J Appl Physiol 114: 1482–1489, 2013.
First published March 7, 2013; doi:10.1152/japplphysiol.00925.2012.
Calpain and caspase-3 play required roles in immobilization-induced limb
muscle atrophy
Erin E. Talbert, Ashley J. Smuder, Kisuk Min, Oh Sung Kwon, and Scott K. Powers
Department of Applied Physiology and Kinesiology, University of Florida, Gainesville, Florida
Submitted 27 July 2012; accepted in final form 27 February 2013
tBid; calpastatin; muscle wasting; ubiquitin proteasome system; disuse muscle atrophy
muscle inactivity due to spaceflight, bed rest, or limb immobilization result in muscle atrophy
in both animals and humans. Due to both the invasive nature of
human muscle biopsies and the desire to use pharmacological
interventions not approved for humans, animal models are
commonly used to explore the mechanisms responsible for
disuse atrophy, with hindlimb immobilization (i.e., casting) of
rodents often used to mimic human limb immobilization (19).
Disuse skeletal muscle atrophy results from both decreased
protein synthesis and increased protein degradation, with degradation playing a major role (28). Four major proteolytic
systems [autophagy, the ubiquitin protease system (UPS),
calpains, and caspase-3] are active during atrophy in skeletal
muscle (19). However, the degree to which each system contributes to disuse atrophy remains debatable. It has been argued
that the UPS plays a dominant role during inactivity-induced
muscle atrophy because the UPS is responsible for degrading
most sarcomeric proteins (7, 16). Nonetheless, the UPS cannot
degrade intact actin and myosin because of the closely packed
arrangement of these proteins in the sarcomeres of striated muscle
PROLONGED PERIODS OF SKELETAL
(6, 27). Therefore, the UPS may not be the rate-limiting step in
muscle atrophy, as release of myofilaments could be a prerequisite
for proteasome-mediated degradation of both actin and myosin.
Calpain and caspase-3 are two proteases that may assist the
UPS by releasing myofilaments. For example, calpain can
degrade the structural proteins ␣-II-spectrin (␣-fodrin), titin,
and nebulin (9). Breakdown of these cytoskeletal proteins
releases actin and myosin from sarcomeres, allowing musclespecific E3 ligases to ubiquitinate myofilament proteins, targeting them for degradation by the UPS. Furthermore,
caspase-3 has been reported to degrade intact actomyosin (6).
Nonetheless, definitive evidence that calpain and/or caspase-3
plays a required role in limb disuse muscle atrophy does not
currently exist. Indeed, disagreement exists regarding the role
that calpain plays in disuse-induced atrophy in limb muscles
(24, 29), and incomplete data are available regarding the part
that caspase-3 plays in limb muscle atrophy (18).
Recent work in mechanical ventilation-induced diaphragm
atrophy suggests that a regulatory “cross talk” exists between
calpain and caspase-3 in respiratory muscles. Indeed, inhibition
of calpain prevented caspase-3 activation in the diaphragm
during mechanical ventilation, suggesting that calpain regulates caspase-3 activity. Caspase-3 inhibition also prevented
calpain activation, demonstrating caspase-3 regulation of calpain activity (17). Nevertheless, mechanical ventilation-induced
diaphragmatic atrophy is an unusually rapid and unique type of
muscle atrophy, and it remains unclear if this same regulatory
cross talk between calpain and caspase-3 exists in limb skeletal
muscles during immobilization-induced atrophy (10, 12).
Therefore, because of limited knowledge regarding the role that
calpain and caspase-3 play in disuse atrophy in limb skeletal
muscles, these experiments were designed to achieve two goals:
1) to determine whether inhibition of calpain or caspase-3 is
sufficient to protect against immobilization-induced limb muscle
atrophy; and 2) to determine whether a regulatory cross talk exists
between calpain and caspase-3 in limb skeletal muscle. We
hypothesized that both calpain and caspase-3 play important roles
in disuse limb muscle atrophy, and that a regulatory cross talk
exists between these two proteases. Our findings support these
hypotheses and demonstrate, for the first time, that both calpain
and caspase-3 play a required role in inactivity-induced atrophy of
limb muscle. Furthermore, our results also reveal that a calpain/
caspase-3 regulatory cross talk exists in limb skeletal muscle
whereby calpain can promote caspase-3 activation, and active
caspase-3 can induce calpain activation in the soleus muscle
during prolonged immobilization.
METHODS
Address for reprint requests and other correspondence: S. K. Powers, Dept.
of Applied Physiology and Kinesiology, Center for Exercise Science, Rm. 3,
PO Box 118206, Univ. of Florida, Gainesville, FL 32611 (e-mail: spowers
@hhp.ufl.edu).
1482
Animals and Experimental Design
Animal groups. Female 300-g Sprague-Dawley rats (Charles River,
Wilmington, MA) were assigned to one of six groups: 1) noncasted
8750-7587/13 Copyright © 2013 the American Physiological Society
http://www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
Talbert EE, Smuder AJ, Min K, Kwon OS, Powers SK. Calpain
and caspase-3 play required roles in immobilization-induced limb
muscle atrophy. J Appl Physiol 114: 1482–1489, 2013. First published
March 7, 2013; doi:10.1152/japplphysiol.00925.2012.—Prolonged
skeletal muscle inactivity results in a rapid decrease in fiber size,
primarily due to accelerated proteolysis. Although several proteases
are known to contribute to disuse muscle atrophy, the ubiquitin
proteasome system is often considered the most important proteolytic
system during many conditions that promote muscle wasting. Emerging evidence suggests that calpain and caspase-3 may also play key
roles in inactivity-induced atrophy of respiratory muscles, but it
remains unknown if these proteases are essential for disuse atrophy in
limb skeletal muscles. Therefore, we tested the hypothesis that activation of both calpain and caspase-3 is required for locomotor muscle
atrophy induced by hindlimb immobilization. Seven days of immobilization (i.e., limb casting) promoted significant atrophy in type I
muscle fibers of the rat soleus muscle. Independent pharmacological
inhibition of calpain or caspase-3 prevented this casting-induced
atrophy. Interestingly, inhibition of calpain activity also prevented
caspase-3 activation, and, conversely, inhibition of caspase-3 prevented calpain activation. These findings indicate that a regulatory
cross talk exists between these proteases and provide the first evidence
that the activation of calpain and caspase-3 is required for inactivityinduced limb muscle atrophy.
Calpain and Caspase-3 Cause Casting-Induced Atrophy
Pharmacological Inhibitors
Calpain inhibition. To inhibit calpain activity in the soleus muscle
during hindlimb immobilization, we administered SJA-6017 (Calpain
inhibitor IV, EMD Chemicals, Gibbstown, NJ) at a dose of 3 mg·kg
body wt⫺1·day⫺1. SJA-6017 was dissolved in 88% propylene, 10%
ethyl alcohol, and 2% benzyl alcohol. Animals were injected subcutaneously immediately before casting and then every 24 h for 7 days.
Caspase-3 inhibition. To inhibit caspase-3 activity during hindlimb
immobilization, we administered the caspase-3 inhibitor Ac-DEVDCHO (Enzo Life Sciences, Farmingdale, NY) at a dose of 3 mg·kg
body wt⫺1·day⫺1. Ac-DEVD-CHO was dissolved in 0.9% sterile
saline. Animals were injected subcutaneously in the neck immediately
before casting and then every 24 h for 7 days.
Specificity of calpain and caspase-3 inhibition. Our laboratory has
previously performed experiments inhibiting calpain activity in skeletal muscle using SJA-6017 and inhibiting caspase-3 with Ac-DEVDCHO. In this regard, these in vitro experiments demonstrate that these
inhibitors target only the intended protease without off-target effects
(17). More specifically, the calpain inhibitor SJA-6017 does not
inhibit the activity of either caspase-3 or caspase-9, a caspase responsible for the activation of caspase-3. Furthermore, these data show that
Ac-DEVD-CHO does not inhibit the activity of calpain-1 or calpain-2
(17).
Antioxidant properties of the inhibitors. Because oxidative stress
can promote disuse atrophy (21, 22), we used a Trolox equivalent
antioxidant capacity assay (23) to determine whether either inhibitor
possessed antioxidant properties. Our results reveal that neither protease inhibitor (e.g., SJA-6017 or Ac-DEVD-CHO) quenched the
oxidants used in the assay (data not shown).
Tissue Harvesting
At the completion of the experimental period, animals were again
anesthetized using isoflurane. Once animals reached a surgical plane
of anesthesia, both solei were removed. One soleus was divided in
half, with one half frozen for cross-sectional area (CSA) analysis and
the other permeabilized to measure mitochondrial function. The remaining soleus was snap frozen in liquid nitrogen and saved for
Western blotting and RT-PCR analysis. Tissue was stored at ⫺80°C
until analysis. After tissue removal, animals were killed by removal of
the heart.
Talbert EE et al.
1483
Biochemical Measures
Preparation of permeabilized muscle fibers. We measured mitochondrial oxygen consumption in permeabilized muscle fibers as
previously described (15). Briefly, ⬃10-mg pieces of soleus muscle
were teased apart on ice in cold buffer X (60 mM K-MES, 35 mM
KCl, 7.23 mM K2EGTA, 2.77 mM CaK2EGTA, 20 mM imidazole,
0.5 mM DTT, 20 mM taurine, 5.7 mM ATP, 15 mM phosphocreatine,
and 6.56 mM MgCl2, pH 7.1). After dissection, soleus fiber bundles
were rotated in buffer X containing 50 ␮g/ml saponin for 30 min at
4°C to permeabilize the fiber membranes. The permeabilized bundles
were washed three times for 5 min by rotation in buffer Z (110 mM
K-MES, 35 mM KCl, 1 mM EGTA, 5 mM K2HPO4, 3 mM MgCl2,
0.05 mM glutamate, 0.02 mM malate, and 0.5 mg/ml BSA, pH 7.1).
Measurement of mitochondrial function. Oxygen consumption by
mitochondria in permeabilized soleus fibers was determined as previously described (15). Mitochondria respired on 5 mM pyruvate and
2 mM malate. State 3 respiration was stimulated by 0.25 mM ADP.
After all ADP was consumed, 10 ␮g/ml oligomycin were added to
stimulate state 4 respiration. The respiratory control ratio (RCR) was
computed as the ratio of state 3 oxygen consumption to state 4 oxygen
consumption.
Myofiber CSA. Pieces of soleus muscle were frozen in optimal
cutting temperature medium in liquid nitrogen-cooled isopentane, as
previously described (14). Soleus sections were cut at 10 ␮m using a
cryotome (Shandon, Pittsburgh, PA) and stained for dystrophin and
myosin heavy chain isoforms, as described previously (14). Fiber
CSA was determined by tracing membrane boundaries using Scion
Image software (Scion, Frederick, MD). At least 100 fibers were
analyzed for each animal.
Western blotting. Soleus muscles were homogenized using a motorized glass-on-glass technique with 1:10 (mg wt/␮l buffer) 5 mM
Tris/5 mM EDTA (pH 7.5) containing protease inhibitor cocktail 1:20
(vol/vol) (Sigma-Aldrich). Following homogenization, samples were
centrifuged at 1,500 g for 10 min at 4°C. Protein concentration in the
supernatant was determined using the method of Bradford (2). Equal
concentrations of protein were mixed 1:1 (vol/vol) with 5% ␤-mercaptoethanol in Laemmli buffer (Bio-Rad, Hercules, CA). Membranes
were probed for cleaved (active) calpain-1, cleaved caspase-3 (Cell
Signaling, Danvers, MA), Bid-to-(Bid) ratio (tBid/Bid) (Imgenex, San
Diego, CA), total calpain, calpastatin, and ␣-II spectrin (Santa Cruz
Biotechnology, Santa Cruz, CA). Images were visualized using the
Odyssey system (Licor Biosciences, Lincoln, NE). To control for
protein loading and transfer differences, each protein was normalized
to the level of ␣-tubulin (Santa Cruz) detected on the same membrane.
Real-time reverse transcriptase polymerase chain reaction. Messenger RNA was isolated, and cDNA synthesized, as previously
described (26). Briefly, TRIzol reagent (Life Technologies, Carlsbad,
CA) was used according to the manufacturer’s directions to isolate
total RNA. RNA content (␮g/muscle) was determined by spectrophotometry. cDNA was produced using the Superscript III First Strand
Synthesis System (Life Technologies). Three micrograms of RNA
were reverse-transcribed using the manufacturer’s protocol.
Taqman chemistry (Applied Biosystems, Foster City, CA) was
used to determine the relative expression of calpastatin. The ABI
Prism 7000 Sequence Detection system (Applied Biosystems) was
used to perform the computed tomography method (Applied Biosystems, user bulletin no. 2, ABI PRISM 7700 Sequence Detection
System). Twenty-five microliters of reactions containing 1 ␮l of
cDNA were used to determine calpastatin expression (Entrez gene ID
25403) and expression of the calibrator sample [housekeeping gene, in
this case ␤-glucuronidase (Entrez gene ID 244434)]. ␤-Glucuronidase, a lysosomal glycoside hydrolase, was chosen as the reference
gene based on previous work showing unchanged expression in
another model of disuse atrophy, mechanical ventilation (4, 5).
Statistical analysis. For CSA, comparisons between groups were
made using two-way ANOVA with Bonferroni posttests used to
J Appl Physiol • doi:10.1152/japplphysiol.00925.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
receiving saline injections (n ⫽ 10); 2) noncasted receiving the
calpain inhibitor SJA-6017 (n ⫽ 6); 3) noncasted receiving the
caspase-3 inhibitor Ac-DEVD-CHO (n ⫽ 10); 4) hindlimbs casted
receiving saline (n ⫽ 8); 5) hindlimbs casted receiving the calpain
inhibitor SJA-6017 (n ⫽ 7); and 6) casted receiving the caspase-3
inhibitor Ac-DEVD-CHO (n ⫽ 8). All experiments were approved by
the University of Florida Institutional Animal Care and Use Committee.
Immobilization. Gaseous isoflurane (2% for induction of anesthesia, 0.5–1.5% to maintain a surgical plane of anesthesia) was used
during casting. After a surgical plane of anesthesia was reached, a
layer of Medipore Dress-it (3M Health Care, St. Paul, MN) was
applied to the entire area to be casted to prevent skin abrasions. A
plaster-of-Paris cast (Gypsona, Reynosa, Tamaulipas, Mexico) was
applied from the last rib down through both legs with the ankle
plantar-flexed so as to induce maximum soleus atrophy. To prevent
casted animals from damaging the cast by chewing, a single layer of
fiberglass (Scotchcast Plus, St. Paul, MN) was applied on top of the
plaster-of-Paris. Animals were allowed to recover from anesthesia and
were monitored carefully to ensure unhindered access to food and
water within their cages. Animals were removed from their cages
daily to check for any damage to the cast or swelling or abrasions to
the feet.
•
1484
Calpain and Caspase-3 Cause Casting-Induced Atrophy
determine differences in muscle fiber size between casted/noncasted
animals for each of the three treatment groups. For the rest of the
analysis, comparisons between groups for each dependent variable were
made by a one-way analysis of variance, and, when appropriate, Tukey’s
honestly significant difference test was performed post hoc. Significance
was established at P ⬍ 0.05. Data are presented as means ⫾ SE.
RESULTS
Independent Inhibition of Calpain and Caspase-3 Prevents
Casting-induced Muscle Atrophy
These experiments used a rat model of hindlimb immobilization to mimic the atrophy that occurs in humans during the
casting of a limb. Analysis of the CSA of type I soleus fibers
by two-way ANOVA revealed that an interaction effect exists
•
Talbert EE et al.
between our casted/noncasted and drug treatment variables
following 7 days of treatment. A Bonferroni posttest concluded
that casting only had a significant effect in the saline-treated
group (Fig. 1A). In contrast, casting did not induce significant
changes in the CSA of animals treated with either our calpain
inhibitor (SJA-6017) or our caspase-3 inhibitor (Ac-DEVDCHO), demonstrating that both calpain and caspase-3 play
important roles in inactivity-induced muscle atrophy. An experimental concern with pharmacological inhibition of proteases is that prolonged protease inhibition will induce muscle
hypertrophy, potentially masking atrophy. To determine
whether our protease inhibitors promoted muscle hypertrophy,
we treated “control” ambulatory animals with either the calpain
or caspase-3 inhibitor for 7 days.
J Appl Physiol • doi:10.1152/japplphysiol.00925.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
Fig. 1. Soleus muscle fiber cross-sectional area (CSA) by
fiber type. A: quantification of type I myofibers. Con,
noncasted receiving saline injections; Con-Calp, noncasted
receiving the calpain inhibitor SJA-6017; Con-Cas-3, noncasted receiving the caspase-3 inhibitor Ac-DEVD-CHO;
Cast, hindlimbs casted receiving saline; Cast-Calp,
hindlimbs casted receiving the calpain inhibitor SJA-6017;
Cast-Cas-3, casted receiving the caspase-3 inhibitor AcDEVD-CHO. Values are means ⫾ SE. †Significantly
different (P ⬍ 0.05). B: quantification of type IIa myofibers. Values are means ⫾ SE. C: representative staining of
myosin heavy chain (MHC) I (blue), MHC IIa (green), and
dystrophin (red) proteins in a control cross section.
D: representative staining of Con-Calp. E: representative
staining of Con-Cas-3. F: representative staining of Cast.
G: representative staining of Cast-Calp. H: representative
staining of Cast-Cas-3. Scale bars ⫽ 100 ␮m.
Calpain and Caspase-3 Cause Casting-Induced Atrophy
•
Talbert EE et al.
1485
calpain activation in the soleus muscle was successful. Interestingly, independent inhibition of caspase-3 also prevented
activation of calpain in the immobilized soleus muscle. The
prevention of calpain activation by inhibition of caspase-3
demonstrates that caspase-3 plays a regulatory role in the
activation of calpain.
Casting Activates Calpain in the Soleus During 7 Days of
Immobilization
Casting Activates Caspase-3 in the Soleus During 7 Days of
Immobilization
Casting significantly increased the presence of the cleaved
(active, 76 kDa) band of calpain-1 following 7 days of cast
immobilization (Fig. 2A). Casting also significantly increased
the presence of a calpain-specific cleavage product of ␣-IIspectrin that appears at 145 kDa on a Western blot of ␣-IIspectrin (Fig. 2B). This calpain-specific cleavage fragment of
␣-II-spectrin is an index of in vivo calpain activity (30). Daily
treatment with the calpain inhibitor SJA-6017 prevented the
activation of calpain-1 and the increase in the calpain-specific
␣-II-spectrin fragment, demonstrating that our inhibition of
Seven days of cast immobilization also significantly activated caspase-3 in the soleus muscle, demonstrated by an
increase in cleaved (active) caspase-3 (Fig. 2C). Casting also
significantly increased the caspase-3-specific breakdown product of ␣-II-spectrin, a fragment of ␣-II-spectrin that can be
detected at 120 kDa on a Western blot (Fig. 2D). Importantly,
treatment of animals with the caspase-3 inhibitor Ac-DEVDCHO blunted the immobilization-induced activation of
caspase-3 and completely prevented the increase in the 120kDa product of ␣-II-spectrin in the soleus muscle. Calpain
Fig. 2. Activation and activity of the proteases calpain-1 (A and B) and caspase-3 (C
and D) were determined via Western blotting. A: active calpain-1. B: specific ␣-IIspectrin cleavage product of calpain (145
kDa). C: active caspase-3. D: specific ␣-IIspectrin cleavage product of caspase-3 (120
kDa). Representative Western blots are
shown below the graphs. Note that active
calpain-1 is the smaller (76 kDa), cleaved
band underneath the larger (80 kDa) band in
A. Values are presented as fold control of
mean arbitrary units ⫾ SE. Blots were normalized to ␣-tubulin, which is shown below
the representative blot. *Significantly different vs. all other groups (P ⬍ 0.05). †Significantly different vs. Con (P ⬍ 0.05).
J Appl Physiol • doi:10.1152/japplphysiol.00925.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
We found neither an interaction effect nor main effects when
analyzing our type IIA CSAs (Fig. 1B), demonstrating that no
significant changes occurred in type IIA soleus fibers in response to either daily treatment with either of our protease
inhibitors, or casting. Representative muscle cross sections of
all experimental groups appear in Fig. 1, C–H.
1486
Calpain and Caspase-3 Cause Casting-Induced Atrophy
inhibition also prevented the full activation of caspase-3 and
completely prevented the increase in the caspase-3-specific
␣-II-spectrin fragment, demonstrating that calpain plays a
regulatory role in the activation of caspase-3. Together, these
•
Talbert EE et al.
results indicate that a regulatory cross talk exists between
calpain and caspase-3 in the soleus muscle during immobilization-induced fiber atrophy.
Potential Mechanism(s) Responsible for Regulatory Cross
Talk Between Calpain and Caspase-3
Inhibition of Both Calpain and Caspase-3 Protect Against
Immobilization-induced Mitochondrial Dysfunction
No significant differences in oxygen consumption between groups were found during active state 3 respiration or
Fig. 3. Truncated Bid (tBid; A), Bid (B), and the tBid-to-Bid ratio (C). The top
band represents Bid in the representative blot, while tBid is the bottom
fragment. Note that Bid and tBid were measured on the same blot, but were
analyzed under different exposures, as shown here. Values are mean arbitrary
units ⫾ SE. *Significantly different from all other groups (P ⬍ 0.05).
†Significantly different vs. Con (P ⬍ 0.05). ‡Significantly different vs. Con
and Cast (P ⬍ 0.05).
J Appl Physiol • doi:10.1152/japplphysiol.00925.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
Following the observation that a regulatory cross talk exists
between calpain and caspase-3, we sought to determine the
mechanism by which active calpain promotes caspase-3 activation. Evidence in nonmuscle cells indicates that calpain can cleave
Bid, a proapoptotic protein, to tBid. tBid then translocates to the
mitochondria, leading to the release of cytochrome c followed by
caspase-3 activation (3). Similar results have been reported in
diaphragm muscle during prolonged mechanical ventilation (17).
Our results reveal that immobilization of the soleus muscle results
in an increase in both tBid and tBid/Bid (Fig. 3). The change in
tBid/Bid was driven by an increase in tBid, as all three casted
groups demonstrated no differences in the abundance of Bid (Fig.
3B). Importantly, this inactivity-induced increase in tBid/Bid was
blunted by inhibition of calpain activity in the soleus muscle (Fig.
3C). Although these observations do not provide definitive proof,
our results are consistent with the notion that the calpain-mediated
activation of caspase-3 can occur, in part, by the formation of the
proapoptotic protein tBid. This is a testable hypothesis worthy of
further research.
We then sought to determine the mechanism by which active
caspase-3 promotes the activation of calpain. Calpastatin, the
endogenous inhibitor of calpain, is a known substrate of
caspase-3. Active caspase-3 may degrade calpastatin, removing
calpastatin’s inhibition of calpain (31). Our results reveal that
calpastatin levels decreased in the soleus muscle during prolonged
immobilization (Fig. 4A). The decline in calpastatin levels decreased the calpastatin-to-calpain ratio (calpastatin/calpain),
which is an index of the ability of calpastatin to inhibit calpain.
Inhibition of caspase-3 activity led to calpastatin levels statistically similar to those of both control and casted animals (Fig. 4A).
Total calpain expression was not different between any of the
groups (Fig. 4B). Similar to calpastatin expression, calpastatin/
calpain in the soleus muscle of casted animals treated with our
caspase-3 inhibitor was similar to that in both control and casted
animals (Fig. 4C). Hence, our results do not provide firm evidence
that caspase-3 regulates calpain activity by degrading calpastatin
in limb muscles during inactivity. To ensure that casting simply
was not increasing the turnover of calpastatin, we measured the
mRNA expression of calpastatin. Calpastatin mRNA was decreased by casting and not rescued by either protease inhibitor
(Fig. 4D).
Calpain and Caspase-3 Cause Casting-Induced Atrophy
•
Talbert EE et al.
1487
basal state 4 respiration. However, hindlimb casting resulted
in a ⬃50% decrease in the RCR of mitochondria in permeabilized fibers. Independent treatment of animals with a
calpain or caspase-3 inhibitor partially prevented this decrease in mitochondrial coupling, with calpain inhibition
providing a strong trend toward complete protection (P ⫽
0.056) (Table 1).
Table 1. Mitochondrial function
Parameter
Con
Cast
Cast-Calp
Cast-Cas-3
State 3 V̇O2
State 4 V̇O2
RCR
5.40 ⫾ 1.43
1.24 ⫾ 0.30
4.45 ⫾ 1.18
3.73 ⫾ 1.09
1.88 ⫾ 0.52
2.17 ⫾ 1.05*
4.88 ⫾ 1.09
1.52 ⫾ 0.63
4.07 ⫾ 1.90†
5.10 ⫾ 1.26
1.53 ⫾ 0.36
3.385 ⫾ 0.68
Values are means ⫾ SD. Con, noncasted receiving saline injections; Cast,
hindlimbs casted receiving saline; Cast-Calp, hindlimbs casted receiving the
calpain inhibitor SJA-6017; Cast-Cas-3, casted receiving the caspase-3 inhibitor Ac-DEVD-CHO. Oxygen consumption (V̇O2) from permeabilized soleus
fibers was determined during both state 3 (ADP-stimulated) and state 4
(following ADP stimulation). The respiratory control ratio (RCR) is the ratio
of these two values and is a measure of mitochondrial coupling. *Significantly
different vs. Con (P ⬍ 0.05). †Note that a trend toward significant differences
exists between Cast and Cast-Calp, P ⫽ 0.056.
DISCUSSION
Overview of Major Findings
This is the first investigation to demonstrate that independent
inhibition of calpain or caspase-3 in locomotor skeletal muscle is
sufficient to prevent immobilization-induced type I fiber atrophy.
Furthermore, our findings also reveal that a regulatory cross talk
exists in limb skeletal muscle, whereby calpain and caspase-3 can
activate one another. Finally, these results also provide new
information about the impact of calpain and caspase-3 activation
on mitochondrial function in locomotor skeletal muscle.
Independent Inhibition of Calpain or Caspase-3 Activity
Prevents Immobilization-induced Soleus Muscle Atrophy
Similar to previous reports in diaphragm muscle (13, 14,
17), the present study reveals that independent pharmacological inhibition of calpain or caspase-3 is sufficient to prevent
disuse muscle atrophy. However, note that our results indicate
that 7 days of limb immobilization promoted atrophy in type I
fibers only, as this duration of muscle disuse was not sufficient
to achieve significant atrophy in the type IIa fibers within the
soleus muscle. Nonetheless, in the adult Sprague-Dawley rat,
⬃85% of the soleus fibers are type I (8), and, therefore, our
J Appl Physiol • doi:10.1152/japplphysiol.00925.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
Fig. 4. Calpastatin (A), total calpain (B),
calpastatin-to-calpain ratio (C), and calpastatin mRNA (D). Representative images are
shown in the middle. Values are mean arbitrary units ⫾ SEM. †Significantly different
vs. Con (P ⬍ 0.05).
1488
Calpain and Caspase-3 Cause Casting-Induced Atrophy
Regulatory Cross Talk Exists Between Calpain
and Caspase-3
Our data provide the first evidence that a regulatory cross
talk exists between calpain and caspase-3, as inhibition of
calpain is sufficient to prevent activation of caspase-3 in limb
skeletal muscle. Similarly, inhibition of caspase-3 prevented
calpain activation in the inactive soleus muscle. To investigate
the mechanism responsible for these findings, we tested the
hypothesis that calpain can potentially activate caspase-3
through cleavage of Bid to form tBid. tBid is a membrane
Talbert EE et al.
pore-forming protein that can release cytochrome c from the
mitochondria, resulting in the subsequent activation of
caspase-3 (3). Our results reveal that inhibition of calpain
activity results in a significant attenuation of the immobilization-induced increase in tBid/Bid in the soleus muscle (Fig.
3C). Thus it is feasible that calpain activates caspase-3 in
skeletal muscle disuse atrophy via a tBid-dependent mechanism. Nonetheless, this evidence is not definitive, as active
caspase-3 can also lead to Bid cleavage, leading to a feedforward activation of caspase-3 (25). Therefore, additional
work will be required to determine the specific mechanism(s)
by which active calpain promotes caspase-3 activation.
To investigate the mechanism responsible for the caspase3-mediated activation of calpain, we measured the protein
abundance of both calpain and the endogenous calpain inhibitor, calpastatin. In this regard, our laboratory’s prior work
revealed that caspase-3 inhibition prevented the decrease in
calpastatin and calpastatin/calpain associated with mechanical
ventilation induced diaphragm atrophy (17). The present experiments also demonstrate that caspase-3 inhibition blunts the
casting-induced decrease in calpastatin levels in the soleus
muscle (Fig. 4A), and either calpain inhibition or caspase-3
inhibition is sufficient to provide very modest protection
against immobilization-induced decreases in calpastatin/calpain (Fig. 4C). Nonetheless, because the inhibition of
caspase-3 does not completely prevent the immobilizationinduced decrease in both calpastatin and calpastatin/calpain in
the soleus muscle, it is possible that caspase-3-mediated activation of calpain may involve other mechanisms in skeletal
muscle exposed to long periods of inactivity. An important
difference between the present study and our laboratory’s
previous work (17) in diaphragm muscle is the duration of
muscle inactivity. Indeed, Nelson et al. (17) investigated mechanical ventilation-induced diaphragm atrophy following
only 12 h of inactivity, while the present study investigated
limb muscle atrophy following 7 days of immobilization. Thus
caspase-3 inhibition may be sufficient to prevent calpastatin
degradation in skeletal muscle in the short term, but this may
not be an important protective mechanism over longer time
periods. Supporting this prediction is the observation that a
decrease in calpastatin gene expression following 7 days of
casting was not rescued by either calpain or caspase-3 inhibition (Fig. 4D).
Protection of Mitochondrial Function by Protease Inhibition
Prolonged muscle inactivity is associated with mitochondrial dysfunction in both limb and diaphragm muscle (11, 15,
20). Consistent with previous findings, the present investigation shows that 7 days of immobilization induced a significant
decrease in the RCR of mitochondria in permeabilized soleus
fibers (Table 1). RCR is an index of mitochondrial coupling,
and a decrease in RCR indicates that mitochondria in the casted
muscle are less coupled than mitochondria within the soleus
muscles of weight-bearing animals. Interestingly, caspase-3
inhibition prevented a significant decline in mitochondrial
function, and inhibition of calpain activity in the soleus muscle
almost completely rescued the immobilization-induced decline
in the mitochondrial RCR. Although our experiments do not
provide a mechanism to explain this observation, one potential
explanation is that calpain-10, a mitochondrial calpain linked
J Appl Physiol • doi:10.1152/japplphysiol.00925.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
observed protection against disuse fiber atrophy is physiologically important.
Differences exist in the level of protection against disuse
muscle atrophy between the present study, which used a highly
selective pharmacological inhibitor of calpain, and previous
reports using the overexpression of calpastatin to retard calpain
activity. The two published reports using calpastatin overexpression to prevent calpain activation reported only partial (29)
or minimal (24) protection against hindlimb suspension-induced muscle atrophy. The explanation for these divergent
findings is unclear but could be due to several factors. First, the
duration of muscle inactivity and the experimental model of
muscle disuse differed between the present study and these
earlier studies. Indeed, the present study used 7 days of
immobilization to induce muscle atrophy, whereas 10 –14 days
of hindlimb suspension were utilized in both of the calpastatin
overexpression studies. These studies using hindlimb suspension saw greater atrophy than our casting model, which may
play a role in the level or type of protease activation. Second,
neither of the calpastatin overexpression investigations evaluated muscle atrophy by assessing fiber-type-specific CSAs,
which may account for some of the differences between the
present study and these previous reports. Additionally, the
present study utilized a rat experimental model, while both
calpastatin studies used a murine model to investigate disuse
muscle atrophy. Finally, the study of Tidball and Spencer (29)
acknowledges that they did not achieve complete inhibition of
calpain activity in skeletal muscle, whereas our data (Fig. 2B,
␣-II-spectrin levels) indicate that the calpain activity in the
soleus muscle of our casted animals receiving the calpain
inhibitor did not differ from the calpain activity in the soleus
muscle of an ambulatory control animal.
Importantly, our results also reveal that pharmacological
inhibition of caspase-3 provides protection against immobilization-induced muscle atrophy in type I fibers of the soleus
muscle. Similar results have been reported in ventilator-induced atrophy of diaphragm muscle (14, 17). Moreover, a
recent report also concluded that denervation-induced gastrocnemius muscle atrophy is diminished in caspase-3 knockout
mice (18). However, the present experiment reveals that
caspase-3 inhibition results in complete protection against
disuse-induced soleus muscle atrophy, whereas Plant et al. (18)
report only a modest reduction in gastrocnemius muscle atrophy in the wild-type vs. the caspase-3 knockout mice. The
explanation for these divergent findings is unclear, but species
differences, duration of experiment, fiber-type differences between gastrocnemius and soleus muscles, and/or differences in
atrophy model (i.e., denervation vs. immobilization) could be
responsible for these disparities.
•
Calpain and Caspase-3 Cause Casting-Induced Atrophy
to mitochondrial dysfunction (1), might be inhibited by our
calpain inhibitor. If this is the case, prevention of calpain-10
activation could protect mitochondrial electron transport chain
proteins from degradation and thus preserve mitochondrial
function. This is another testable hypothesis worthy of future
experimentation.
11.
12.
Conclusions
13.
14.
15.
16.
17.
18.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
19.
AUTHOR CONTRIBUTIONS
Author contributions: E.E.T. and S.K.P. conception and design of research;
E.E.T., A.J.S., K.M., and O.-S.K. performed experiments; E.E.T. analyzed
data; E.E.T., A.J.S., and S.K.P. interpreted results of experiments; E.E.T.
prepared figures; E.E.T. drafted manuscript; E.E.T., A.J.S., and S.K.P. edited
and revised manuscript; E.E.T., A.J.S., K.M., O.-S.K., and S.K.P. approved
final version of manuscript.
20.
REFERENCES
22.
1. Arrington DD, Van Vleet TR, Schnellmann RG. Calpain 10: a mitochondrial calpain and its role in calcium-induced mitochondrial dysfunction. Am J Physiol Cell Physiol 291: C1159 –C1171, 2006.
2. Bradford MM. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye
binding. Anal Biochem 72: 248 –254, 1976.
3. Chen M, He H, Zhan S, Krajewski S, Reed JC, Gottlieb RA. Bid is
cleaved by calpain to an active fragment in vitro and during myocardial
ischemia/reperfusion. J Biol Chem 276: 30724 –30728, 2001.
4. Deruisseau KC, Kavazis AN, Powers SK. Selective downregulation of
ubiquitin conjugation cascade mRNA occurs in the senescent rat soleus
muscle. Exp Gerontol 40: 526 –531, 2005.
5. DeRuisseau KC, Shanely RA, Akunuri N, Hamilton MT, Van Gammeren D, Zergeroglu AM, McKenzie M, Powers SK. Diaphragm
unloading via controlled mechanical ventilation alters the gene expression
profile. Am J Respir Crit Care Med 172: 1267–1275, 2005.
6. Du J, Wang X, Miereles C, Bailey JL, Debigare R, Zheng B, Price SR,
Mitch WE. Activation of caspase-3 is an initial step triggering accelerated
muscle proteolysis in catabolic conditions. J Clin Invest 113: 115–123,
2004.
7. Hasselgren PO, Fischer JE. The ubiquitin-proteasome pathway: review
of a novel intracellular mechanism of muscle protein breakdown during
sepsis and other catabolic conditions. Ann Surg 225: 307–316, 1997.
8. Hirofuji C, Ishihara A, Itoh K, Itoh M, Taguchi S, Takeuchi-Hayashi
H. Fibre type composition of the soleus muscle in hypoxia-acclimatised
rats. J Anat 181: 327–333, 1992.
9. Huang J, Forsberg NE. Role of calpain in skeletal-muscle protein
degradation. Proc Natl Acad Sci U S A 95: 12100 –12105, 1998.
10. Hudson MB, Smuder AJ, Nelson WB, Bruells CS, Levine S, Powers
SK. Both high level pressure support ventilation and controlled mechan-
21.
23.
24.
25.
26.
27.
28.
29.
30.
31.
Talbert EE et al.
1489
ical ventilation induce diaphragm dysfunction and atrophy. Crit Care Med
40: 1254 –1260, 2012.
Kavazis AN, Talbert EE, Smuder AJ, Hudson MB, Nelson WB,
Powers SK. Mechanical ventilation induces diaphragmatic mitochondrial
dysfunction and increased oxidant production. Free Radic Biol Med 46:
842–850, 2009.
Levine S, Nguyen T, Taylor N, Friscia ME, Budak MT, Rothenberg P,
Zhu J, Sachdeva R, Sonnad S, Kaiser LR, Rubinstein NA, Powers SK,
Shrager JB. Rapid disuse atrophy of diaphragm fibers in mechanically
ventilated humans. N Engl J Med 358: 1327–1335, 2008.
Maes K, Testelmans D, Powers S, Decramer M, Gayan-Ramirez G.
Leupeptin inhibits ventilator-induced diaphragm dysfunction in rats. Am J
Respir Crit Care Med 175: 1134 –1138, 2007.
McClung J, Kavazis A, DeRuisseau K, Falk D, Deering M, Lee Y,
Sugiura T, Powers S. Caspase-3 regulation of diaphragm myonuclear
domain during mechanical ventilation-induced atrophy. Am J Respir Crit
Care Med 175: 150 –159, 2007.
Min K, Smuder AJ, Kwon OS, Kavazis AN, Szeto HH, Powers SK.
Mitochondrial-targeted antioxidants protect skeletal muscle against immobilization-induced muscle atrophy. J Appl Physiol 111: 1459 –1466, 2011.
Mitch WE, Goldberg AL. Mechanisms of muscle wasting. The role of
the ubiquitin-proteasome pathway. N Engl J Med 335: 1897–1905, 1996.
Nelson WB, Smuder AJ, Hudson MB, Talbert EE, Powers SK. Crosstalk between the calpain and caspase-3 proteolytic systems in the diaphragm during prolonged mechanical ventilation. Crit Care Med 40:
1857–1863, 2012.
Plant PJ, Bain JR, Correa JE, Woo M, Batt J. Absence of caspase-3
protects against denervation-induced skeletal muscle atrophy. J Appl
Physiol 107: 224 –234, 2009.
Powers S, Kavazis A, McClung J. Oxidative stress and disuse muscle
atrophy. J Appl Physiol 102: 2389 –2397, 2007.
Powers SK, Hudson MB, Nelson WB, Talbert EE, Min K, Szeto HH,
Kavazis AN, Smuder AJ. Mitochondria-targeted antioxidants protect
against mechanical ventilation-induced diaphragm weakness. Crit Care
Med 39: 1749 –1759, 2011.
Powers SK, Smuder AJ, Criswell DS. Mechanistic links between oxidative stress and disuse muscle atrophy. Antioxid Redox Signal 15:
2519 –2528, 2011.
Powers SK, Smuder AJ, Judge AR. Oxidative stress and disuse muscle
atrophy: cause or consequence? Curr Opin Clin Nutr Metab Care 15:
240 –245, 2012.
Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C.
Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med 26: 1231–1237, 1999.
Salazar JJ, Michele DE, Brooks SV. Inhibition of calpain prevents
muscle weakness and disruption of sarcomere structure during hindlimb
suspension. J Appl Physiol 108: 120 –127, 2010.
Slee EA, Keogh SA, Martin SJ. Cleavage of BID during cytotoxic drug
and UV radiation-induced apoptosis occurs downstream of the point of
Bcl-2 action and is catalysed by caspase-3: a potential feedback loop for
amplification of apoptosis-associated mitochondrial cytochrome c release.
Cell Death Differ 7: 556 –565, 2000.
Smuder AJ, Hudson MB, Nelson WB, Kavazis AN, Powers SK.
Nuclear factor-␬B signaling contributes to mechanical ventilation-induced
diaphragm weakness. Crit Care Med 40: 927–934, 2012.
Solomon V, Goldberg AL. Importance of the ATP-ubiquitin-proteasome
pathway in the degradation of soluble and myofibrillar proteins in rabbit
muscle extracts. J Biol Chem 271: 26690 –26697, 1996.
Thomason DB, Biggs RB, Booth FW. Protein metabolism and betamyosin heavy-chain mRNA in unweighted soleus muscle. Am J Physiol
Regul Integr Comp Physiol 257: R300 –R305, 1989.
Tidball JG, Spencer MJ. Expression of a calpastatin transgene slows
muscle wasting and obviates changes in myosin isoform expression during
murine muscle disuse. J Physiol 545: 819 –828, 2002.
Wang KK. Calpain and caspase: can you tell the difference? Trends
Neurosci 23: 20 –26, 2000.
Wang KK, Posmantur R, Nadimpalli R, Nath R, Mohan P, Nixon RA,
Talanian RV, Keegan M, Herzog L, Allen H. Caspase-mediated fragmentation of calpain inhibitor protein calpastatin during apoptosis. Arch
Biochem Biophys 356: 187–196, 1998.
J Appl Physiol • doi:10.1152/japplphysiol.00925.2012 • www.jappl.org
Downloaded from http://jap.physiology.org/ by 10.220.33.4 on July 12, 2017
In conclusion, this study revealed that inhibition of either
calpain or caspase-3 is sufficient to prevent casting-induced
atrophy of the soleus, providing evidence that both calpain and
caspase-3 are important proteases in disuse limb muscle atrophy. Furthermore, we have demonstrated for the first time in
limb muscle that a regulatory cross talk exists between calpain
and caspase-3, whereby calpain can stimulate caspase-3 activation, and active caspase-3 can promote calpain activation.
Finally, our results reveal that active calpain and, to a lesser
extent, active caspase-3 can promote mitochondrial uncoupling
in skeletal muscle. The mechanism(s) responsible for this
finding is unknown and warrants further research.
•