.9 s, Fig 13B, p < 0.05). The SOL from Tg males showed a similar decrease in fatigue
susceptibility, losing 50% of its original force significantly sooner than WT SOL (Tg versus WT:
38.6+3.2 s versus 46.9+1.8 s , p < 0.05) but the effect of disease on fatigability in the SOL seems
much less marked than in the EDL (Fig 12C, D).

Motor dysfunction correlates with muscle dysfunction in myogenic Tg males.
As expected, 141 Tg mice also exhibited profoundly compromised motor function, showing
deficits on all three measures – rearing, grip strength, and hang times (Fig 11D, E and F; pvalues < 0.001 for all tests). These data confirm findings from previous studies (Monks et al.,
2007). Body and muscle weights were also decreased in Tg males compared to WTs but serum
T levels were comparable (Table 2), indicating that abnormal T levels do not explain the motor
phenotype in 141 Tg male mice.
The EDL and SOL of motor-impaired 141 Tg males showed similar functional deficits as
muscles from diseased 97Q males (Fig 14), with both the EDL and SOL showing significant
deficits in both raw twitch and tetanic force production compared to WT EDL and SOL muscles

53

(p-values < 0.001). Significant deficits were also evident once muscle mass was taken into
account (Fig 14B, H and K), with the one exception that tetanic force in the Tg EDL was no
longer less than WT controls (Fig 14D vs. 4E). Given that the EDL from chronically impaired
141 Tg males contains about 30% fewer fibers than normal (Johansen et al., 2011; Monks et al.,
2007), we suspect that this deficit in fiber number in Tg EDL underlies the deficit in raw tetanic
force and that the maximal contractile force produced by each fiber is comparable to that of WT
fibers. However, because the Tg EDL showed deficits (compared to normal) in normalized
twitch but not tetanic force, the twitch/tetanus ratio in this muscle was also significantly reduced
(p < 0.005) to about half that of WT muscle (Fig 14F). The twitch/tetanus ratio for Tg SOL was
also significantly decreased compared to WT controls although the magnitude of the effect was
less (Fig 14L). In short, both EDL and SOL muscles in diseased myogenic males show primary
dysfunction that is independent of muscle mass, sharing similar defects as muscles of diseased
97Q males. We again found no evidence of a drop in force production from the first to the last
recording of peak tetanic tension (Fig 16) prior to fatiguing stimulation.
Our analysis of twitch kinetics reveals that rise time of twitches in the myogenic EDL was
significantly prolonged compared to normal (Table 3; p < 0.001). However, relaxation time in
the EDL was not affected by disease nor was contraction or relaxation time affected in the SOL
of myogenic Tg males.
During fatiguing stimulation, the EDL from myogenic motor-impaired males lost less force
overall than the WT EDL (71% force loss in Tg versus 95% in WT; Fig 15A), suggesting that
the Tg EDL had greater endurance than the WT EDL, consistent with an apparent shift toward a
more oxidative phenotype in diseased EDL (Monks et al., 2007). However, the time it took to
reach a 50% drop in force was the same for Tg and WT EDL muscles (Fig 15B). The fatigue

54

profile in the SOL of diseased myogenic Tg males was similar to that of the 97Q EDL, with little
apparent loss in force overall during the period of fatiguing stimulation. Nonetheless, the
diseased SOL in myogenic Tg males fatigued faster, at least initially, since it took significantly
less time to reach a 50% drop in force compared to WT SOL (Tg versus WT: 9.8+0.9 s versus
42.1+2.4 s, p < 0.05).

Histology.
Finding similar muscle dysfunction in both the 97Q and myogenic Tg models prompted us to
examine muscles directly in histological sections stained with hematoxylin and eosin to evaluate
whether dysfunction correlates with any known histological markers of disease. We counted the
number of muscle fibers in each muscle and the number of fibers with centralized nuclei,
excepting the EDL from 141 myogenic males, which have been analyzed previously (Johansen et
al., 2011).
97Q model. Even though the EDL muscles from diseased 97Q males were about half the
mass of those from age-matched WT males (Table 2), the number of EDL fibers did not differ
between diseased 97Q Tg males and healthy WTs. These data suggest that EDL fibers from
diseased 97Q Tg males are on average significantly smaller. Consistent with this speculation,
some fibers in cross section of EDL are visibly atrophic (Fig 17). Regardless, even when
differences in muscle mass are taken into account, force capacity of the diseased EDL of 97Q
males is ~6 times less than that of WT EDL during both twitch and tetanus contractions,
indicating that fiber size alone cannot explain the loss of intrinsic strength. The SOL of diseased
97Q males also contained a normal number of fibers (Table 4), in this case paralleling its normal
mass (Table 2). The percentage of SOL fibers with centralized nuclei was also the same as

55

normal. Cross sections of stained SOL also look remarkably normal (Fig 17). On the other
hand, the 97Q EDL contains significantly more fibers with centralized nuclei than normal (Table
4), paralleling the greater force deficit in the EDL of the 97Q model.
141 myogenic model. Previous work shows that the EDL of myogenic males contains about
a third fewer fibers than WT EDL (Johansen et al., 2011). In contrast, the SOL of 141 diseased
males shows no change in total fiber number even though its mass is significantly less than WT
SOL. While variability in fiber size appears relatively normal, fibers in general appear smaller in
size but otherwise remarkably normal (Fig 17). However, diseased SOL of 141 males contains a
significantly higher proportion of fibers with centralized nuclei compared to WT SOL (Table 4).
This pattern of differences is similar to that in the 97Q model, where the incidence of centralized
nuclei correlates with the most severe force deficit, with decreases in muscle mass but no change
in overall fiber number (EDL in the 97Q model and SOL in the myogenic 141 model).

Knock-in model.
Although motor dysfunction was observed in KI mice relative to WT controls, with both
forelimb grip strength and hang times significantly less (Fig 6A, B; grip p < 0.002; hang p <
0.03), we found no significant deficits in the contractile properties of the EDL or SOL of these
KI males (Fig 6C – N). Still, the EDL of KIs produced consistently less force than did the WT
EDL (Fig 6C-G). Neither did we find significant deficits in the twitch kinetics of either muscle
from KI males (data not shown).

Discussion

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We set out to determine whether skeletal muscle dysfunction could underlie motor
dysfunction in spinal and bulbar muscular atrophy (SBMA). To this end, the contractile
properties of isolated fast twitch EDL and slow twitch SOL were examined in vitro from motorimpaired male mice of three different mouse models of SBMA. The KI and 97Q Tg models each
broadly express a CAG expanded AR allele linked to SBMA (Katsuno et al., 2002; Yu et al.,
2006) while the third ‘myogenic’ (141) model expresses a rat WT AR transgene only in skeletal
muscle fibers but nonetheless, recapitulates the same androgen-dependent loss of motor function
as seen in the other two models (Monks et al., 2007). In vitro studies of muscle function using
acutely diseased Tg females in the myogenic model indicate that muscles of diseased mice
undergo profound losses in contractile strength that are independent of muscle mass (Oki et al.,
2013), suggesting that primary muscle dysfunction may contribute to the loss of motor function
in SBMA. These data led us to ask whether similar muscle defects are evident in affected
SBMA male mice. We find that the EDL and SOL of motor-impaired 97Q and myogenic male
mice generally show striking deficits in their ability to produce contractile force and resist
fatigue. While KI males show motor impairments, albeit less severe than in the other two
models, only subtle deficits in force production were detected in the EDL, suggesting that other
mechanisms underlie motor impairments in this model. We are the first to directly examine the
function of skeletal muscles isolated from the nervous system in any SBMA model. Consistent
with other reported findings implicating skeletal muscle in motoneuron disease (Dobrowolny et
al., 2005; Johansen et al., 2011; Kieran et al., 2005; Monks et al., 2007; Palazzolo et al., 2009;
Perlson et al., 2009; Wong and Martin, 2010; Yu et al., 2006), these data suggest that therapies
that target skeletal muscle may be effective in protecting or rescuing motor function in SBMA.

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There are several possible reasons why KI males did not show defects in contractile
properties of muscle despite impaired motor function. The most obvious possibility is that the
motor tests used, hang and grip strength, reflect defects in muscles other than the EDL and SOL.
Since grip strength was based on forepaw grip, it tests the function of only forelimb and not
hindlimb muscles. Moreover, disease susceptibility seems to vary in the KI model depending on
the specific muscle (Yu et al., 2006). Because perineal muscles are enriched for AR (Johansen et
al., 2007; Monks et al., 2004), these muscles are affected first and more severely in KI males
than limb muscles. Hence, it is possible that marked dysfunction would have been detected if
perineal muscles had been studied rather than the EDL and SOL. Another possibility is that
presynaptic dysfunction underlies the loss in motor function in KI males with the muscle
themselves functionally intact. Retrograde axonal transport is severely impaired in diseased KI
males (Kemp et al., 2011), indicating that motoneurons are indeed dysfunctional in KI males.
Since defects in axonal transport are often associated with defects in nerve terminal function
(Pigino et al., 2009), it seems likely that KI males have impairments in synaptic function.
Finally, the apparent deficits in muscle strength suggested by shortened hang times and reduced
grip strengths may reflect nonspecific effects of disease in KI males. Dysfunction of the perineal
muscles in KI males leads to occlusion of the urethra, uremia and early morbidity (Yu et al.,
2006). Poor motor performance could reflect effects of this condition rather than direct toxic
effects of AR on limb neuromuscular. Regardless, these data underscore the fact that defects in
motor function do not always translate to defects in muscle contractile properties in SBMA
models.
Muscle atrophy triggered by the loss of innervation has long been assumed to underlie the
progressive loss of muscle strength in SBMA. Current data does not support this view. While

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muscles from both 97Q and 141 male mice generally have less mass and produce less absolute
force than muscles from WT controls, significant deficits in twitch and tetanic force remain even
once force measures are normalized to muscle weight (Figs 2 and 4). The one exception is the
myogenic (141) EDL in which a deficit in raw tetanic force drops out once muscle mass is taken
into account. However, the myogenic EDL is also the only muscle among the two models that
contains significantly fewer muscle fibers (Johansen et al., 2011), suggesting that the deficit in
raw tetanic force in the myogenic EDL reflects the deficit in fiber number. For the EDL and
SOL of 97Q males and the SOL of myogenic males, most of the deficit before normalization is
also evident after normalization, indicating that muscle atrophy accounts for only a small portion
of the deficit in contractile strength. These data point to other mechanisms besides the loss of
actin and myosin contractile proteins that contribute to the loss of muscle strength in SBMA
perhaps mediated by altered function of calcium-handling proteins. Changes in the
twitch/tetanus ratio in the two Tg models supports this view and implicates mechanisms that
release, bind and/or sequester calcium.
How toxic AR causes a loss in contractile force is not known and warrants future studies.
However, recent microarray results assessing changes in gene expression in hindlimb muscle
from males of these three SBMA models suggests possible candidate molecules (Mo et al.,
2010). For example, calpain 2 is upregulated in hindlimb muscles of both 97Q and 141 Tg males
but not KI males. Calpain 2 is a calcium-dependent protease which resides in the sarcoplasm of
muscle fibers (Vermaelen et al., 2007). Calpain activity is thought to be an important player in
promoting muscle atrophy (Talbert et al., 2013). While enhanced calpain activity may account
for loss of muscle strength due to atrophy, it may also be involved in regulating muscle strength
independent of mass. Calpain promotes the activity of caspase 3 (Talbert et al., 2013), with

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caspase 3 activity during sepsis implicated in the rapid loss of force without mass (Supinski and
Callahan, 2006).
Another possibility is that toxic AR causes muscle weakness by impairing the function of the
ryanodine receptor (RyR1), a critical player in the excitation-contraction pathway. The RyR1
opens in response to activity, releasing calcium from intracellular stores to bind troponin-C
which triggers muscle contraction. While this receptor is ordinarily closed during inactivity, in
some pathological states such as muscular dystrophy or aging, the RyR1 stays partially open
(Bellinger et al., 2009), leaking calcium into the cytosol of muscle fibers in the absence of action
potentials. This slow and constant leak of calcium leads to an elevated concentration of cytosolic
calcium and is thought to underlie muscle weakness in both muscular dystrophy and aging
(Andersson et al., 2011; Bellinger et al., 2009). Importantly, this defect in RyR1 function
involves post-translational modifications which can develop within hours and is readily
reversible (Bellinger et al., 2008). Recent evidence from acutely diseased Tg 141 females also
points to leaky RyR1 underlying the loss of muscle strength in SBMA (Oki et al., 2013).
Trophic factors may also be involved in regulating the strength of muscles. One molecule of
particular interest is insulin like growth factor-1(IGF-1). In addition to IGF-1 likely being a
main mediator of the anabolic effects of androgens on skeletal muscle (Gentile et al., 2010), IGF1 has also been implicated in SBMA. When overexpressed exclusively in skeletal muscle, IGF-1
rescues 97Q mice from disease (Palazzolo et al., 2009). IGF-1 expression is also lower in
muscles of affected KI males (Yu et al., 2006). While studies often focus on the effects of
muscle-derived trophic factors on motoneurons, the role of such factors in regulating muscle
function in disease merits further attention, particularly because skeletal muscles express the
receptors for many muscle-derived trophic factors (Chevrel et al., 2006).

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One significant aspect of these data is that losses in force occur without evidence of
histopathology. For example, the SOL in the 97Q males appears largely protected from disease
based on morphological attributes: its mass is unaffected (Table 2), the number and apparent size
and uniformity of muscles fibers is normal (Table 3, Fig 17), and the frequency of fibers with
centralized nuclei is also normal (Table 3). Despite the lack of histopathology, its function is
significantly impaired. It exerts significantly less twitch and tetanic force (Fig 2H, K), has a
significantly reduced twitch/tetanus ratio (Fig 2L) and fatigues more rapidly, losing 50% of its
starting force sooner than normal SOL (Fig 13D). The SOL muscle in myogenic males presents
a similar picture. While stained cross sections of SOL look remarkably normal, with fibers
appearing relatively uniform in size, albeit smaller, and fiber number normal (Table 4), the
diseased SOL produces considerably less force (~65–70% less) than the WT SOL (Fig 14H, K).
The only apparent morphological correlate of muscle dysfunction that we found was the
incidence of fibers containing centralized nuclei but this relationship is not strict. Only muscles
that lost the most amount of force in each model (EDL in the 97Q males and SOL in the
myogenic males) contained a significantly higher percentage of fibers with centralized nuclei
compared to their respective WT controls. However, the 97Q SOL and 141 EDL also both show
significant force deficits, albeit not as severe. The marked absence of muscle histopathogy in
motor-impaired mice is reminiscent of reports for another mouse model of SBMA (ChevalierLarsen et al., 2004) and reinforces the likelihood that cellular dysfunction well precedes diseaserelated changes in cell morphology.
While motor function appears comparably affected in the two different SBMA Tg mouse
models, the severity of the deficit in muscle force differed for each model. The EDL was more
severely affected in the 97Q model while the SOL was more severely affected in the myogenic

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141 model. Given that the EDL is a fast twitch muscle while the SOL is slow, these results
suggest that the toxic potential of AR in each model depends on the fiber type composition of the
muscle although the factors behind this fiber-type dependence are not clear. Fiber-type
differences have also been found to play a role in ALS models where fast-twitch motor units are
more severely affected (Atkin et al., 2005; Hegedus et al., 2008). One possible explanation for
the differing susceptibilities to disease is that AR expression levels also vary, paralleling the
more toxic effects in select muscles. We think this explanation is unlikely. In the myogenic
model, for example, we find the same pattern of deficits in acutely diseased Tg females, with
force being more severely affected in the SOL than in the EDL (Oki et al., 2013). However, AR
expression appears comparable in the EDL and SOL muscles of 141 Tg females (Oki et al.,
2013). While enhanced AR expression might explain the greater force deficits in the 97Q EDL
compared to the SOL, it does not readily account for the deficit in the twitch/tetanus ratio that
occurs only in the 97Q SOL. Microarray data also offer a possible explanation, indicating that
the slow isoform of troponin-C, which binds calcium to trigger skeletal muscle contractions, is
downregulated in limb muscle of 141 males. Slow troponin-C is found primarily in slow-twitch
and cardiac muscles, and its downregulation could potentially contribute to the more severe loss
of force in 141 Tg SOL. However, the fact that the force deficit is fully established within three
days of disease onset in the SOL of acutely diseased 141 Tg females but the half life of slow
troponin-C is more than five days (Martin, 1981) challenges this idea.
Contraction kinetics were largely normal in diseased muscles with the exception of the EDL
from myogenic 141 Tg males where twitch rise time was significantly prolonged (Table 3).
These data in addition to deficits in the twitch/tetanus ratios also implicate calcium handling
mechanisms, particularly the RyR1. Both defects could readily be explained by altered channel

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properties in RyR1 that lead to inefficient or reduced calcium efflux from the sarcoplasmic
reticulum. Parvalbumin also represents a candidate molecule that may underlie the selective
effects on contractions kinetics in the 141 EDL. This protein is expressed only in fast-twitch
muscles where it buffers calcium upon its release from the sarcoplasmic reticulum to regulate
twitch kinetics. For example, parvalbumin overexpression has been shown to shorten twitch rise
time in the SOL (Chin et al., 2003) while reducing its expression in the EDL leads to prolonged
rise times (Schwaller et al., 1999). Microarray data indicate that parvalbumin mRNA level is
downregulated ~17-fold in hindlimb muscle of affected myogenic male mice, whereas it is only
modestly downregulated (3-4 fold) in hindlimb muscles of 97Q males and it is normal in KI
males (Mo et al., 2010). Thus, the large decrease in parvalbumin expression seen only in the
EDL of myogenic males may explain the prolonged rise time of the twitch in this muscle.
However, reduced parvalbumin also predicts prolonged relaxation time (Schwaller et al., 1999)
which we do not find (Table 3). Moreover, decreased parvalbumin also increases the force of the
twitch (Schwaller et al., 1999), just the opposite of how disease affects twitch force. Thus,
changes in parvalbumin expression in the myogenic EDL are unlikely to explain the prolonged
rise time seen in this muscle.
Skeletal muscles of affected 97Q and myogenic 141 Tg males may also have altered
metabolic properties as suggested by the fatigue data. The EDL of myogenic Tg males exhibited
greater resistance to fatigue than WT controls, losing less total force over the course of the
experiment (Fig 15A). These data are consistent with previous findings of a more oxidative
profile (Fernando et al., 2010; Monks et al., 2007). The SOL of myogenic males, on the other
hand, exhibited little drop in force output with repeated stimulation, but we suspect this is
because force output was near basement to begin with (Fig 14H, K). Nonetheless, what force was

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lost, occurred at a faster rate, with the SOL from 141 Tgs losing 50% of their original force ~4
times faster than WT EDL. Both the EDL and SOL muscles of 97Q males show less resistance
to fatigue compared to WT controls (Fig 13). While mitochondrial function in muscles of 97Q
mice have not been studied, the effects of the 97Q AR transgene in neuron-like cell cultures
suggest that mitochondria are defective in 97Q muscles (Ranganathan et al., 2009).
The current work suggests that primary defects in muscle function may underlie the loss of
motor function in SBMA. Finding elevated levels of serum creatine kinase consistently in
SBMA patients also implicates muscle as a primary site of AR action in this disease (Battaglia et
al., 2003; Chahin and Sorenson, 2009; Soraru et al., 2008; Sorenson and Klein, 2007). In
summary, data from isolated fast and slow twitch muscles indicate that defects in skeletal muscle
function may contribute to deficits in motor function in two different mouse models of SBMA.
While muscle strength may well be reduced by reductions in mass, significant losses in strength
occur independent of muscle mass. Muscles from SBMA models affected by disease are also
generally more susceptible to fatigue. Future studies aimed at developing therapies that block
the toxic effects of AR in skeletal muscle may offer significant benefits to SBMA patients.

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GENERAL DISCUSSION
General Findings
The current findings confirm that skeletal muscles exhibit androgen-dependent dysfunction
in models of spinal and bulbar muscular atrophy (SBMA). In general, force loss or inability to
produce normal force in skeletal muscles may underlie the motor dysfunction observed in
diseased mice modeling SBMA. This was demonstrated in testosterone (T)-treated transgenic
(Tg) female mice of a myogenic (141) model and in male Tg mice of the 141 and the CAGexpanded 97Q model that exhibit androgen-dependent motor dysfunction. The current data add
to clinical data suggesting primary skeletal muscle involvement in SBMA. Previous reports find
elevated serum creatine kinase levels in SBMA patients comparable to what are seen in patients
suffering from primary myopathies, such as Duchenne’s muscular dystrophy or myotonic
dystrophy (Arenas et al., 1988; Ebashi et al., 1959; Heatwole et al., 2006; Vassella et al., 1965)
but notably, not in patients who have amyotrophic lateral sclerosis (ALS) (Chahin and Sorenson,
2009). However, this is the first line of research that examines directly the function of skeletal
muscles in diseased SBMA mice compared to healthy controls using an isolated in vitro
preparation that is separated from the nervous system.

Overview of findings
In vitro muscle mechanics in all tested muscles measured force, kinetics, and fatigue.
Muscle force was measured by inducing twitch and tetanus in the predominantly fast-twitch
extensor digitorum longus (EDL) and predominantly slow-twitch soleus (SOL). All
measurements were normalized to skeletal muscle mass, and the deficits in muscle force still
remained, suggesting mechanisms aside from atrophy likely mediate muscle dysfunction. Force

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production of skeletal muscles at the beginning and end of experiments suggest force loss was
not due to decay of the preparations in vitro. Although previous studies suggest atrophy genes
are upregulated in muscles of SBMA mice (Mo et al., 2010), the current physiological data,
particularly from female SBMA mice, indicate cell dysfunction precedes cell atrophy or death
(Oki et al., 2013) and likely underlies the expression of early disease symptoms. Thus, atrophy
and death of skeletal muscle fibers and, possibly, motoneurons are likely when symptoms are
advanced in SBMA rather than being the underlying cause of the disease.
Although muscle weakness was present in both sexes of the 141 model and in males of the
97Q model, the force loss in skeletal muscles was different between sexes within the 141 model
and between males across Tg models. The different muscle weakness was based on fiber-type
composition between the fast-twitch EDL and the slow-twitch SOL. Differences in motor unit
vulnerability has been observed in other disease models as fast motor units tend to be more
affected in ALS models (Atkin et al., 2005; Hegedus et al., 2007; Hegedus et al., 2008) while
slow motor units may be more vulnerable in SMA models (Murray et al., 2008). In the current
SBMA studies, females 141 Tg mice treated with T exhibited loss of force and endurance in both
the EDL and SOL, but the SOL showed a more severe force loss than the EDL. Like diseased
Tg females, male 141 Tg mice exhibited loss of force and endurance in SOL, which was more
severe than what was measured in the EDL. However, male 141 Tg mice did not exhibit loss of
tetanus force in the EDL and showed higher endurance in the Tg EDL compared to WT EDL. It
is uncertain if the difference in males versus females in the 141 model occurs due to the time
period of disease symptoms or due to differences in development. It is possible that genetic
adaptations in skeletal muscles may have occurred in male mice surviving longer with chronic
disease symptoms and is reinforced by microarrays showing a different gene expression profile

66

in male 141 Tg mice compared to male WT mice (Mo et al., 2010). Alternatively, because the
disease is present in male 141 Tg mice from a prenatal stage, skeletal muscle and motor unit
development could have been altered in these mice. In cell cultures, myoblasts expressing AR
with a reduced or expanded CAG region had abnormal muscle development, suggesting this
could occur with muscles in SBMA subjects during early development (Sheppard et al., 2011).
Although the 141 Tg mice have a normal CAG repeat numbers, they overexpress the protein and
have a similar phenotype to the CAG-expanded SBMA models. Thus, it is possible that the
disease phenotype compromises muscle development in male 141 Tg mice compared to WT
mice. In contrast, acutely diseased females were treated with T in adulthood and likely did not
have altered development. The period of T treatment (3-5 days) and subsequent muscle
weakness is likely too short of duration to induce significant genetic changes or adaptations in
skeletal muscles and could have contributed to the differences seen between the sexes in Tg mice
of the 141 model. In male mice of the 97Q model, the EDL and SOL exhibited weakness during
twitch and tetanus, but unlike the 141 model, the EDL was more severely weakened rather than
the SOL. Although differences in muscle weakness between fiber types could be due to different
in vivo functions of the EDL and SOL in the animals (i.e. sporadically activated for movement
vs. chronically activated for posture), such an effect would likely result in a similar pattern of
muscle weakness across SBMA models. As this was not observed, it is likely that different
effects are due to AR toxicity having different effects due to fiber-specific properties of the EDL
and SOL across the two Tg models.
Examination of skeletal muscle histology during the disease phenotype suggests pathological
markers in the skeletal muscles were not always accurate predictors for muscle dysfunction.
Although the frequency of fibers with internalized nuclei was correlated with the severity of

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force loss in the 97Q and 141 myogenic models, the SOL from T-treated female 141 Tg mice
had little to no pathological markers in skeletal muscle cross sections but exhibited severe force
loss. Conversely, EDL from male 141 Tg mice had greater loss of mass and a greater presence
of internalized nuclei than WT controls but exhibited the same output of normalized maximal
force as EDL’s from male WT control. This suggests that standard histological markers of
disease are not very informative about the mechanisms underlying functional deficits in disease.
Although mice from two of the investigated models exhibited skeletal muscle dysfunction,
intact male mice in the third model, the knock-in’s (KI) with the diseased exon 1 allele
containing 113 repeats of the codon CAG, similar to humans (Thomas et al., 2005; Yu et al.,
2006), did not exhibit differences in skeletal muscle function between KI and WT controls in
spite of motor dysfunction in KI mice. The loss of motor function could be due to non-specific
effects due to an inability to evacuate their bladders, presynaptic rather than postsynaptic effects
underlying disease symptoms in this particular model (Kemp et al., 2011), or skeletal muscle
dysfunction not being present in the particular muscles investigated because they are not
androgen-responsive in rodents (Souccar et al., 1982). In fact, the muscle effects observed in the
KI model in a previous study were in the androgen-dependent bulbocavernosus/levator ani
muscles, suggesting mechanics may be affected based on androgen-dependence of skeletal
muscles.
In addition to force measurements, kinetic measurements were recorded during the time to
peak force (rise time) and time to return to baseline (relaxation time) during a twitch stimulation.
Rise time is dependent on the release of Ca from the sarcoplasmic reticulum (SR) initiated by
dihydropyridine receptors and ryanodine receptor 1 (RyR1), and relaxation time is dependent on
Ca reuptake into the SR by sarcoendoplasmic reticulum ATPase (SERCA). Alterations to either

68

measurement suggest impaired Ca handling in the skeletal muscles and would warrant further
investigation into the level and function of the main proteins during the separate contraction
events. Only the 141 model exhibited differences in the rise and relaxation measurements.
Delays in rise and relaxation times were recorded in T-treated female Tg mice, and delayed rise
time was recorded in male Tg mice.
Finally, fatigue measurements were induced by repetitive tetanic stimulation in the EDL and
SOL. This test indicates whether the disease phenotype could be due to differences between
susceptibility to fatigue in muscles of symptomatic and asymptomatic mice. Based on the
susceptibility to fatigue, inferences can also be made on the fiber type composition of each
muscle as done in Burke et al. (1971). Histochemical staining, particularly stains for myosin
types, mitochondrial complexes, and periodic acid Schiff could be done on sections tested during
the fatigue protocol to validate speculation on alterations to muscle fiber composition in these
mice. This would be relevant as some disease conditions result in changes to motor units that
affect their endurance (Kieran et al., 2005).

Mechanisms of Muscle Dysfunction
It is still unclear what mechanisms are involved in underlying the skeletal muscle
dysfunction. The heterogeneity of the muscle weakness across fiber types in spite of similar
androgen-dependent disease phenotypes suggests there may be more than one universal
mechanism underlying skeletal muscle dysfunction in the different groups. However, several
lines of evidence suggest that examining calcium (Ca) as handling mechanisms, trophic factors,
and metabolic pathways in muscle could provide insight into the mechanisms causes skeletal
muscles to become dysfunctional.

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Calcium Mechanisms
Ionic concentrations in general may underlie the motor and skeletal muscle dysfunction
observed in the SBMA mice. Yu et al (2006) noted that in the KI model of SBMA, chloride (Cl)
channel expression was decreased in the skeletal muscles of the perineum. This is in line with
features in myotonic dystrophy where decreased Cl channels are thought to underlie the
hyperexcitability and myotonia in skeletal muscles. One would predict that this would require a
lower stimulating voltage to initiate muscle contractions in diseased EDL and SOL as decreased
Cl channels would raise the resting membrane potential of muscles closer to the threshold for
activation. Differences in stimulation voltages were not observed in the current studies, but this
may be due to the method involving parallel electrodes where the distance between the
electrodes has a greater influence on the stimulation voltage rather than inherent properties of the
skeletal muscle. Future experiments could examine Cl channel expression levels in weaker
skeletal muscles and the voltage required to elicit a muscle response during direct stimulation on
the surface of the muscle membrane. This could determine whether Cl channel loss/dysfunction
is a possible mechanism of muscle weakness.
Possible involvement of Ca mechanisms has been indicated by the current and previous
research into mice with weaker muscles. Nearly all Tg muscles in the 97Q and 141 models had
lower twitch to tetanus ratios compared to WT controls, indicating a greater deficit in twitch
force compared to tetanic force in diseased muscles. These findings are suggestive of
abnormalities in Ca handling mechanisms during contractions. The delays in rise and relaxation
times in T-treated female Tg mice and rise time in male Tg mice of the 141 model also suggest
impaired Ca handling.

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Previous microarrays in the 141 model indicate decreased parvalbumin (PV) gene (17-fold
downregulation) in muscles from Tg compared to WT controls (Mo et al., 2010). This also
suggests protein levels for PV could be decreased in diseased muscles. PV buffers Ca levels
during muscle contractions and shortens rise and relaxation times in rodent muscles with a
greater proportion of fast-twitch muscle fibers (Heizmann et al., 1982). PV decreases could lead
to the delay in rise and relaxation times in EDL’s from T-treated 141 Tg females and the delay in
rise time of the 141 Tg male EDL’s, but there were no effects on relaxation times in 141 Tg male
EDL’s. Additionally, aside from the kinetic effects, the decline in PV expression in muscle
would not explain the loss of force (Schwaller et al., 1999). Thus, if Ca handling mechanisms
are affected, it would likely occur during the phases when Ca acts as the messenger during
contraction: 1) release from the sarcoplasmic reticulum, which is mediated by dihydropyridine
receptors (DHPR) and ryanodine receptor 1 (RyR1), 2) Ca binding of troponin C (TnC) to
remove tropomyosin from actin binding sites, and 3) relaxation with sarcoendoplasmic reticulum
ATPases (SERCA’s) returning Ca back into the sarcoplasmic reticulum (SR). Each of these
specific mechanisms could be impaired and contribute the deficits in contractile function of
diseased muscles.
Involvement of RyR1 was implicated by the severe force loss concomitant with delay in
kinetics, specifically in the 141 Tg EDL’s. Although their studies did not exhibit changes in rise
time, Bellinger et al. (2008, 2009) suggest leaks in RyR1 may underlie the disease phenotype in
Duchenne muscular dystrophy (DMD). As Ca leaks out of the SR, it is hypothesized that protein
degradation and cell death occur, leading to aberrant skeletal muscle morphology and function.
Additionally, leaks may activate Ca-dependent proteases, such as calpains (Spencer et al., 1995),
that lead to the muscle pathology seen in the EDL of 141 Tg mice (Johansen et al., 2011). The

71

hypothesis for RyR1 leaks leading to muscle damage and weakness was reinforced when S107, a
compound that prevents RyR1 leaks, was given to mice modeling DMD and resulted in overall
improvement in motor function and skeletal muscle structure as well as decreased calpain
activity compared to mice receiving vehicle (Bellinger et al., 2009). In the SBMA models,
defective RyR1 would likely lead to increased basal tension, a measurement not tested in the
mechanics studies but important to find in future studies, and would likely manifest as
hypertonia or rigidity in mice. This is observed in humans and pigs with cases of malignant
hyperthermia (MH). The underlying genetic basis for MH is a mutation on the RyR1 gene in
skeletal muscles, and the condition is triggered by certain anesthetics and/or nondepolarizing
muscle relaxants. The binding of the compounds results in a continuous release of Ca ions,
leading to rigidity, and increased metabolic activity leading to higher body temperatures
(Mickelson and Louis, 1996). Although 141 Tg mice may exhibit muscle rigidity, they did not
exhibit differences in body temperatures compared to WT controls (data not shown). One way
of testing whether RyR1-related Ca leaks underlie the weakness would be to treat diseased 141
Tg mice with S107, a compound that prevents RyR1 leaks (Bellinger et al., 2008), and measure
whether that leads to motor improvements (at the behavioral level) and return of muscle
function. However, this would only explain the results in the fast-twitch EDL’s of the 141 Tg
mice. The greatest force deficits seen in the EDL’s of 97Q mice and SOL’s of 141 Tg mice
occur in the absence of any kinetic changes and may be due to other Ca-related mechanisms.
Evidence for the involvement of TnC is inferred from microarrays showing that expression
of the slow TnC gene is downregulated in limb muscles from male 141 Tg mice. Slow TnC is
primarily found in slow-twitch and cardiac muscles, and its decrease could explain the force loss
in the SOL of myogenic 141 Tg mice. To assess this possibility, one could estimate the amount

72

of TnC protein in muscle by Western blotting (Hartner and Pette, 1990). However, loss of
protein may not explain the loss of force in T-treated 141 Tg females due to the rapid onset of
weakness: after 3 days of T exposure, force is maximally reduced in the SOL but the TnC half
life (Martin, 1981) is much longer. Moreover, the fast TnC isoform found in fast-twitch skeletal
muscles was not reported to be decreased in male 97Q Tg mice (Mo et al., 2010) but nonetheless,
it was the EDL that showed the most severe loss in force in this model. However, before TnC is
ruled out as a possible mechanism underlying muscle weakness, in T-treated 141 Tg females and
97Q males, it would be necessary to examine not just TnC levels but also its function. TnC is
pH-sensitive and has decreased affinity to bind Ca at lower pH during conditions such as
acidosis (Parsons et al., 1997). Thus, function can be affected without changes in protein levels.
If TnC does not properly bind Ca, it is likely that less binding sites on actin are available for
myosins, leading to decreased force. A way to investigate whether TnC binding affinity is
affected is to examine skeletal muscle function using skinned fiber experiments. In brief, this
method involves either chemically or mechanically “skinning” the fibers to remove their plasma
membrane and then image Ca binding using Ca-sensitive dyes. After the initial imaging, TnC
can be extracted to deplete muscle fibers of TnC. Comparisons of the initial and final images
indicate whether TnC was functional in the skeletal muscles (Patel et al., 1997). This approach
could be done in conjunction with Western blots or ELISAs to estimate the amount of specific
TnC isoforms in diseased and healthy muscle.
Finally, SERCA involvement with RyR1 dysfunction may also result in muscle weakness.
Slowed relaxation was observed in T-treated female 141 Tg mice also suggesting possible
SERCA defects in those mice. Due to the rapid occurrence of the phenotype, it may be possible
that part of the initial onset of disease is due to SERCA dysfunction. Apart from the continuous

73

contraction leading to ATP hydrolysis in the actin-myosin interactions due to a RyR1 leak,
SERCA also utilizes ATP during reuptake of Ca against the Ca gradient (MacLennan et al.,
1997). With SERCA constantly activated by Ca presence due to RyR1 leaks, it may be possible
that SERCA function is compromised due to the metabolic stress. To determine whether this is
an issue of SERCA downregulation vs. dysfunction, SERCA Western blots could be run to
indicate or eliminate quantity as the underlying issue (Chami et al., 2001). Additionally,
missplicing of SERCA and RyR1 has been observed in mice modeling myotonic dystrophy
(Kimura et al., 2005) and may be worth investigating if alterations to protein structure lead to
functional deficits in the current SBMA models. This could be done using the RT-PCR analysis
described by Kimura et al. (2005) to determine splice variants within EDL and SOL of SBMA
mice.

Trophic Factors
Several muscle-derived trophic factors have been found to be decreased in skeletal muscle of
diseased SBMA mice. In addition to Ca mechanisms, it is of interest to investigate whether
trophic factors in skeletal muscles may alter the disease phenotype. One trophic factor, insulinlike growth factor I (IGF-1) has been shown to improve motor function and increase survival in
ALS and SMA models when given directly to skeletal muscles (Bosch-Marce et al., 2011;
Dobrowolny et al., 2005). Those results have been replicated in SBMA models by crossing mice
with skeletal muscle-restricted IGF-1 overexpression with 97Q mice (Palazzolo et al., 2009) or
by injecting 97Q mice with IGF-1 (Rinaldi et al., 2012). It is currently unknown what would
occur if IGF-1 were overexpressed by crossing in the 141 Tg mice or were injected into the 141
Tg mice. Another trophic factor, glial cell line-derived neurotrophic factor (GDNF) has also

74

produced improvement in neurodegenerative mouse models. Grafting myoblasts modified to
secrete GDNF into hindlimb muscles of mice modeling ALS with a SOD1 mutation resulted in
improvement of motor function and motoneuron survival (Mohajeri et al., 1999). Furthermore,
similar to the IGF-1 study, crossing mice with muscle-specific GDNF overexpression with G93A
ALS mice improves disease phenotype in the resulting offspring (Li et al., 2007). Vascular
endothelial growth factor (VEGF) has also been shown to be consistently decreased in skeletal
muscles from diseased mice (Monks et al., 2007). However, VEGF levels are still decreased
upon recovery of motor function and may proceed from, rather than underlie, muscle weakness
(Johansen et al., 2009). Thus, experiments manipulating trophic factor levels in diseased mice
could lead to a better understanding of their roles during disease and improvement. Additionally,
recovery experiments in Tg mice could be used to examine trophic factor levels in skeletal
muscles during disease phenotype and at several different timepoints during recovery.

Metabolic Mechanisms
Data from the 141 model have consistently shown that metabolic properties are altered in
rodents overexpressing WT AR in skeletal muscle fibers. The EDL of male Tg mice consistently
exhibit a more oxidative profile with NADH-TR staining (Johansen et al., 2011; Monks et al.,
2007). This is in line with functional alterations observed in the isolated EDL from 141 Tg
males showing greater endurance during the fatigue stimulation protocol. Additionally, mice and
rats overexpressing WT AR in muscles have decreased fat pads and increased mitochondrial
complex activities (Monks, unpublished observations; (Fernando et al., 2010; Musa et al.,
2011)). However, it is unknown whether the shift in metabolic properties precedes or proceeds
from alterations in muscle function as metabolic mechanisms may also be related to Ca-related

75

mechanisms. Increased PV expression has been shown to decrease activity of succinate
dehydrogenase, a mitochondrial marker enzyme (Chin et al., 2003) while mice with decreased
PV have increased cytochrome C oxidase activity and mitochondrial volume (Chen et al., 2001).
This is relevant to the current studies as male 141 Tg mice exhibited PV gene downregulation in
microarrays (Mo et al., 2010). Overexpressing activated calcineurin, a Ca-dependent
phosphatase, leads to higher levels of slow-twitch fibers and lower PV levels (Chin, 2004; Naya
et al., 2000; Wu et al., 2001). Increased Ca levels also activate calcium/calmodulin-dependent
kinases leading to mitochondrial biogenesis (Fluck et al., 2000; Wu et al., 2002) and a slowtwitch phenotype. Thus, while metabolic alterations may underlie the muscle weakness and
motor dysfunction, metabolic alterations may also be a consequence of Ca handling issues in the
skeletal muscles. Aside from possible mechanisms inducing changes in muscle phenotype, if Ca
is unable to bind TnC in 141 Tg SOL, as stated previously, it is possible that muscles are not
producing enough force to induce fatigue during testing, another line of reasoning that suggests
deficits in Ca handling may underlie some of the metabolic alterations seen in skeletal muscles.

Future Directions
Given all of the data, it would be prudent to investigate Ca mechanisms using an initial
immunoblot of relevant Ca handling proteins and proceeding to examine function of the proteins
of interest. Trophic factor experiments focusing on IGF-1 and GDNF could be conducted by
manipulating levels in diseased mice, or by monitoring their levels during different stages of
disease and recovery. Finally, metabolic mechanisms should also be investigated by examining
mitochondrial activity and levels in muscles. However, such experiments should be done in

76

conjunction with Ca-related experiments to determine the extent of or to rule out overlap
between the two mechanisms.

Conclusions
While previous studies have suggested skeletal muscle involvement in SBMA, the current
findings indicate unequivocally that primary skeletal muscle dysfunction is an important event
during the loss of motor function in SBMA. While it is still unclear what mechanisms may
underlie the disease phenotype, several experiments have been suggested that target potential
mechanism(s) that cause the loss of muscle strength. Because contraction kinetics were not
uniformly affected across muscles from the different SBMA models, it is not clear whether to
expect slower contraction kinetics in muscles of SBMA patients. Nonetheless, it seems likely
that mutant AR exerts direct toxic effects in muscle that impair its function independent of
regressive changes in the strength and viability of its motoneuronal inputs.

77

APPENDIX
Myonuclei

Myofibrils

Tendon

Figure 1. For interpretation of the references to color in this and all other figures, the reader is referred to the electronic version of
this dissertation. The whole skeletal muscle is comprised of multiple fascicles with capillaries between fascicles providing a
blood supply. Fascicles are then comprised of multiple myofibers. Myofibers are large, multinucleated individual cells of a
skeletal muscle and are comprised of myofibrils.

78

Figure 2. Large multinucleated myofibers contain the mitochondria on the muscle periphery to aid in muscle energetic. Myofibrils
comprise the myofiber and are, in turn, comprised of myofilaments. Along myofibrils are sarcomeres, the most basic unit of a skeletal
muscle that allows for a contraction. Upon electrical stimulation, multiple sarcomeres shorten within a muscle resulting in whole
muscle shortening to produce a contraction.

79

Figure 3. The myofilaments that comprise myofibrils and sarcomeres are shown when the overlying sarcoplasmic reticulum (SR) is
removed from the myofibril. Myosin and actin form an interdigitating pattern throughout the myofibril to allow contractions. Myosin
are anchored at the M line while actin is anchored by Z disks. Regions of the SR closer to the T tubules are termed the terminal
cisternae. T-tubules that run on either side of the interdigitating region contain openings that form a triad with the terminal cisternae.
These regions are where the electrical stimulus initiates muscle contractions.

80

Figure 4. 0) The first image at the top right lists the key proteins involved during a skeletal
muscle contraction. A T-tubule is in contact with the sarcoplasmic reticulum (SR) containing
calcium (Ca) ions that will initiate contraction. Ryanodine receptor 1 (RyR1) is closed, and
sarcoendoplasmic reticulum Ca ATPase (SERCA) is inactive while Ca is within the SR. On the
actin filament, tropomyosin covers the binding site preventing myosin heads from binding to
actin. 1) With a voltage change, T-tubules conduct the electrical depolarization to initiate the
opening of RyR1, releasing Ca from the SR. Upon Ca release from the SR, it binds to the Casensitive subunit of the troponin complex. This also initiates SERCA function, but the large
release of Ca into the cytosol is greater than the rate at which Ca is replaced into the SR. 2) Ca
binding of troponin induces a conformational change in troponin removes the troponintropomyosin complex away from the actin filament, exposing the actin binding sites. 3) A
myosin head to binds to the actin binding site, and 4) ATP is hydrolyzed, resulting in ADP and
inorganic phosphate. Once phosphate is released, the myosin head turns. 5) With the release of
ADP, the myosin head is turned even further, moving the actin filament, shortening the
sarcomere, and resulting in a power stroke to initiate skeletal muscle contraction. This
shortening in the sarcomeres produces the contractile force generated by muscles. In cases
where ATP is depleted, the myosin head remains in this locked position on the actin filament. 6)
As long as ATP is available, another ATP binds to the myosin head. 7) This releases the myosin
head from the actin filament, returning the myosin head to its original position (same as step 2).
8) Once the skeletal muscle returns to resting potential, RyR1 closes, and SERCA, its function
initiated when Ca entered the cytosol, is able to return Ca back to the SR against the Ca
concentration gradient (dashed arrow). Consequently, TnC no longer binds Ca, and the troponintropomyosin complex returns to its resting state where tropomyosin covers the myosin binding
sites on actin. 0) With Ca return to the SR, the entire region is ready for another contraction.

81

Figure 4 (cont’d)

82

Muscle Weights
(mg)
Plasma T Levels
(nmol/L)

EDL
SOL

Treatment Groups
Wild Type
Transgenic
B
T
B
T
8.0+0.4
8.6+0.2
9.5+0.3
8.6+0.3
7.6+0.2
8.3+0.6
9.3+0.4
9.4+0.4
1.4+0.3
29.3+1.8
1.2+0.1
49.5+5.9

Normalized Peak
Twitch Force
(N/g)

EDL
SOL

6.67+0.73 7.48+0.42 7.20+0.47 4.21+0.40*
3.22+0.20 3.38+0.23 3.25+0.19 0.45+0.04*

Twitch/Tetanus
Ratio
(5 Day T)

EDL
SOL

0.21+0.01 0.24+0.01 0.21+0.01 0.24+0.01
0.17+0.01 0.18+0.01 0.21+0.00 0.12+0.01*

(3 Day T)

EDL
SOL

Protein
Content(mg)/Muscle
weight(g)

EDL
SOL

NA
NA

0.27+0.02
0.21+0.02

NA
NA

47.6+8.8 66.0+14.8 53.1+7.4
52.2+18.5 66.9+12.1 43.5+13.2

0.23+0.02
0.13+0.02*

41.5+6.6
55.9+10.1

Table 1. Biometric data for female 141 mice. T, testosterone; mg, milligrams; nmol/L,
nanomolar per liter; N/g, newtons/gram. Values are means + standard error of means. * P<0.05.

83

Figure 5. Motor function declines rapidly in testosterone (T)-treated adult female transgenic
(Tg) mice that express a wild-type androgen receptor (AR) transgene only in skeletal muscle
fibers. Baseline performance at Day 0 before hormone treatment did not differ across treatment
groups. T treatment induced a rapid decline in grip strength (A), hang time (B), and body weight
(C) only in Tg females. By Day 5, motor performance on both tests was decreased significantly
for T-treated Tg (T Tg) females compared to baseline or compared to the other groups, including
asymptomatic females that were blank-treated wild-type (B WT), testosterone-treated wild-type
(T WT), and blank-treated transgenic (B Tg). Each asymptomatic group maintained pretreatment performance levels on both the grip strength and hang tests across the 5 days of
treatment, indicating that only the combination of AR transgene expression in muscle fibers and
male levels of androgens instigates motor dysfunction. Values are means + standard errors of
means based on N = 5-6 mice/muscle and group. *P<0.05, T Tg versus B Tg or T WT.
84

Figure 6. Tetanic force produced by both EDL and SOL of motor-impaired female mice is
reduced significantly compared to tetanic force produced by corresponding muscles of
asymptomatic controls after 5 days of testosterone (T) treatment. Representative traces for T
WT muscles and T-treated transgenic (T Tg) muscles are shown (A, B). Tetanic force produced
by the EDL of T Tgs is about half of control females (C), whereas tetanic force produced by the
SOL of T Tg females is about a quarter of T WT controls (D). Tetanic force produced by
muscles of control-treated Tg females (B/Tg) is equivalent to that of WTs, indicating that AR
transgene expression per se does not impair muscle strength. Comparable force deficits are
observed at the beginning and end of experiments (indicated as first and last tetanus) for both
EDL and SOL of T Tg mice, suggesting that force deficit is present in vivo and is not due to a
more rapid deterioration of these muscles in vitro. Force (in Newtons-N) was normalized to
muscle wet weight (grams-g). Values in histogram are means + standard errors of means, N = 56 mice/group. *P<0.001, T Tg versus B Tg or T WT.

85

Figure 7. Neither histopathology nor expression of the AR transgene explains the severe deficit
in SOL force production of T-treated Tg mice. Cross sections of SOL muscle (scale bar = 500
μm) from Tg females after 5 days of T treatment stained with H&E exhibit no signs of abnormal
morphology (e.g. fiber atrophy/hypertrophy, centralized nuclei, A), nor alterations in fiber
number (B) compared to the SOL from other treatment groups (N = 4-5 mice/group). Bars
represent means + standard errors of means. AR immunoblot (C) shows AR expression levels in
the EDL and SOL from asymptomatic (B Tg) versus symptomatic (T Tg) mice. We find no
consistent differences in AR expression (~110 kD) between EDL and SOL muscles, indicating
that a difference in AR protein content between the EDL and SOL is not likely to underlie the
relatively greater force deficit in the SOL compared to the EDL in T-treated Tg mice. Actin
(~40 kD) loading control shows no consistent differences in protein loading between the EDL
and SOL of Tg mice.

86

Figure 8. Twitch kinetics are slowed only in the EDL of motor-impaired Tg mice after 5 days of
T treatment. Representative twitches from the fast-twitch EDL and the slow-twitch SOL (A, B)
reveal decreased peak twitch force for both muscles of T-treated Tg mice (see Table 1 for mean
values) but altered twitch kinetics only for the EDL. Quantitative analysis of individual twitches
(C – F) confirms a significant prolongation in the time it takes to reach peak twitch force (C) and
to relax (E) in EDL, but neither parameter is significantly altered by T in the SOL muscle of Tg
mice (D, F). Plotted values are means + standard errors of means, with N = 5-6 mice/group.
*P<0.001 T Tg versus B Tg or T WT.

87

Figure 9. The SOL but not the EDL of motor-impaired mice shows the same deficit in peak
tetanic force after 3 days of T treatment. Tetanic force in the slow-twitch SOL from T Tg mice
was significantly decreased after 3 days of T treatment (A), comparable to the magnitude of the
effect of 5 days of treatment (Fig 6). A subtle, but not significant difference was seen in EDL
from the same motor-impaired mice (T Tg versus T WT mice, p = 0.07). However, peak twitch
force in both muscles was significantly reduced in T Tg mice compared to T WT mice (B).
Twitch kinetics were largely unaffected in T-treated Tg mice after 3 days of treatment (C, D)
except that relaxation time was slowed significantly in EDL, consonant with the effect of T on
this parameter after 5 days of treatment (Fig 8). N = 5-7/group. *P<0.05.

88

Figure 10. The EDL but not the SOL from motor-impaired Tg mice showed compromised
resistance to fatigue after 5, but not 3, days of T treatment. Fatigue resistance was measured as
time in seconds from onset of tetanizing stimulation to when the muscles decreased in force by
50% of maximal tetanic force. The EDL from Tg mice after 5 days of T treatment fatigued
significantly faster, dropping to half maximal force several seconds sooner than the EDL from
either T-treated WT or blank-treated Tg mice (A). Interestingly, the EDL from blank-treated Tg
mice showed a greater resistance to fatigue than the EDL from blank-treated WT (B Tg versus B
WT), indicating a beneficial effect of the transgene itself on resistance to fatigue in the EDL.
Five days of T treatment had no effect on fatigue resistance in SOL (B), and there was no effect
of T on fatigue resistance after 3 days of treatment in either muscle from Tg mice (C and D). 5day EDL: N = 5-6 mice/group; 5-day SOL, N = 5-6 mice/group; 3-day EDL, N = 6 mice/group;
3-day SOL, N = 4-7 mice/group. *P<0.05.

89

Biometric Table for male SBMA mice

WT
97Q Tg

Body Mass (g)
21.6 + 1.0
16.5 + 1.2*

EDL (mg)
9.3 + 0.6
4.7 + 0.7*

SOL (mg)
7.2 + 0.5
8.0 + 0.6

T (nmol/L)
26.1+2.4
31.0+3.6

WT
141 Tg

26.8 + 0.9
16.8 + 0.6*

12.0 + 0.7
6.7 + 0.3*

9.8 + 0.7
7.8 + 0.4*

2.7+1.2
2.2+0.9

Table 2. Body weight, muscle mass and plasma testosterone (T) levels at end stage. Motorimpaired Tg male mice in each SBMA model show significantly decreased body weights
compared to their respective WT controls. All but the soleus (SOL) muscle in 97Q males had
significantly less mass. Circulating T levels were comparable between Tg and WT males within
each line but notably higher for Tg and WT males in the 97Q model because these animals were
castrated and given exogenous T starting at puberty. *P<0.05 difference between transgenic
mice and their respective WT controls, values = mean + standard error of mean

90

Rise time (ms)
WT
97Q Tg

EDL
18+0
21+1

SOL
33+2
38+3

Relaxation Time (ms)
EDL
SOL
65+4
115+12
85+10
121+8

WT
141 Tg

19+1
27+1*

36+3
39+2

78+7
77+9

118+6
103+38

Table 3. Kinetics analysis of individual twitch traces for motor-impaired Tg males and their WT
controls. Analyses revealed few significant effects of disease on twitch kinetic; only rise time
(time to peak force) in the EDL muscle of 141 Tg males was significantly prolonged compared
to WT controls. Note however a consistent trend toward slower twitch kinetics in diseased
muscles of both models. *P<0.05, values = mean + standard error of mean

91

Muscle Morphology
EDL
WT
97Q Tg
WT
141 Tg

Fibers
1169+94
979+80

0.023+0.007*

1

2

1055+75
753+72

Cent Nuclei
0.003+0.001

0.005+0.002
0.013+0.003

Fibers
976+24
988+36

SOL
Cent Nuclei
0.003+0.001
0.004+0.002

869+43

0.003+0.001

922+59

0.017+0.004*

Table 4. Muscle morphology for male mouse muscles. Total number of muscle fibers and
percent of total number of fibers containing centralized (cent) nuclei for EDL and SOL of the
two Tg SBMA models (141 myogenic and 97Q). Only the EDL from myogenic males had
significantly fewer fibers than WT controls. Fiber number was unaffected in both the EDL and
SOL of 97Q Tg males and also in the SOL of 141 Tg males. The EDL of 97Q males and the
SOL of 141 males contain a higher percentage of fibers with centralized nuclei, indicative of an
abnormal muscle state. Note that these two muscles also showed the severest loss in force (Figs
2, 4). n=5/group in all except 141 EDL (n=3) *P<0.05, values = mean + standard error of mean.
1
2
values from (Johansen et al., 2011); unpublished data (Johansen and Jordan).

92

Figure 11. Transgenic (Tg) males in both the 97Q and 141 ‘myogenic’ models of SBMA show
the expected impairment in motor function. Mean number of rearings in an open field, taken as
an indirect measure of hindlimb strength, was decreased significantly in Tg males of both the
97Q (A) and myogenic 141 (D) models of SBMA compared to their respective WT controls.
Similar significant deficits were also found in mean frontpaw grip strength (B, E) and hang times
(C, F). These measures indicate that Tg males in both models show the expected SBMA
phenotype. *p < 0.05 compared to WT controls. Error bars represent standard error of the mean,
with n = 6-7 mice/group.

93

Figure 11 (cont’d)

94

Figure 12. Both the fast twitch extensor digitorum longus (EDL) and slow twitch soleus (SOL)
muscles from motor-impaired 97Q Tg males show deficits in contractile force. Raw twitch and
tetanic force (A,D, and G) were significantly reduced, with the one exception that raw tetanic
force was unaffected in the SOL (J). However, twitch and tetanic forces once normalized to
muscle mass were significantly decreased in both muscles, including tetanic force produced by
the SOL (B, E, H, and K), indicating primary defects in contractile function of muscles of
diseased 97Q Tg males that are independent of their mass. The force deficit is also readily
apparent in examples of individual traces of EDL (C) and SOL (I) twitches. Although somewhat
reduced, the twitch/tetanus (Tw/Tet) ratio in the diseased EDL was not significantly different
than WT (F). In contrast, the Tw/Tet ratio in the SOL of diseased males was significantly
reduced (L), suggesting defects in calcium handling mechanisms. Such defects in muscle
function may contribute to if not underlie defects in motor function. *p < 0.05 compared to WT
controls. Error bars represent the standard error of the mean, with n = 4-7 mice/group.

95

Figure 12 (cont’d)

96

Figure 13. Both the EDL and SOL from motor-impaired 97Q Tg males fatigue more rapidly.
The fatigue profile for both the EDL and SOL indicates that both muscles in Tg males produce
less force per gram muscle at the start of stimulation (A, C) followed by a drop in force for all
muscles indicating muscle fatigue during tetanizing stimulation. While the Tg EDL loses less of
its original force than the WT EDL over the stimulation period (A), the Tg EDL appears to
fatigue faster, losing 50% of its original force (time to half of maximal force loss) significantly
sooner than WT EDL (B). The Tg SOL also appears to have a faster time course of fatigue than
WT SOL, losing 50% of its original force significantly sooner than WT SOL (D). *p < 0.05
compared to WT controls. Plotted values are means + standard error of the mean, with n = 4-7
mice/group.

97

Figure 14. Both the fast twitch EDL and slow twitch SOL muscles from motor-impaired
myogenic (141) Tg males show deficits in contractile force. Raw and normalized twitch and
tetanic force were significantly reduced in both the EDL and SOL of motor-impaired males (A,
B, D, G, H, J and K) with the exception of normalized tetanic force in Tg EDL (E). On the other
hand, the deficit in raw tetanic force in the EDL probably is due to the deficit in fiber number in
this muscle (Table 4). Samples of twitch traces reveal such deficits in twitch force (C, I). The
Tw/Tet ratio is also significantly reduced in both the Tg EDL (F) and SOL (L), also suggesting
that that calcium mobilization from intracellular stores may be perturbed. Muscles of myogenic
141 males show similar defects in contractile properties as 97Q males, although disease affects
force most in the SOL of 141 males while affecting most the EDL in 97Q males. Nonetheless,
these results suggest that motor dysfunction likely involves primary defects in muscle function.
*p < 0.05 compared to WT controls. Error bars represent the standard error of the mean, with n
= 4-7 mice/group.

98

Figure 14 (cont’d)

99

Figure 15. Only the SOL from motor-impaired 141 Tg males fatigues more rapidly. The fatigue
profile indicates that the Tg EDL shows increased resistance to fatigue, losing less total force
than WT EDL overall (A). However, both Tg and WT EDL appeared to fatigue at about the
same rate, reaching a 50% drop at about the same time (B). The apparent increase in oxidative
metabolism in the diseased EDL (Johansen et al., 2011; Monks et al., 2007) may help stave off
some of the later losses in force exhibited by WT EDL. The SOL of diseased 141 Tg males on
the other hand fatigues rapidly, losing 50% of its starting force in less than 9.8+0.9 s compared to
42.1+2.4 s in WT SOL. The marked increase in fatigue susceptibility of the SOL muscle in 141
Tg males also likely contributes to motor dysfunction in this model. *p < 0.05 compared to WT
controls. Plotted values are means + standard error of the mean, with n = 4-6 mice/group.
100

Figure 16. Muscles from diseased and healthy mice remain stable in vitro during the course of
the experiment. Viability of each skeletal muscle was monitored with tetanus stimulations
throughout an experiment. Force produced by tetani at the beginning of the experiment (open
bar) was comparable to force produced at the end (filled bar). These data suggest that the force
deficits found in vitro in diseased muscles likely reflect a genuine force deficit in vivo, rather
than a defect that emerges in vitro. In vitro measurements were taken during a 2-3 hour window.
Plotted values are mean + standard error of the mean.
101

Figure 17. Representative photomicrographs of cross sections of muscle used for fiber counts.
The EDL from the myogenic model was previously characterized (Johansen et al., 2011) and
therefore not included. EDL muscle fibers in diseased 97Q Tg males are markedly
heterogeneous in size, with some fibers notably smaller than the rest (arrow). EDL fibers from
97Q males also contain centralized nuclei (arrow). Since mass of the EDL from diseased 97Q
males is reduced by half (Table 2) without loss of fibers (Table 4), it is likely that atrophy of
individual fibers underlies the deficit in EDL mass. Note however that decreases in muscle mass
contribute little to the deficits in twitch and tetanic force (Fig 2A, B, D and E) since a large
proportion of the deficit persists once force measures are normalized to muscle mass. No major
histological changes were observed in cross section of SOL from either 97Q males (middle
panels) or myogenic (141) males (bottom panels) except for centralized nuclei which were more
frequent in the SOL of 141 Tg males compared to WT SOL. Despite the lack of striking
histopathology, SOL function is impaired in both models, but particularly in the 141 myogenic
males, which show a three-fold loss of normalized force (Fig 14H, K). These data indicate that
impairments in muscle function need not be accompanied by marked histopathology. Scale bar
= 50 µm.
102

Figure 18. KI males with mild motor deficits show negligible deficits in muscle function.
Measurements of forepaw grip strength and hang times are significantly decreased in KI males
relative to WT controls (A, B), showing the expected impaired motor function in KI males.
While force measures in the KI EDL suggest small deficits in twitch and tetanic force consistent
with the mild motor phenotype in this model, these apparent deficits are not significant (C – H).
Likewise, we found no significant deficits in twitch and tetanic forces produced by the SOL from
KI males (I - N). These data raise the possibility that motor deficits in KI males may reflect
synaptic defects upstream to the skeletal muscles. *p < 0.05 compared to WT controls. Plotted
values are mean + standard error of the mean, with n = 5-7 mice/group.

103

Figure 18 (cont’d)

104

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105

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