Transgenic overexpression of γ-cytoplasmic actin protects against eccentric contraction-induced force loss in mdx mice.

Abstract

γ-cytoplasmic (γ-cyto) actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The purpose of this study was to determine whether further elevation of γ-cyto actin levels improve or exacerbate the dystrophic phenotype of mdx mice.
We transgenically overexpressed γ-cyto actin, specifically in skeletal muscle of mdx mice (mdx-TG), and compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different ages. We investigated the mechanism by which γ-cyto actin provides protection from force loss by studying the role of calcium channels and stretch-activated channels in isolated skeletal muscles and muscle fibers. Analysis of variance or independent t-tests were used to detect statistical differences between groups.
Levels of γ-cyto actin in mdx-TG skeletal muscle were elevated 200-fold compared to mdx skeletal muscle and incorporated into thin filaments. Overexpression of γ-cyto actin had little effect on most parameters of mdx muscle pathology. However, γ-cyto actin provided statistically significant protection against force loss during eccentric contractions. Store-operated calcium entry across the sarcolemma did not differ between mdx fibers compared to wild-type fibers. Additionally, the omission of extracellular calcium or the addition of streptomycin to block stretch-activated channels did not improve the force-generating capacity of isolated extensor digitorum longus muscles from mdx mice during eccentric contractions.
The data presented in this study indicate that upregulation of γ-cyto actin in dystrophic skeletal muscle can attenuate force loss during eccentric contractions and that the mechanism is independent of activation of stretch-activated channels and the accumulation of extracellular calcium.

Full text

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RESEARCH
Transgenic overexpression of g-cytoplasmic actin
protects against eccentric contraction-induced
force loss in mdx mice
Open Access
Kristen A Baltgalvis1, Michele A Jaeger1, Daniel P Fitzsimons2, Stanley A Thayer3, Dawn A Lowe4and
James M Ervasti1*
Abstract
Background: g-cytoplasmic (g-cyto) actin levels are elevated in dystrophin-deficient mdx mouse skeletal muscle. The
purpose of this study was to determine whether further elevation of g-cytoactin levels improve or exacerbate the
dystrophic phenotype of mdx mice.
Methods: We transgenically overexpressed g-cytoactin, specifically in skeletal muscle of mdx mice (mdx-TG), and
compared skeletal muscle pathology and force-generating capacity between mdx and mdx-TG mice at different
ages. We investigated the mechanism by which g-cytoactin provides protection from force loss by studying the
role of calcium channels and stretch-activated channels in isolated skeletal muscles and muscle fibers. Analysis of
variance or independent t-tests were used to detect statistical differences between groups.
Results: Levels of g-cytoactin in mdx-TG skeletal muscle were elevated 200-fold compared to mdx skeletal muscle
and incorporated into thin filaments. Overexpression of g-cytoactin had little effect on most parameters of mdx
muscle pathology. However, g-cytoactin provided statistically significant protection against force loss during
eccentric contractions. Store-operated calcium entry across the sarcolemma did not differ between mdx fibers
compared to wild-type fibers. Additionally, the omission of extracellular calcium or the addition of streptomycin to
block stretch-activated channels did not improve the force-generating capacity of isolated extensor digitorum
longus muscles from mdx mice during eccentric contractions.
Conclusions: The data presented in this study indicate that upregulation of g-cytoactin in dystrophic skeletal
muscle can attenuate force loss during eccentric contractions and that the mechanism is independent of activation
of stretch-activated channels and the accumulation of extracellular calcium.
Keywords: stretch-activated channels, calcium, skeletal muscle injury, dystrophin, costamere
Background
Duchenne muscular dystrophy (DMD) is a severe mus-
cle-wasting disease caused by mutations in the dystro-
phin gene. Dystrophin localizes primarily to costameres,
where it links the cortical actin cytoskeleton to the sar-
colemma and the extracellular matrix [1]. It is part of
the dystrophin-glycoprotein complex (DGC), a large oli-
gomeric complex of proteins thought primarily to
stabilize the sarcolemma during muscle contraction. In
the absence of a functional dystrophin protein, myofi-
bers are susceptible to injury, leading to muscle degen-
eration, inflammation and fibrosis.
Dystrophin binds to filamentous actin with high affi-
nity via two distinct actin-binding domains: an N-term-
inal tandem calponin homology domain and a region of
basic spectrin-like repeats in the middle rod domain [1].
In skeletal muscle, dystrophin forms a mechanically
strong link with the cortical actin cytoskeleton, which is
composed of g-cytoplasmic (g-cyto) and b-cytoplasmic
actins [2,3]. The tight association between dystrophin
and actin has been demonstrated with inside-out peeled
* Correspondence: jervasti@umn.edu
1Department of Biochemistry, Molecular Biology and Biophysics, University of
Minnesota, 6-155 Jackson Hall, 321 Church Street SE, Minneapolis, MN 55455,
USA
Full list of author information is available at the end of the article
Baltgalvis et al. Skeletal Muscle 2011, 1:32
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Skeletal Muscle
© 2011 Baltgalvis et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.

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sarcolemma from wild-type (WT) myofibers in which g-
cytoactin was retained in a riblike costameric pattern
but absent from the dystrophin-deficient mdx mouse
sarcolemma [2]. The importance of dystrophin-actin
binding in vivo is also evident in transgenic mdx mice
that express various dystrophin deletion constructs.
Despite restoration of DGC members at the sarco-
lemma, at least one actin-binding domain is essential for
partial or complete rescue of the dystrophic phenotype
in transgenic mdx mice [4-6].
A robust phenotype of the mdx mouse is increased
susceptibility to injury following eccentric muscle con-
tractions [7,8], a standard procedure to determine the
efficacy of a drug or therapy in preclinical trials [9].
Force loss following eccentric contractions depends on
the number of contractions, speed of contraction, overall
length change, muscle type and age of the mice. Overall
force loss in WT mice ranges from 2% to 29%, but force
loss can range from 27% to 69% in mdx mice [10]. The
mechanism of force loss following eccentric contractions
in healthy muscle has been investigated. The majority of
force loss following eccentric contractions in an in vivo
rodent model was due to excitation-contraction uncou-
pling, and about 25% of the force loss can be attributed
to physical damage [11]. After the initial bout of injury,
the loss of contractile protein accounts for strength loss
during 14 to 28 days of recovery. It is unknown whether
excitation-contraction uncoupling, physical damage, loss
of contractile protein or a combination of different
mechanisms explains the rapid force loss in mdx mice
following eccentric contractions.
Recent studies have suggested that stretch-activated
cation channels (calcium or sodium) at the sarcolemma
contribute to force loss during eccentric contractions
[12-15]. The removal of calcium in the muscle-bathing
media or the addition of streptomycin to block stretch-
activated channels has been shown to reverse or attenu-
ate force loss during injurious muscle contractions in
dystrophic muscle [12,15-17]. Store-operated calcium
entry (SOCE) is an important mechanism whereby cal-
cium is brought into muscle cells during sarcoplasmic
reticulum (SR) calcium depletion [18]. It has been pro-
posed that SOCE occurs through either (1) an interac-
tion between transient receptor potential C (TRPC)
channels and either the ryanodine receptor or inositol
trisphosphate receptors or (2) the STIM1/Orai1 pathway
[19]. TRP channels are plasma membrane channels that
vary in cation selectivity and sensitivity to mechanical
stress. While TRP gene transcripts are generally in low
abundance in skeletal muscle compared to some other
tissues, TRPC3 is the most abundant, with TRP vanilloid
3 (TRPV3), TRPV4, TRPV6, TRP melastatin 3 (TRPM3),
TRPM4 and TRPM7 also present [20]. In fact, the over-
expression of dominant-negative TRPC3 or TRPV2
channels in muscle ameliorates pathological changes
[13,21,22], and TRPC3 overexpression in WT mice is
sufficient to induce muscular dystrophy [22]. mdx mice
that overexpress a dominant-negative TRPV2 channel
are also protected from extreme force loss following
eccentric muscle contractions [13].
Although g-cytoactin comprises only 1/4, 000th of the
actin population in skeletal muscle [23], it is essential
for the maintenance of normal muscle function in adults
[24]. We previously reported that g-cytoactin levels are
elevated fivefold in dystrophin-deficient skeletal muscle
[23,25,26] and that muscle-specific ablation of g-cyto
actin does not exacerbate the dystrophic condition [26].
As many costameric components are actin-binding pro-
teins, it has been proposed that this increased g-cyto
actin expression may contribute to a compensatory
remodeling response to reinforce the compromised sar-
colemma in mdx mice [23]. Understanding the function
of increased g-cytoactin in dystrophic muscle may iden-
tify a unique pharmacological target for the treatment of
muscular dystrophies. The purpose of this study was to
determine whether further elevation of g-cytoactin levels
improves or exacerbates the dystrophic phenotype of
mdx mice. We generated a line of transgenic mdx mice
(mdx-TG) that expressed g-cytoactin 200-fold above
mdx g-cytoactin levels and showed substantial incor-
poration of g-cytoactin into thin filaments. Interestingly,
mdx-TG mice showed significantly attenuated force loss
during eccentric muscle contractions through a mechan-
ism independent of extracellular calcium and stretch-
activated channels.
Methods
Generation of transgenic mice
Generation of Actg1-TG mice has been previously
described [27]. To generate dystrophin-deficient mdx-
TG mice carrying the Actg1 transgene, Actg1-TG male
mice were crossed with mdx females. TG-positive males
were subsequently crossed with mdx females to generate
mdx-TG mice. Animals were housed and treated in
accordance with the standards set by the Institutional
Animal Care and Use Committees at the University of
Minnesota and the University of Wisconsin.
Determination of g-cytoplasmic actin concentration in
skeletal muscle
Known amounts of purified bovine brain actin [27] and
SDS extracts from mdx-TG gastrocnemius or quadriceps
femoris muscles were run side-by-side on a 3% to 12%
SDS-polyacrylamide gel and transferred to nitrocellulose
membrane. Two g-cytoactin antibodies (monoclonal
mouse 2 to 4 and polyclonal affinity-purified rabbit
7577) were used for Western blot analysis, each at a 1:1,
000 dilution. To determine the concentration of actin
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(in micrograms) in the SDS extracts, a concentration
standard curve was derived from the known values of
purified bovine g-cytoactin using the densitometry and
concentration standards feature in the LI-COR Odyssey
Application Software version 2.1.12 (LI-COR Bios-
ciences, Lincoln, NE, USA).
Skeletal muscle histology and confocal microscopy
Tibialis anterior (TA) and gastrocnemius muscles from
WT, mdx and mdx-TG mice were frozen in melting iso-
pentane and mounted in O.C.T. mounting medium for
cryosectioning [27]. Ten-micrometer sections were
stained with hematoxylin and eosin. Centrally nucleated
fibers (CNFs) were counted on whole sections and
expressed as a percentage of the total number of fibers.
Isolation of myofibrils and confocal microscopy were
both performed as previously described [27]. Cross-sec-
tions of gastrocnemius muscles or myofibrils were
stained using the following antibodies: affinity-purified
g-cytoactin rabbit 7577 (1:75 dilution; Sigma, St Louis,
MO, USA), fast myosin heavy chain clone MY-32 (1:100
dilution; Sigma) and laminin a-2 rat clone 4H8-2 (1:200
dilution; Sigma). All images were obtained using a Fluo-
View FV1000 single-photon confocal microscope
equipped with a PlanAPO 60×/1.40 na oil immersion
lens objective, a UPlan Fluorite N 1.30 NA 40× oil
immersion lens objective and a UPLSAPO 0.40 NA 10×
lens objective (all from Olympus America Inc, Melville,
NY, USA). Images were collected with Olympus Fluo-
View FV1000 version 1.7b software and assembled and
processed using ImageJ software (National Institutes of
Health, Bethesda, MD, USA) and Adobe Photoshop ver-
sion 8.0 software (Adobe Systems Inc, San Jose, CA,
USA).
Serum creatine kinase analysis
Serum was collected and stored at -80°C until analysis.
Ten microliters of serum were applied to a VITROS
DT60 II MicroSlide (Ortho-Clinical Diagnostics, Inc,
Rochester, NY, USA) and creatine kinase (CK) activity
measured using a Kodak Ektachem DT60 Analyzer
(Eastman Kodak Co, Rochester, NY, USA) and a
VITROS DTSC II Module (Ortho-Clinical Diagnostics,
Inc).
Ex vivo contractility measurements and eccentric injury
protocol
The extensor digitorum longus (EDL) muscles were
assessed for contractile function and susceptibility to
eccentric contraction-induced injury as previously
described [26,28]. Age-matched male mdx and mdx-TG
mice (n = 5 or 6 per group) were anesthetized with
sodium pentobarbital (100 mg/kg body weight). Their
muscles were dissected and mounted onto a dual-mode
muscle lever system (model 300B-LR Dual-Mode Lever
Arm; Aurora Scientific Inc, Aurora, ON, Canada) with
4-0 suture in a 1.5-ml bath assembly filled with Krebs-
Ringer bicarbonate buffer that was maintained at 25°C
and perfused with 95% O2. The muscles were adjusted
to their anatomic optimal length (Lo) based on resting
tension of 0.4 g, and then muscle length was measured.
The muscles remained quiescent in the bath for ten
minutes. Passive stiffness, twitch force (Pt), tetanic force
(Po) and active stiffness were measured prior to initiat-
ing the eccentric injury protocol. First, passive stiffness
of the muscle was determined by stretching the muscle
sinusoidally from 97.5% Loto 102.5% Loat 0.5 Hz while
measuring the resulting force [29,30]. Two twitches (Pt)
separated by 30-second intervals were elicited, and the
resulting force was measured. Maximal isometric tetanic
contractions (Po) were obtained by stimulating muscles
for 400 ms at 180 Hz and 150 V (Grass S48 Square
Pulse Stimulator; Grass Technologies, West Warwick,
RI, USA) delivered through a SIU-V stimulus isolation
unit (Grass Technologies). Two minutes later a second
tetanic isometric contraction was elicited, and at peak
force a sinusoidal length oscillation of 0.01% Loat 500
Hz was imposed to determine active stiffness, an indir-
ect marker of strong-binding myosin [29,30]. Two min-
utes after that an injury protocol consisting of five to
ten eccentric contractions was begun. For these contrac-
tions, muscles were passively shortened from Loto 0.95
Loover 3 seconds, stimulated tetanically for 200 ms as
the muscle lengthened to 1.05 Loat 0.5 Lo/second and
then passively returned to Lo. Each eccentric contraction
was separated by three minutes of rest. The degree of
injury was calculated as the percentage change in
eccentric force production from the first to the last
eccentric contraction. Specific force was calculated by
dividing isometric force by the anatomical cross-sec-
tional area of the muscle. The cross-sectional area of
the muscle was calculated by dividing the mass of the
muscle by the product of fiber length (Lf) (Lo× 0.44)
and the density of muscle (1.06 g/mm3). Contractility
and injury protocols were followed to study both the
right and left sides and averaged as a single data point
for each mouse when comparing mdx and mdx-TG
mice.
To determine the effects of extracellular calcium and
stretch-activated channels on the susceptibility to
eccentric contraction-induced injury, three different
conditions were tested, all using male mdx mice, with
the contralateral muscle from the same mouse used as a
control (n = 3 to 5 per group). In the first condition,
Krebs-Ringer bicarbonate buffer was made without
CaCl2but replaced with the same molar ratio of MgCl2
(that is, calcium-free). In the second condition, 1 mM
ethylene glycol tetraacetic acid (EGTA) was added to
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the calcium-free buffer. Pilot work revealed that EGTA
blunted isometric force production, so buffer containing
1 mM EGTA was introduced only after the preinjury
contractile measurements were completed. The muscle
remained quiescent for ten minutes after the addition of
the calcium-free buffer with EGTA before the eccentric
contraction protocol was begun. In the third condition,
2 mM streptomycin was added to Krebs-Ringer bicarbo-
nate buffer for the entire protocol. Pilot work revealed a
greater effect on postinjury isometric force production
with the 2 mM concentration instead of the previously
reported 0.2 mM [12].
Flexor digitorum brevis myofiber isolation
Flexor digitorum brevis (FDB) fibers were isolated from
WT and mdx mice as previously described [31-33]. The
right and left FDB muscles were dissected from WT,
mdx and mdx-TG mice and placed in 5 ml of sterile
physiological rodent saline (138 mM NaCl, 2.7 mM KCl,
1.8 mM CaCl2, 1.06 mM MgCl2, 12.4 mM 4-(2-hydro-
xyethyl)-1-piperazineethanesulfonic acid (HEPES), 5.6
mM glucose, pH 7.3). The muscles were then trans-
ferred to a Petri dish containing 2 ml of media (DMEM,
10% fetal bovine serum, and 0.5% penicillin/streptomy-
cin) containing 2 mg/ml collagenase. Following this
step, they were incubated at 37°C (5% CO2) for three
hours and then gently triturated in fresh media and
allowed to settle at 1 × g. All but 1 ml of the media was
aspirated, and the fiber pellet was gently resuspended.
Next the fibers were placed into six 35-mm Petri dishes,
each containing a glass coverslip coated with 1 μg of
laminin supplemented with 2 ml of media. The fibers
were allowed to adhere to the coverslips overnight at
37°C. They were analyzed for SOCE within 24 to 48
hours after dissection.
Store-operated calcium entry
SOCE was measured in FDB fibers as previously
described [22,34]. Coverslips containing adhered FDB
fibers were briefly washed in physiological saline solu-
tion (PSS) (130 mM NaCl, 5.6 mM KCl, 1 mM MgCl2,
1.7 mM CaCl2, 11 mM glucose, 10 mM HEPES, pH
7.4). Indo-1 AM (Invitrogen, Carlsbad, CA, USA) was
mixed 1:1 with Pluronic F-127 solution (Invitrogen) and
added to myofibers at a final concentration of 1 μM.
Myofibers were loaded with dye for 30 minutes at 37°C
and washed with PSS for 15 minutes at 37°C. Instru-
mentation and recording of the calcium signal was per-
formed as previously described [35]. Myofibers were
perfused with PSS at a rate of 1 to 2 ml/minute for two
minutes before being moved to calcium-free PSS. The
calcium-free PSS did not contain CaCl2, had a MgCl2
concentration of 2 mM, contained 1 mM EGTA and
contained 30 μM cyclopiazonic acid (CPA) to block SR
calcium-ATPase. Calcium-free PSS was perfused for
four to eight minutes before 5 mM caffeine was added
to release calcium from the SR. After the indo-1 ratio
had returned to baseline (8 to 12 minutes), the muscle
was perfused with PSS and SOCE was calculated as the
difference in the minimum and maximum indo-1 ratios
averaged over a 30-second period. During the recording
of the indo-1 signal, 50 μM N-benzyl-p-toluene sulfona-
mide (BTS) was added to all solutions to prevent myofi-
ber contractions.
Data analysis
Two-way analysis of variance (ANOVA) was used to
assess differences in CNFs and serum CK with mouse
strain (WT vs mdx vs mdx-TG) and age as the factors.
One-way ANOVA was used to compare intracellular
calcium concentration ([Ca2+]i) between WT, mdx and
mdx-TG mice. Post hoc analyses were performed using
the Holm-Sidak test. t-tests were used to analyze differ-
ences between all other variables. Data are reported as
means ± SEM. Significance was set at P < 0.05.
Results
To examine the role of elevated g-cytoactin in dystro-
phin-deficient muscle, Actg1-TG male mice were
crossed with mdx female mice. Levels of g-cytoactin in
mdx-TG skeletal muscle SDS extracts were analyzed
alongside a purified g-cytoactin calibration curve on
SDS-PAGE gels and blotted with g-cytoactin-specific
antibodies. Mean (± SEM) mdx-TG g-cytoactin levels in
muscle were 397 ± 53 μM, which is approximately 200-
fold that in mdx skeletal muscle and similar to that in
skeletal muscles of Actg1-TG mice (Figure 1A).
To determine the localization of g-cytoin mdx-TG ske-
letal muscle, cross-sections were stained with an affinity-
purified g-cytoactin antibody. Capillaries, blood vessels,
nerves and immune cells stained brightly in mdx skeletal
muscle (Figure 1B). In addition, the internal structure of
mdx-TG muscle showed intense g-cytoactin localization
as previously described [27] for Actg1-TG mice (Figure
1B). Internal staining of g-cytoactin was prominent in
the majority of fibers. Staining of isolated mdx-TG myo-
fibrils showed g-cytoactin incorporation into the thin
filament that was absent in mdx myofibrils (Figure 1C).
Colocalization of g-cytoactin with fast myosin heavy
chain is consistent with reports that the human a-skele-
tal actin promoter used to drive transgene expression is
preferentially active in fast-twitch muscle fibers [27,36].
To determine whether elevated levels of g-cytoactin
were beneficial or detrimental to dystrophin-deficient
skeletal muscle, several well-established parameters of
dystrophic pathology were examined. Hematoxylin and
eosin-stained skeletal muscle cross-sections from mdx
and mdx-TG mice were indistinguishable, showing
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characteristic fiber size variations, necrotic cells, fibrosis
and mononuclear cell infiltration (Figure 2A). In addi-
tion, mdx mice exhibited high levels of CNFs and ele-
vated serum CK. CNF levels were quantified in
gastrocnemius and TA muscles at various ages (Figure
2B). As expected, mdx and mdx-TG mice had greater
CNFs in the TA muscle (P < 0.001) and the gastrocne-
mius muscle (P < 0.001) compared to WT mice. In the
TA muscle, there was a significant interaction between
mouse group and age (P < 0.001). mdx-TG mice had
37% fewer CNFs in the TA muscle at one month of age
compared to mdx mice, but there were no differences in
CNFs between mdx and mdx-TG mice at any other ages
for the TA muscle (P > 0.155) or the gastrocnemius
muscle (P = 0.793). Serum CK levels were elevated in
dystrophic compared to WT mice (P ≤ 0.013) but were
Figure 1 g-cytoplasmic actin expression in skeletal muscle and incorporation into thin filaments. (A) Quantitative Western blot analysis of
g-cytoplasmic (g-cyto) actin levels in SDS extracts of mdx-TG murine skeletal muscle. Values are means +/- SEM. *Previously published values
[23,27]. (B) g-cytoactin expression in mdx and mdx-TG gastrocnemius muscle cross-sections. (C) g-cytoactin expression in thin filaments. Isolated
myofibrils were costained with antibodies against g-cytoactin and fast myosin heavy chain.
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