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Contents lists available at ScienceDirect

Comparative Biochemistry and Physiology, Part A

j ourna l homepage: www.e lsev ie r .com/ locate /cbpa

Changes in calpains and calpastatin in the soleus muscle of Daurianground squirrels during hibernation

OFChen-Xi Yang a,c,1, Yue He a,1, Yun-Fang Gao a,⁎, Hui-Ping Wang a, Nandu Goswami b

a Key Laboratory of Resource Biology and Biotechnology in Western China, Northwest University, Ministry of Education, Xi'an 71069, Chinab Institute of Physiology, Center of Physiological Medicine, Medical University Graz, Austriac College of Life Science and Technology, Longdong University, Qingyang, Gansu, 745000, China

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⁎ Corresponding author at: College of Life Sciences, NorChina. Tel.: +86 29 88303935.

E-mail address: gaoyunf@nwu.edu.cn (Y.-F. Gao).1 These authors contributed equally to the study.

http://dx.doi.org/10.1016/j.cbpa.2014.05.0221095-6433/© 2014 Published by Elsevier Inc.

Please cite this article as: Yang, C.-X., et alhibernation, Comp. Biochem. Physiol., A (20

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Article history:Received 12 February 2014Received in revised form 3 May 2014Accepted 27 May 2014Available online xxxx

Keywords:Hindlimb suspensionDisuse atrophyReloadingForced exerciseUltrastructuralZ-disk

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RWe investigated changes in muscle mass, calpains, calpastatin and Z-disk ultrastructure in the soleus muscle(SOL) ofDaurian ground squirrels (Spermophilus dauricus) after hibernation or hindlimb suspension to determinepossiblemechanisms bywhichmuscle atrophy is prevented in hibernators. Squirrels (n= 30)were divided intofive groups: no hibernation (PRE, n=6); hindlimb suspension (HLS, n=6); twomonths hibernation (HIB, n=6);two days after 90±12days of hibernation (POST, n=6); and forced exercise (one time forced,moderate-intensitytreadmill exercise) after arousal (FE, n= 6). Activity and protein expression of calpains were determined by caseinzymography and western blotting, and Z-disk ultrastructure was observed by transmission electron microscopy.The following results were found. Lower body mass and higher SOL muscle mass (mg) to total body mass (g)ratio were observed in HIB and POST; calpain-1 activity increased significantly by 176% (P = 0.034) in HLScompared to the PRE group; no significant changes were observed in calpain-2 activity. Protein expression ofcalpain-1 and calpain-2 increased by 83% (P = 0.041) and 208% (P = 0.029) in HLS compared to the PRE group,respectively; calpastatin expression increased significantly by 180% (P b 0.001) and 153% (P= 0.007) in HIB andPOST, respectively; the myofilaments were well-organized, and the width of the sarcomere and the Z-disk bothappeared visually similar among the pre-hibernation, hibernating and post-hibernation animals. Inhibition ofcalpain activity and consequently calpain-mediated protein degradation by highly elevated calpastatin proteinexpression levels may be an important mechanism for preventing muscle protein loss during hibernation andensuring that Z-lines remained ultrastructurally intact.

© 2014 Published by Elsevier Inc.

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R1. Introduction

Mammalian skeletal muscles exhibit significant loss in contractileproteins during prolonged unloading or disuse (e.g. hindlimb suspen-sion, bed rest and spaceflight), which possibly result from an early de-crease in protein synthesis and a late increase in protein degradation(Booth and Seider, 1979; Thomason et al., 1989; Fitts et al., 2001;Haddad et al., 2003; Trappe et al., 2009). Increased protein catabolismhas been identified as a major step in muscle atrophy (Thomasonet al., 1987). Although there are at least four proteolytic pathways(calpain, ubiquitin–proteasome, lysosomal, and caspase) involved inthe protein degradation of muscle during disuse atrophy (Jackmanand Kandarian, 2004), calpain and ubiquitin–proteasome appear to bethemost important. The ubiquitin–proteasome system is themajor pro-teolytic machinery involved in protein breakdown, and calpains play an

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initiating role in the process (Kitagaki et al., 2000; Costelli et al., 2005).Calpains tend to be concentrated in the Z-disk where disassemblybegins (Bird et al., 1980) and are activated by calcium, with calciumtreatment of purified myofibrils causing rapid and complete loss of theZ-disk (Busch et al., 1972). Calpains are initiators of myofibrillar proteindegradation as they aid in the release of molecules suitable for furtherdegradation by proteasomes (Jackman and Kandarian, 2004). Calpainsinitiate the digestion of individual myofibrillar proteins, includingdesmin, filament, C-protein, tropomyosin, troponin T, troponin I, titin,nebulin, vimentin, gelsolin, and vinculin (Goll et al., 2003). Therefore,changes in calpains are important indicators for the study of proteinmetabolism in disuse muscle atrophy.

The calpain system is mainly comprised of three molecules:two Ca2+-dependent proteases, calpain-1 (mu-calpain) and calpain-2(m-calpain) (Goll et al., 2003), and a widely distributed polypeptide,calpain-specific inhibitor (calpastatin), which has four internallyrepetitive inhibitory domains. A single domain or even a truncated27-mer fragment thereof, possesses inhibitory activity against calpains(Murachi, 1990). Calpain-1 and calpain-2 become proteolytically activein the presence of 2–200 microM Ca2+ (Goll et al., 2003).

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Daurian ground squirrels (Spermophilus dauricus) are rodents of thefamily Sciuridae. Our previous research indicated that Daurian groundsquirrels exhibit onlymarginal loss in extensor digitorum longusmusclemass, increased wet muscle/total body mass ratio and unchanged fibercross-sectional area over their three to fourmonths of winter dormancy(Gao et al., 2012). Hibernators of varying sizes, torpor depths, and lifehistories (from bats to bears to rodents) may have different mecha-nisms for emerging from prolonged inactivity with muscle mass andcontractile proteins and function remarkably intact (Tinker et al.,1998; Rourke et al., 2006; Lee et al., 2008; Nowell et al., 2011). The resis-tive properties of the torpid bat (Murina leucogaster ognevi) muscleagainst atrophy might be attained by relatively constant proteolysis incombination with oscillatory anabolic activity (e.g., p-mTOR) and up-regulation or at least balanced regulation of the chaperones (HSPs)(Lee et al., 2008, 2010); transcription factor FOXO1 and MAFbx mRNAare down-regulated in ground squirrels (Callospermophilus lateralis),whichmay contribute to reduced atrophy (Nowell et al., 2011). Despiteresearch on the molecular mechanisms of anti-disuse muscle atrophyin hibernators, themolecularmechanisms in this naturalmuscle systemof hibernators have not been clarified. We investigated changes incalpains, calpastatin and Z-disk ultrastructure in the SOL muscle ofDaurian ground squirrels to determine the possible mechanisms bywhich muscle atrophy in hibernators is prevented.

Muscle atrophy does not appear to continue linearly throughouthibernation, with hibernators exhibiting different phases of atrophy.We therefore examined changes in the calpain system in three phasesof hibernation (pre-hibernation, hibernation for two months and twodays after arousal). In addition, we examined changes in two othergroups (two week hindlimb suspension group as a disuse atrophymodel and forced exercise after arousal group as a reloading subsequentto muscle disuse model).

2. Materials and methods

2.1. Animals and groups

Thirty Daurian ground squirrels of both sexes were obtained fromthe Weinan region in the Shaanxi province of China. The LaboratoryAnimal Care Committee of the China Ministry of Health approved allprocedures, animal care and handling.

The animals were sorted by sex into plastic cages (0.35 × 0.3 ×0.45 m3), with 3 to 4 animals per cage. Specifically, the animalswere kept in the laboratory for three to four months after collection foracclimation purposes, before they were split into groups. The animalcolony room was maintained at a temperature range of 18–20 °C, andlighting was changed daily to coincide with local sunrise and sunset.Animals were given wood chips. Squirrels were provided with waterand rodent food blocks, and supplemented with fresh fruit and vegeta-bles. In November, three groups were placed in a cold room hibernacu-lum at (4–6 °C) (2L:22D dark). Hibernators were provided with foodand water. The dates of entering torpor were determined by puttingsawdust on the back of each animal and by them reaching a body tem-perature below 9 °C. Body temperature was determined by thermalimaging using a visual thermometer (Fluke VT04Visual IR Thermometer,USA). Based on our year-by-year records, most animals return to hiber-nation after 1 to 2 days of inter-bout arousals. Animals that arousedstayed awake for more than two days and were assigned to POST. Dailyobservations were made during the experimental period.

Animals were matched for body mass and were randomly assignedto five groups (n = 6): PRE: Control (no hibernation) animals investi-gated in late-autumn; HLS: no hibernation but with two weeks ofhindlimb suspension in autumn; HIB: Animals after two months ofhibernation; POST: Animals two days after arousal from 90 ± 12 daysof hibernation; and FE: Animals two days after arousal from 90 ±12 days of hibernation that experienced forced exercise (one timeforced, moderate-intensity treadmill exercise: 10 m/min, 0% grade,

Please cite this article as: Yang, C.-X., et al., Changes in calpains and chibernation, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016

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45 min) using a programmed motor-driven wheel cage (Chae andKim, 2009), with the SOL muscle collected 24 h after exercise. The SOLmusclewas in a disuse state in the hibernation and hindlimb suspensiongroups, but was not in a disuse state in the pre-hibernation, post-hibernation and forced exercise after arousal groups.

2.2. Muscle collection

Animals were anesthetizedwith 90 mg/kg sodium pentobarbital i.p.Hindlimb SOLmuscleswere excised from both legs; bodymass andwetmass of SOL were recorded, with the muscles then fixed in glutaralde-hyde for Z-disk ultrastructure examination using a transmission elec-tron microscope. The remaining samples were subsequently stored at−80 °C until further processing. At the end of surgical intervention,the animals were sacrificed by an overdose injection of sodium pento-barbital. The appropriate ethics committee (Northwest UniversityEthics Committee) reviewed and approved all procedures in the animalstudies.

2.3. Transmission electron microscopy (TEM)

Both longitudinally and transversely orientated myofiber sections(1 mm thick) were stained with toluidine blue, and areas of interestwere chosen for ultrastructural examination by light microscopy.Using diamond knives, 60 nm thick sections were cut, then collectedon uncoated 200 mesh copper grids, and double stained with bothReynolds lead citrate (Reynolds, 1963) and ethanolic uranyl acetate.The stained sections were then observed by TEM (JEM-100SX, Japan).

2.4. Calpain activity assay

The non-Ca2+-dependent component of calpain activity wasmeasured by casein zymography (Raser et al., 1995), using 0.2% alkali-denatured casein (Sigma-Aldrich) co-polymerized in 10% mini-gels(0.75 mm thick). Gels were pre-run for 15 min at 4 °C usingzymography running buffer (25 mM Tris (pH 8.3), 192 mM glycine,1 mM EGTA, and 1 mM dithiothreitol (DTT)). Each sample and purifiedcalpain-1 or calpain-2 were subsequently loaded and run. Gels werethen incubated overnight at 25 °C in development buffer (25 mMTris–HCl (pH 7.3), 10 mM DTT, and 5 mM CaCl2). After incubation, thegels were stained with Coomassie brilliant blue R-250. After destaining,the activity of calpain-1 and calpain-2 developed as clear bands againsta dark background, which demonstrated casein proteolysis. The calpainactivities were quantified with NIH Image J software. Using standardbovine albumin fraction V (Sigma-Aldrich), we ascertained the proteinconcentration in the muscle homogenate by the Bradford method.The specific activities of purified calpain-1 (1200 units/mg protein)and calpain-2 (1000 units/mg protein) (Calbiochem) showed linearrelationships with their optical densities. Consequently, the activitiesof calpain-1 and calpain-2 were determined by their optical densities(units/mg protein) on the gels.

2.5. Western blot analysis

Total protein was extracted from the SOL muscle of groundsquirrels by homogenization in SDS-PAGE sample buffer (1% SDS),with muscle protein extracts resolved by SDS-PAGE using Laemmligels (10% gel with an acrylamide/bisacrylamide ratio of 37.5:1 forcalpain-1, calpain-2, and calpastatin; and 12% gel with an acrylamide/bisacrylamide ratio of 29:1 for β-actin, a reference protein). After elec-trophoresis, the proteins were electrically transferred to nitrocellulosemembranes (0.45 um pore size) using Bio-Rad semi-dry transfer appa-ratus. The blotted nitrocellulose membranes were blocked with 1%bovine serum albumin (BSA) in Tris-buffered saline (TBS; 150 mMNaCl, 50 mM Tris–HCl, pH 7.5) and incubated with rabbit polyclonalanti-calpain-1 large subunit (1:1000; Cell Signaling Technology, Inc.

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(CST), Danvers, MA, USA), rabbit polyclonal anti-calpain-2 large subunit(1:1000; CST), rabbit polyclonal anti-calpastatin (1:1000; CST), rabbitanti-desmin (1:1000; CST), mouse monoclonal anti-b-actin (1:4000;Sigma-Aldrich), or mouse monoclonal anti-TnI (1:4000; Courtesy ofDr. Jin (Yu et al., 2001)) in TBS containing 0.1% BSA at 4 °C overnight.The nitrocellulose membranes were incubated with IRDye 680 CWgoat-anti mouse or IRDye 800 CW goat-anti rabbit secondary anti-bodies (1:10,000) for 90 min at room temperature, and visualized withan Odyssey scanner (LI-COR Biosciences, Lincoln, NE, USA). Quantifica-tion analysis of the blots was performed using NIH Image J software.

2.6. Statistical analysis

One-way ANOVA was used to determine statistically significant dif-ferences (SPSS10.0). Fisher's LSD post hoc test was used to determinegroup differences. The ANOVA–Dunnett's T3 method was used whenno homogeneity was detected. A paired-sample t-test was appliedto test for body weight loss. Statistical significance was assumed atP b 0.05. With 0.05 N P b 0.1, a tendency was assumed.

3. Results

In comparison with the PRE group, the body weights were (−24%,P = 0.042 and −38%, P = 0.001) in HIB and POST, respectively; SOLmuscle wet weights were (−9%, P = 0.042 and −16%, P = 0.001) inHIB and POST, respectively. The SOL muscle mass (mg) to total bodymass (g) ratio was significantly higher in the HIB (+33%, (P b 0.001)and POST (+59%, P b 0.001) groups in comparison with that of thePRE group (Table 1 and Figs. 1 and 2).

Compared to the PRE group, calpain-1 activity increased significantlyin HLS 176% (P = 0.034), with no significant changes observed incalpain-2 activity (see Fig. 3).

Protein expression of calpain-1 increased by 83% (P= 0.041) in HLScompared to the PRE group; calpain-2 expression increased by 208%(P = 0.029) in HLS; calpastatin expression increased significantly by180% (P b 0.001) and 153% (P = 0.007) in HIB and POST, respectively;and calpastatin expression decreased significantly by 133% (P = 0.013)in FE compared to the POST group (Fig. 4).

Compared to the PRE group, the myofilaments were well organizedand the width of the sarcomere and Z-disk both appeared visuallysimilar between groups (see Fig. 5).

4. Discussion

4.1. Lower body mass and higher SOLmuscle mass (mg) to total body mass(g) ratio in hibernators and post-hibernators

Our previous study showed that after 14 days of hindlimb suspen-sion in rats, the SOL muscle mass (mg) to total body mass (g) ratiosdecreased by 41% (Zhang et al., 2007). In contrast higher SOL musclemass (mg) to total body mass (g) ratios were observed in HIB andPOST groups (+33%, P b 0.001 and+59%, P b 0.001, respectively) com-pared to the PRE group in this study, because muscle wet weightdecreased less than body weight in hibernating animals. During hiber-nation, adipose tissue is the primary source of metabolic energy andwaste is consumed to a large extent, mostly explaining the loss in

Table 1Body mass and SOL muscle-to-body mass ratio (mean ± s.d.; n = 6 each).

Group BW before hibernation (g) BW at experiment time (g) BW loss (%)

HLS 325 ± 24 290 ± 22(P = 0.003) 10.1 ± 1.1PRE 331 ± 15 331 ± 15 0HIB 338 ± 18 258 ± 19 (P b 0.001) 23.6 ± 2.6POST 331 ± 20 208 ± 21 (P b 0.001) 37.7 ± 2.3FE 327 ± 18 205 ± 15 (P b 0.001) 37.5 ± 2.9

Please cite this article as: Yang, C.-X., et al., Changes in calpains and chibernation, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016

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body weight (Lee et al., 2008). Changes in muscle mass during hiberna-tion clearly differ from what happened with disuse atrophy in variousspecies of non-hibernators. Seven days of weightlessness on aSoviet biosatellite induced a 23% decrease in SOL muscle mass in rats(Desplanches et al., 1990). The soleus muscles of wild-type miceshowed a 25% decline in mass after 14 days of hindlimb suspension(Salazar et al., 2010). After two or threemonths of dormancy, onlymar-ginal SOL muscle mass loss was observed in the HIB and POST groups(−9%, P = 0.042 and −16%, P = 0.001, respectively) compared tothe PRE group. In consistent with our results, the gastrocnemius andsoleus muscles exhibited a 14–20% reduction in their relative massover 6 months of hibernation in the golden-mantled ground squirrel(C. lateralis) (Wickler et al., 1991). The muscle wet weight decreasedless than the body weight in hibernating animals, resulting in a steadyincrease in muscle-to-body mass ratio, which suggested a protectiveeffect that reduces or avoids muscle atrophy during hibernation inDaurian ground squirrels.

4.2. Maintenance of calpain activity during hibernation

Disuse induced atrophic muscle fibers show elevated intracellularCa2+ concentrations (calcium overload), activated calpains (calcium-dependent cysteine proteases) and increased Ca2+-dependent proteol-ysis (Ingalls et al., 2001; Goll et al., 2003; Jackman andKandarian, 2004).Research on rat SOL muscle showed that five days of immobilizationincreased autolyzed calpain-1 in the particulate fraction and increasedautolyzed calpain-2 in the soluble fraction after immobilization(Vermaelen et al., 2007), and calpain-1 activity showed a progressiveincrease in 1-, 2-, and 4-weeks of unloaded SOL muscles (Ma et al.,2011). Other research in our laboratory showed that hindlimb unloadedrats demonstrated 1.5 fold and 4.3 fold increases in the activities ofcalpain-1 and calpain-2, respectively (He et al., 2012). In the presentstudy, calpain-1 activity increased by 176% (P = 0.034) in the 14-dayhindlimb suspended ground squirrels, but no significant changes wereobserved in calpain-2 activity. Calpain-1 activity might be related toan elevation in intra-cellular Ca2+ concentration (Costelli et al., 2005).Because calpain-1 and calpain-2 are optimally activated by micromolar(μM) and millimolar (mM) Ca2+ concentrations, respectively (Carafoliand Molinari, 1998), calpain-1 was more susceptible than calpain-2and thus more easily activated in response to elevated intracellularCa2+ concentrations (Branca et al., 1999). Hibernating cells are charac-terized by down-regulation in the activity of Ca2+ channels in the cellmembrane, which helps to prevent excessive Ca2+ entry (Wang et al.,2002). We previously observed that Ca2+ concentration decreasedby 7% in HIB compared to the PRE group (Wu et al., unpublisheddata). Furthermore, in our present study, elevation in the activities ofcalpain-1 and calpain-2 was not observed during hibernation in HIB.Taken together, these results suggest that calpain activity was notactivated during 90± 12 days of hibernation. This may be due to bettercapability in squirrels to maintain intracellular Ca2+ homeostasis,thereby preventing calpain activity (Wu et al., unpublished data).

4.3. Calpains and calpastatin protein expression

Calpastatin is an endogenous calpain-inhibitor protein of calpains(Murachi, 1990), and its over-expression can inhibit calpain activity

SOL MWW at experiment time (mg) SOL MWW/BW at experiment time (mg/g)

128 ± 12 0.44 ± 0.03 (P = 0.002)129 ± 7 0.39 ± 0.01129 ± 9 0.52 ± 0.01 (P b 0.001)127 ± 8 0.62 ± 0.04 (P b 0.001)125 ± 9 0.61 ± 0.03 (P b 0.001)

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Fig. 1. Thermal imaging of ground squirrel body temperature detected by a visual thermometer (Fluke VT04 Visual IR Thermometer, USA). A. No hibernation state. B. Hibernating state.C. Arousing from hibernation. D. Arousal from hibernation.

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and decrease proteolysis (Goll et al., 2003). In the present study, theexpression of calpastatin was not changed, but the protein expres-sion of calpain-1 and calpain-2 in HLS significantly increased by83% (P = 0.041) and 208% (P = 0.029), respectively, suggesting thatmuscle atrophy can occur in hibernators during non-hibernating states.

In this study, the protein expression of calpastatin increased signifi-cantly (180%, P b 0.01) in HIB. A similar protein expression of calpastatinalso occurred in the POST group (153%, P= 0.007). According to a priorreport, calpastatin-overexpression can lead to reduced muscle fiberatrophy in hindlimb suspension mice (Tidball and Spencer, 2002). Thus,the increased expression of calpastatin wasmaintained from hibernationuntil at least a few days post-hibernation, which likely enabled theground squirrel to reduce or avoid hibernation-related muscle atrophyin the SOLmuscle. In summary, increased expressionof calpastatin duringand after hibernation is an effective mechanism for ground squirrels toprevent or lessen muscle atrophy.

In this study, however, the protein expression of calpastatindecreased significantly (133%) after forced exercise compared to thatin the POST group. This indicated that the elevated calpastatin levelsoccurred during hibernation, and only lasted a short time after hiberna-tion. The forced exercise may accelerate the protolysis of elevatedcalpastatin in hibernation. However, further research is needed todetermine the detailed mechanism involved.

4.4. Slight changes in Z-disk structure

Ultrastructural changes in the myofibrils constitute an importantparameter for judging the degree of disuse atrophy (Fitts et al., 2001;

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Fig. 2. Lower bodymass and higher SOLmusclemass (mg) to total bodymass (g) ratio ob-served in hibernators and post-hibernators in the SOL muscle of Daurian ground squirrels(mean ± SD, N = 6 each). BW = body mass, MWW = muscle wet mass. **P b 0.01,***P b 0.001, compared to BW before hibernation (paired-samples T test); ##P b 0.01,###P b 0.001, compared to PRE (ANOVA-LSD). PRE: no hibernation group; HLS: twoweek hindlimb suspension group; HIB: two month hibernation group; POST: arousalfrom hibernation group; FE: forced, moderate-intensity treadmill exercise group.

Please cite this article as: Yang, C.-X., et al., Changes in calpains and chibernation, Comp. Biochem. Physiol., A (2014), http://dx.doi.org/10.1016

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Volodina and Pozdnyakov, 2004). The skeletal muscles of hibernatorsexperience prolonged inactivity during winter dormancy, consisting ofabout 90% torpor and 10% inter-bout arousals (Sun et al., 2012). Themyofilaments were well organized and the width of the sarcomereand Z-disk both appeared visually similar in the HIB and POST groupscompared to the PRE group. In consistentwith our results, muscle fibersdid not undergo any significant ultrastructural alterations in hibernatedbats (Myotis myotis) (Klika and Zaitsova, 1984); and myocardial sarco-lemma, mitochondria, Golgi zones, nuclei and myofibrils appearedunchanged during hibernation in golden-mantled ground squirrels(C. lateralis) (Aloia and Pengelley, 1971). However, rats experiencing a14-day spaceflight exhibited deleterious changes in extrafusal musclefibers, including extensive sarcomere disruption and disorganizedmyo-filaments, with sarcomere lesions detected in 44% of fibers examined(Fitts et al., 2001).

Hindlimb unloading and spaceflight microgravity induce atrophy ofthe slow adductor longus muscle fibers, which, following reloading,exhibit eccentric contraction-like lesions such as abnormal wideningof sarcomeres with A band disruption and excessively wavy, extractedZ-lines, (Riley et al., 1995; He et al., 2012). Furthermore, unloading

Fig. 3. Calpain activity in hibernators and post-hibernators and in hindlimb suspensionanimals in the SOL of Daurian ground squirrels (mean+ SD, n=6). A. Casein zymograph.B. Changes in activity of calpain-1 and calpain-2. *P b 0.05, compared to the PRE group(ANOVA-LSD). PRE: no hibernation group; HLS: two week hindlimb suspension group;HIB: two month hibernation group; POST: arousal from hibernation group; FE: forcedexercise group.

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Fig. 4. Calpain protein in hindlimb suspension animals and calpastatin protein in hiberna-tors and post-hibernators in the SOL of Daurian ground squirrels (mean ± SD, n = 6). A.Western Blots. B. Calpains/beta- actin ratio.*P b 0.05, **P b 0.01 compared to the PRE group(ANOVA-LSD); #P b 0.05 compared to the POST group (ANOVA-LSD). PRE: no hibernationgroup; HLS: two week hindlimb suspension group; HIB: two month hibernation group;POST: arousal from hibernation group; FE: forced exercise group.

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can induce muscle atrophy and disrupt the force-generating capability,which decreases muscle performance and increases susceptibility tocontraction-induced injury (Widrick et al., 1999; Trappe et al., 2009).Thus, the antigravity soleus muscle, which is reloaded subsequent tohindlimb unloading, is prone to muscle damage (Prisby et al., 2004). It

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Fig. 5. Changes in the Z-disk ultrastructure in the SOL of Daurian ground squirrels. Slight changmonth hibernation group; C. POST: arousal from hibernation group.

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follows that ground squirrels aroused from hibernation after two days(POST group) should have exhibited eccentric contraction-like lesionswhen they began using their muscles again. However, the structureof the Z-disk remained well-arranged and integrated, and appearedvisually similar among the PRE, HIB and POST groups.

Calpains tend to be concentrated in the Z-disk where disassemblybegins (Bird et al., 1980). Calpains are activated by calcium, and treat-ment of purified myofibrils with calcium causes rapid and completeloss of the Z-disk (Busch et al., 1972). In this study, no significantchanges were observed in calpain-1 and calpain-2 activity in the HIBgroup, and only slight changes were observed in the Z-disk ultrastruc-ture during and after hibernation. This suggests that maintenance ofcalpain activities may be the molecular mechanism ensuring that theZ-disk structure remained well-preserved and intact.

5. Conclusions

The inhibition of calpain activity by highly elevated calpastatinprotein expression mediated cytoskeletal protein degradation andprotected the Z-line ultrastructures. This process was likely responsiblefor preventing skeletal muscle atrophy during hibernation.

6. Uncited reference

Wu, 2013

Acknowledgments and financial support/disclosure

We would like to thank two anonymous reviewers for their com-ments on the original manuscript. This work was supported by fundsfrom the National Natural Science Foundation of China (Grant No.31270455), the Specialized Research Fund for the Doctoral Program ofHigher Education of China (Grant No. 20116101110013), and the Inter-national Scientific and Technological Cooperation Projects in ShaanxiProvince of China (Grant No. 2013KW26-01).

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