Maintenance of muscle mass is not dependent on the calcineurin-NFAT pathway

Esther E. Dupont-Versteegden1, Micheal Knox1, Cathy M. Gurley1, John D. Houlé2, and Charlotte A. Peterson1,3

Departments of 1 Geriatrics and 2 Anatomy and Neurobiology, University of Arkansas for Medical Sciences, and 3 Central Arkansas Veterans Health Care System, Little Rock, Arkansas 72205


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, the role of the calcineurin pathway in skeletal muscle atrophy and atrophy-reducing interventions was investigated in rat soleus muscles. Because calcineurin has been suggested to be involved in skeletal and cardiac muscle hypertrophy, we hypothesized that blocking calcineurin activity would eliminate beneficial effects of interventions that maintain muscle mass in the face of atrophy-inducing stimuli. Hindlimb suspension and spinal cord transection were used to induce atrophy, and intermittent reloading and exercise were used to reduce atrophy. Cyclosporin (CsA, 25 mg · kg-1 · day-1) was administered to block calcineurin activity. Soleus muscles were studied 14 days after the onset of atrophy. CsA administration did not inhibit the beneficial effects of the two muscle-maintaining interventions, nor did it change muscle mass in control or atrophied muscles, suggesting that calcineurin does not play a role in regulating muscle size during atrophy. However, calcineurin abundance was increased in atrophied soleus muscles, and this was associated with nuclear localization of NFATc1 (a nuclear factor of activated T cells). Therefore, results suggest that calcineurin may be playing opposing roles during skeletal muscle atrophy and under muscle mass-maintaining conditions.

cyclosporin; hindlimb suspension; spinal cord transection; soleus


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ADULT SKELETAL MUSCLE is a tissue capable of changing size depending on the functional demands placed on it. Muscle disuse, due to a variety of conditions such as bed rest, spinal cord injury, denervation, immobilization, and microgravity, induces skeletal muscle atrophy. Conversely, stimuli, such as resistance exercise and functional overload, are associated with muscle hypertrophy. Although the underlying mechanisms regulating the changes in muscle mass are largely unknown, intracellular pathways that may be involved are being identified. The Ca2+/calmodulin-dependent protein phosphatase calcineurin appears to regulate a pathway required for skeletal muscle hypertrophy induced by functional overload (16). Calcineurin is a ubiquitously expressed phosphatase that is activated by sustained levels of intracellular Ca2+. It is a heterodimer of a 58- to 64-kDa catalytic and calmodulin-binding subunit, calcineurin A, and a 19-kDa Ca2+-binding regulatory subunit, calcineurin B (for review see Refs. 27 and 36). Cyclosporin A (CsA), an inhibitor of calcineurin, partially blocked the expected increase in plantaris muscle size due to overload on synergist ablation (16). In vitro, calcineurin was shown to mediate the hypertrophic response of muscle cells to insulin-like growth factor I on muscle cells, and this response was inhibited by CsA administration (34, 42). Moreover, when calcineurin was expressed as a constitutively active form, independent of Ca2+ activation, the muscle cells showed a pronounced hypertrophic phenotype (34). In addition to skeletal muscle hypertrophy, it has also been shown that cardiac hypertrophy was mediated by a calcineurin-dependent pathway that was blocked by CsA (32, 46) and by overexpression of endogenous inhibitors of calcineurin, such as Cain and myocyte-enriched calcineurin-interacting protein (15, 40). However, there have also been reports of a lack of change in muscle size on CsA administration in cardiac and skeletal muscles (6, 52). More importantly, transgenic mice overexpressing activated calcineurin did not exhibit skeletal muscle hypertrophy in resting muscles (35) or an exaggerated increase in skeletal muscle mass in response to functional overload (17), indicating that the exact role of calcineurin in skeletal muscle hypertrophy remains to be determined.

NFATs (nuclear factors of activated T cells), a family of transcription factors playing a central role in the immune response, are substrates for calcineurin in skeletal muscle. There are four Ca2+-responsive members of the NFAT family: NFATc1, NFATc2, NFATc3, and NFATc4 (39). Under basal conditions, NFATs are localized in the cytoplasm in a phosphorylated state. In response to increases in intracellular Ca2+, calcineurin becomes activated and dephosphorylates NFATs, promoting their translocation into the nucleus, where they bind to a consensus DNA sequence and stimulate transcription of target genes (39). A number of NFAT isoforms [NFATc1 (NFATc), NFATc2 (NFATp/1), and NFATc3 (NFAT4/x/c3)] have been identified in skeletal muscle cells (24), and in vitro their function is influenced by the differentiation state of the muscle cells (1). In particular, NFATc1 is capable of undergoing Ca2+-dependent nuclear translocation in multinucleated myotubes in vitro (1, 30), suggesting a regulatory role for this isoform in adult skeletal muscle gene expression. Indeed, a recent study showed that NFATc1 was dephosphorylated in response to functional overload (17), indicating that this isoform is capable of responding to intracellular signals in skeletal muscle in vivo.

Recently, myocyte enhancer factor-2 (MEF2) proteins have been identified as substrates for calcineurin in skeletal muscle (37, 50, 51). MEF2 family factors have been shown to play a pivotal role in morphogenesis and myogenesis of skeletal, cardiac, and smooth muscle cells. They are expressed in developing muscle cells, and they bind conserved sequences in the control regions of muscle-specific genes (for review see Ref. 7). In adult skeletal muscle, MEF2-dependent transcription is increased with constitutively active calcineurin (50) and is decreased by inhibition of calcineurin (51). Therefore, MEF2 proteins also appear to be downstream targets for calcineurin.

Whereas the involvement of the calcineurin-NFAT pathway during skeletal muscle hypertrophy is beginning to be elucidated, whether calcineurin is required for maintenance of normal muscle size or the prevention of skeletal muscle atrophy is relatively unexplored. In a recent study, the relationship between calcineurin protein abundance and adult skeletal muscle mass was investigated. A negative correlation was shown to exist between the amount of calcineurin protein present in the vastus intermedius muscle and the extent of atrophy in this muscle in response to spinal cord injury (45). These data indicate that calcineurin may be involved in the maintenance of normal muscle mass. However, recovery from atrophy does not seem to depend on the calcineurin pathway, since CsA did not block this process (8, 43). Therefore, it seems likely that intracellular mechanisms controlling hypertrophy of skeletal muscle are not necessarily identical to mechanisms involved in the maintenance or restoration of muscle mass in the face of an atrophy-inducing stimulus.

The goal of the present study was to investigate the role of the calcineurin-NFAT pathway in muscle atrophy and test the hypothesis that blocking calcineurin activity by CsA administration eliminates beneficial effects of interventions that maintain muscle mass in the face of atrophy-inducing stimuli. Two animal models were used to test the hypothesis. In the hindlimb-suspended and intermittently reloaded rat model, the prevention of weight bearing in the hindlimbs induced by suspending the hindquarters is associated with a decrease in muscle mass mainly of postural muscles such as the soleus. Other hindlimb muscles, such as the plantaris, atrophy less (for review see Ref. 47). A countermeasure known as intermittent weight bearing or reloading was employed with the hindlimb suspension model to reduce the atrophy induced by hindlimb suspension. When animals were allowed to walk around their cage freely with full weight support for a total of 1 h daily, the decrease in soleus muscle mass was ameliorated (5, 14, 38, 49). Results from the hindlimb-suspended animals were verified in a second model, the spinal cord-transected and exercised rat. After spinal cord transection at thoracic level (T10), all hindlimb muscles undergo atrophy, and this loss in muscle mass is ameliorated by exercise on a motor-driven bicycle in the soleus muscle (18, 25, 33). Therefore, soleus muscles in these two animal models were used to investigate the role of the calcineurin-NFAT pathway in the maintenance of muscle mass under atrophy-inducing conditions.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Animals and experimental procedures. All procedures were performed in accordance with institutional guidelines for the care and use of laboratory animals. For the hindlimb suspension experiments, male Sprague-Dawley rats (400-500 g) were separated into six groups: 1) non-hindlimb-suspended rats (control, n = 6), 2) non-hindlimb-suspended rats treated with CsA (25 mg · kg-1 · day-1, n = 6), 3) hindlimb-suspended rats (see below; n = 8), 4) hindlimb-suspended rats treated with CsA (n = 4), 5) hindlimb-suspended and intermittently reloaded rats (see below; n = 9), and 6) hindlimb-suspended and intermittently reloaded rats treated with CsA (n = 5). The dose of CsA has been shown to yield high serum levels of CsA, inhibit calcineurin activity, and produce previously reported changes in skeletal muscle (10, 16, 43, 46, 52). Animals in the hindlimb-suspended and hindlimb-suspended and intermittently reloaded groups with or without CsA were suspended for 14 days as described and outlined below (49). A tail device containing a hook was attached with gauze and glue while the animals were anesthetized with pentobarbital sodium (50 mg/kg). After the animals regained consciousness, the tail device was connected via a thin cable to a pulley sliding on a vertically adjustable stainless steel bar running longitudinally above a high-sided cage with standard floor dimensions. The system was designed in such a way that the rats cannot rest their hindlimbs against any side of the cage. To allow hindlimb-suspended and intermittently reloaded animals and hindlimb-suspended and intermittently reloaded animals treated with CsA to move around their cage freely, the suspension device was unhooked for 1 h at the same time each day (~3 h after lights-on). During the intermittent reloading, the animals were observed constantly and spent a lot of time grooming and exploring the cage. CsA injections resulted in cyclosporin blood levels of 5,000-7,000 ng/ml 20 h after the last injection, and no differences in cyclosporin blood levels between the groups with CsA were observed. Rats were killed with an overdose of pentobarbital sodium, and blood was collected by heart puncture for determination of cyclosporin levels. Soleus muscles were dissected and frozen as described below.

Animals in the spinal cord transection experiment were treated as follows. Adult female Sprague-Dawley rats (225-250 g) were randomly divided into five groups. Control rats (n = 4) did not undergo a spinal cord transection, were not exercised, and did not receive CsA. No control group receiving CsA was added, since the previous experiments in the hindlimb-suspended protocol indicated that CsA did not affect control rats. All other rats underwent a complete transection of the thoracic (T10) spinal cord, as described previously (19, 20). Briefly, a 2- to 3-mm-long aspiration lesion was created while the animals were under anesthesia with ketamine (60 mg/kg) and xylazine (10 mg/kg). After surgery, rats received Penicillin Procaine G and a dextrose-saline injection, and for the duration of the experimental period, expression of the urinary bladder was carried out twice daily. At 4 days after the surgery, rats were assigned to the following four groups: 1) rats that received no further manipulation (n = 4), 2) rats that were exercised on a motor-driven bicycle for 10 continuous days, as described previously (18, 19) and in detail below (n = 3), 3) rats that were treated with CsA (25 mg · kg-1 · day-1, n = 5), and 4) rats that were treated with CsA and started on the exercise protocol described below (n = 3). Exercise was performed using a custom-built motor-driven cycling apparatus. Rats were suspended horizontally in full-body slings, with their feet secured to the pedals. Cycling speed was maintained at 45 rpm, and each exercise bout consisted of two 30-min exercise periods with 10 min of rest between exercise periods. At 10 days after the onset of exercise (14 days after the surgery), rats were killed with an overdose of pentobarbital sodium. Soleus muscles were carefully dissected, weighed, frozen as described below, and stored at -80°C for further analysis. Soleus muscles from one leg were divided into two sections, snap-frozen in liquid nitrogen, and used for Northern and Western blot analysis. Muscles from the other leg were embedded in freezing medium and frozen in liquid nitrogen-cooled isopentane for immunohistochemical analyses.

RNA isolation and Northern blot analysis. RNA isolation and detection were performed as described previously (18, 20). Briefly, total RNA was isolated from soleus muscles using the guanidinium thiocyanate-phenol-chloroform extraction method, as described by Chomczynski and Sacchi (11). Total RNA (10 µg) was electrophoresed through 1% agarose-2% formaldehyde HEPES-EDTA-buffered gels and separated for 2-3 h at 70 V. RNA was then transferred to a nylon membrane (Zeta-Probe, Bio-Rad, Richmond, CA) using a blotting unit (BIOS, New Haven, CT) and ultraviolet cross-linked using a Stratalinker (Stratagene, La Jolla, CA). Membranes were sequentially hybridized with the following cDNA probes: calcineurin A, NFATc1 (NFATc), MEF2C, and 18S rRNA. Calcineurin A and MEF2C probes were kindly provided by E. Olson (University of Texas Southwestern Medical Center, Dallas, TX), NFATc1 probe was a gift from Timothy Hoey (Tularik, S. San Francisco, CA), and 18S rRNA probe was an EcoRI fragment from 18S RNR* (21) obtained from the American Type Culture Collection. The probes were labeled using the random prime method according to the manufacturer's recommendations (Decaprime II kit, Ambion, Austin, TX). Hybridization was performed according to Church and Gilbert (12). Briefly, filters were hybridized overnight in rotating flasks in a hybridization oven (Robbins Scientific, Sunnyvale, CA) at 65°C in buffer containing 0.5% crystalline-grade bovine serum albumin (Calbiochem, San Diego, CA), 1 mM EDTA, 0.5 M NaPO4, pH 7.2, and 3% SDS. Filters were washed sequentially in wash solution A (1 mM EDTA, 40 mM NaPO4, pH 7.2, 5% SDS), wash solution B (1 mM EDTA, 40 mM NaPO4, pH 7.2, 1% SDS), and postwash solution (1.6× saline-sodium citrate, 5 mM Tris, pH 8.0, 1 mM EDTA, 0.1% SDS) at 65°C for 40 min each. Filters were exposed to a PhosphorScreen and scanned using a StormScan (Molecular Dynamics, Sunnyvale, CA), and density analysis of the bands was performed using the ImagiQuant software (Molecular Dynamics). Density of the bands for calcineurin and NFATc1 was normalized to density of the bands for 18S rRNA and expressed as arbitrary density units.

Isolation of protein and Western analysis. Soleus muscles were homogenized in a buffer containing 10 mM MgCl2, 10 mM KH2PO4, 1 mM EDTA, 5 mM EGTA, 1% Igepal, 50 mM beta -glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 1 µg/ml leupeptin, 2 µg/ml antipain, 10 µg/ml benzamidine, 1 µg/ml aprotinin, 1 µg/ml chymostatin, and 1 µg/ml pepstatin. After homogenization, the samples were centrifuged for 10 min at 1,000 g at 4°C. Protein concentrations of the supernatants were determined according to Bradford (9) using the Bio-Rad protein assay reagent. Protein was loaded at 40 µg/lane and separated on a 10% polyacrylamide gel. After electrophoretic separation, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) for Western blot analysis. After transfer, the membrane was incubated in Ponceau S solution (Sigma, St. Louis, MO) for 5 min for visualization of the protein and assurance of equal loading in all the lanes. Membranes were then incubated overnight in blocking solution [5% nonfat powered milk in phosphate-buffered saline (PBS) + 0.5% Tween (PBS-T)]. After overnight blocking, membranes were washed in PBS-T and incubated with calcineurin antibody (Sigma) in blocking solution (1:3,000 dilution) for 1 h at room temperature. This antibody recognizes calcineurin A. The membranes were then washed in PBS-T and further incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (Pierce, Rockford, IL) for 1 h in blocking solution (1:3,000 dilution) at room temperature. Antibody binding was detected by incubating membranes for 5 min at room temperature in the Western substrate kit ChemiGlow (AlphaInnotech, San Leandro, CA). The membranes were scanned using ChemiImager 5500 (AlphaInnotech), and density of the bands was analyzed using FluorChem software (AlphaInnotech). For each blot, the signals were normalized to the band from a control soleus muscle sample, which was run on every blot.

Immunohistochemistry. Consecutive soleus muscle sections were cut on a cryostat (8 µm) and stored at -20°C. NFATc1 localization in soleus muscle sections was determined as follows. Sections were reacted with 0.25% hydrogen peroxide to block endogenous peroxidase activity. Sections were then fixed in 2% paraformaldehyde in PBS and permeabilized using 1% Igepal CA-630 (Sigma) to allow access of antibodies to the nucleus. All subsequent washes and incubations were performed with 0.1% Igepal. NFATc1 antibody (Affinity Bioreagents, Golden, CO; 1:100 dilution) was applied to the sections and incubated for 1 h. An IgG1 horseradish peroxidase-conjugated secondary antibody (Zymed, San Francisco, CA; 1:250 dilution) was applied. After incubation and washes, TrueBlue peroxidase substrate (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was applied for color development, as directed by the manufacturer.

Double staining of NFATc1 and laminin was performed as follows. Sections were cut on a cryostat (8 µm), air-dried, and stored at -20°C. Sections were rehydrated in Tris-buffered saline and reacted with 0.3% hydrogen peroxide in Tris-buffered saline to block endogenous peroxidase activity. Laminin antibody (Sigma; 1:50 dilution) was applied, and the sections were incubated for 1 h at room temperature. An IgG alkaline phosphatase-conjugated secondary antibody (Zymed; 1:200 dilution) was applied, and the sections were incubated for 1 h at room temperature. An alkaline phosphatase substrate (Vector) was applied for color development (red) as directed by the manufacturer. Sections were then permeabilized in 1% Igepal in PBS for 5 min and incubated with NFATc1 antibody (Affinity Bioreagents; 1:200 dilution) at room temperature for 2 h. All incubations and washes after permeabilization were performed in 0.1% Igepal. A biotinylated secondary antibody (Zymed; 1:250 dilution) was applied for 1 h at room temperature, and the sections were incubated in streptavidin-peroxidase (Zymed). TrueBlue peroxidase substrate was applied for color development as directed by the manufacturer. Sections were viewed on a Nikon microscope, displayed on a computer using a videocamera, and analyzed using MetaView image analysis software (Advanced Scientific, Lewisville, TX).

Statistics. To test for statistically significant differences, analysis of variance was used; in the case of significant differences, Tukey's multiple comparisons test was applied. Statistical significance was assumed at P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CsA treatment does not inhibit the beneficial effects of interventions during atrophy. After 2 wk of hindlimb suspension, muscle weight-to-body weight ratio was decreased by 30% in soleus muscle, and intermittent reloading was associated with an attenuation of this atrophy (Fig. 1). CsA treatment did not change muscle size in control rats, nor did it affect the changes seen with hindlimb suspension alone or the combination of hindlimb suspension and intermittent reloading. Because we hypothesized that CsA would inhibit the maintenance of muscle mass with intermittent reloading, the results on the hindlimb-suspended and intermittently reloaded animals were somewhat surprising. Therefore, we wanted to confirm the data in our spinal cord transection and exercise model. We previously showed that short- and long-term exercise started within 5 days after spinal cord transection attenuates soleus muscle atrophy (18, 25). We hypothesized that CsA treatment would block the muscle-sparing effect of exercise after spinal cord transection. Because CsA treatment did not affect control muscles in the previous set of animals, we omitted the control with CsA treatment group from these experiments. Muscle weight-to-body weight ratios of the animals were normalized to those of the control group in the hindlimb-suspended animals. Muscle weight-to-body weight ratios were decreased 37% in transected soleus muscles compared with control, and exercise attenuated this decrease (Fig. 1). CsA treatment did not change the loss in soleus muscle mass due to spinal cord transection, nor did it eliminate the muscle-sparing effects of exercise. These results suggest that calcineurin does not play a role in mediating the beneficial effects of muscle mass-maintaining interventions in the face of an atrophy-inducing stimulus.


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Fig. 1.   Cyclosporin A (CsA) does not inhibit beneficial effects of muscle-maintaining interventions. Muscle weight-to-body weight ratios are shown for soleus muscles from control (Con), hindlimb-suspended (Hs), hindlimb-suspended and intermittently reloaded (HsIr), spinal cord-transected (Tx), and spinal cord-transected and exercised (TxEx) rats with CsA (open bars) or without CsA (solid bars). Bars, means; error bars, SE. * Significantly different from Con; #significantly different from Hs; §significantly different from Tx (P < 0.05).

Changes in calcineurin abundance. Because CsA did not inhibit the beneficial effects of intermittent reloading or exercise, we wanted to investigate whether the level of expression of calcineurin was changed with the interventions. Surprisingly, soleus calcineurin mRNA abundance was elevated about twofold during atrophy due to hindlimb suspension and showed an additional 22% increase with CsA administration (Fig. 2A). With intermittent reloading, calcineurin mRNA abundance returned to levels not significantly different from control. CsA did not affect calcineurin mRNA abundance in soleus muscles from control or hindlimb-suspended and intermittently reloaded rats. Similar results were obtained from the soleus muscles in the transection and exercise model. Atrophied soleus muscles from transected rats showed a twofold increase in calcineurin mRNA abundance and a small but not significant decrease with exercise (Fig. 2B). CsA treatment did not change the calcineurin mRNA abundance in the group subjected to transection alone or the transected and exercised group. The changes in calcineurin mRNA abundance were accompanied by changes in calcineurin protein levels. Western blot analysis of soleus muscles from hindlimb-suspended rats showed that calcineurin was 2.5 times more abundant than in control rats (Fig. 3). There was a trend for higher levels of calcineurin protein in the intermittently reloaded soleus muscles than in controls, but this difference did not reach significance because of the high variability in the data. CsA did not affect calcineurin protein abundance significantly (Fig. 3).


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Fig. 2.   mRNA abundance for calcineurin increased with atrophy. Total RNA from soleus muscles was analyzed by Northern analysis with indicated probes. Insets: composite of representative lanes of each probe. In A, groups are as follows: control (lane 1), control with CsA (lane 2), hindlimb suspended (lane 3), hindlimb suspended with CsA (lane 4), hindlimb suspended with intermittent reloading (lane 5), and hindlimb suspended with intermittent reloading and CsA (lane 6). In B, groups are as follows: control (lane 1), spinal cord transected (lane 2), spinal cord transected with CsA (lane 3), spinal cord transected and exercised (lane 4), and spinal cord transected and exercised with CsA (lane 5). Density of bands was normalized to density of 18S rRNA probe and is summarized in A and B. See Fig. 1 legend for definition of abbreviations. Open bars, with CsA; solid bars, without CsA. Bars, means; error bars, SE. * Significantly different from control; dagger  significantly different from control with CsA (P < 0.05).



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Fig. 3.   Calcineurin protein abundance is elevated with hindlimb suspension. Calcineurin protein abundance is shown in soleus muscles from control, hindlimb-suspended, and hindlimb-suspended and intermittently reloaded rats with (open bars) or without (solid bars) CsA. Samples were normalized to the same soleus control sample loaded on every gel to serve as an internal control. See Fig. 1 legend for definition of abbreviations. Bars, means; error bars, SE. * Significantly different from control (P < 0.05).

Analysis of NFATc1 and MEF2C. Because calcineurin abundance was increased with atrophy in both models, we next examined downstream effectors in the calcineurin pathway: NFATc1 and MEF2C. NFATc1 was chosen because this isoform is abundant in adult skeletal muscle and appears to be one of the downstream targets of calcineurin in skeletal muscles (1, 17, 30). MEF2C was examined, since recent studies indicated that calcineurin regulates MEF2 activity (37, 50). The mRNA abundance of NFATc1 increased 30% in soleus muscles from hindlimb-suspended rats, and CsA treatment attenuated this increase (Table 1). In the spinal cord transection model, NFATc1 mRNA abundance was not different between the groups, except it was slightly elevated in spinal cord-transected and exercised rats treated with CsA (Table 1). MEF2C mRNA abundance showed a very similar trend, because it was elevated in soleus muscles of hindlimb-suspended, but not spinal cord-transected, rats. However, CsA treatment did not change MEF2C mRNA abundance significantly. Therefore, NFATc1 and MEF2C mRNA abundance did not change consistently with atrophy induced by two different experimental models. However, NFATc1 and MEF2C function is not necessarily regulated by abundance, but rather by phosphorylation state. For example, activation of calcineurin leads to dephosphorylation and nuclear translocation of NFATc1, and this process can be studied by immunohistochemistry. Results shown in Fig. 4 indicate that nuclear translocation of NFATc1 occurred in the soleus muscles of hindlimb-suspended rats only (Fig. 4C). Intermittent reloading appeared to block NFATc1 nuclear staining (Fig. 4E). As expected, no nuclear localization of NFATc1 was observed in any of the CsA-treated groups (Fig. 4, B, D, and F). To further analyze which nuclei within the soleus muscle of hindlimb-suspended rats were positive for NFATc1, double staining with laminin and NFATc1 antibodies was performed (Fig. 5). Laminin stains the basal lamina, which surrounds the muscle fiber and includes satellite cells (muscle stem cells). Nuclei present within the basal lamina (inside the laminin-stained fiber) as well as outside the laminin-stained fiber were positive for NFATc1 in soleus muscle from hindlimb-suspended rats (Fig. 5B), but not from control rats (Fig. 5A). These results suggest that, in atrophied soleus muscle, increased calcineurin expression was correlated with NFAT-positive nuclei within the muscle fibers, suggesting activation of the calcineurin-NFAT pathway.

                              
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Table 1.   NFATc1 and MEF2C mRNA abundance



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Fig. 4.   NFATc1 exhibits nuclear localization in soleus muscle from hindlimb-suspended rats. Cross sections of soleus muscles of control rats (A), control rats treated with CsA (B), hindlimb-suspended rats (C), hindlimb-suspended rats treated with CsA (D), hindlimb-suspended and intermittently reloaded rats (E), and hindlimb-suspended and intermittently reloaded rats treated with CsA (F) were immunoreacted with NFATc1 antibody (blue). Nuclear staining for NFATc1 was observed in soleus myofibers from hindlimb-suspended rats only. Arrows, nuclear staining with NFATc1 antibody. Scale bar, 25 µm.



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Fig. 5.   NFATc1 nuclear staining in atrophied soleus is localized to nuclei inside and outside the basal lamina. Cross sections of soleus muscles from control (A) and hindlimb-suspended (B) rats were immunoreacted consecutively with laminin (red) and NFATc1 (blue) antibodies. Nuclear staining in soleus muscle from hindlimb-suspended rats was observed in nuclei in the interstitial space as well as within the basal lamina (muscle nuclei), as indicated by arrows. Scale bar, 25 µm.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The involvement of the calcineurin pathway in skeletal and cardiac muscle hypertrophy has been investigated in a number of studies (15, 16, 32, 34, 40, 42, 46). The present study is the first to report on the potential role of calcineurin during muscle atrophy and interventions that maintain muscle mass under atrophy-inducing conditions. We hypothesized that maintenance of muscle mass was dependent on the calcineurin pathway and that blocking that pathway with CsA would eliminate the beneficial effects of muscle-maintaining interventions. Hindlimb suspension and spinal cord transection induce skeletal muscle atrophy, although molecular and cellular mechanisms resulting in atrophy may differ. In both models, atrophy can be reduced by specific interventions (intermittent reloading and exercise, respectively); therefore, intracellular pathways involved in the maintenance of muscle mass during atrophy can be studied. The major finding of this study was that even though CsA has been shown to attenuate hypertrophy (16), it did not inhibit the beneficial effects of reloading and exercise during hindlimb suspension and spinal cord transection, respectively. Therefore, intracellular mechanisms controlling skeletal muscle mass during hypertrophy may be different from those contributing to maintenance of muscle mass during atrophy-inducing events. These results are consistent with recent findings that CsA did not inhibit the restoration of muscle mass after hindlimb suspension or denervation (8, 43). Moreover, the fact that CsA treatment had no effect on control muscles suggests that the calcineurin pathway is likely not involved in the maintenance of normal muscle size. This observation is consistent with previous reports in which CsA did not change normal muscle weight-to-body weight ratio (6) and in which overexpression of a constitutively active form of calcineurin did not induce hypertrophy in normal muscles (35).

Although CsA did not inhibit the ability of the interventions (intermittent reloading and exercise) to maintain muscle mass, calcineurin abundance changed in the soleus muscles. Unexpectedly, we observed an increase in calcineurin mRNA and protein abundance with atrophy. The increase in calcineurin may represent a compensatory mechanism to maintain muscle mass in the face of an atrophy-inducing event. This idea is supported by the observation that NFATc1 protein was localized to the nuclei only in soleus muscles from hindlimb-suspended animals, indicating that NFATc1 was dephosphorylated, most likely by calcineurin. The translocation of NFATc1 has previously been used to assess calcineurin activity (3). However, the calcineurin pathway does not appear to affect muscle mass in these disuse models, inasmuch as blocking calcineurin activity with CsA, combined with disuse, did not act additively to reduce muscle mass. In fact, CsA had no effect on mass under any conditions, even though the serum levels of CsA were high. This is not unexpected in control and reloaded animals, inasmuch as NFATc1 was found only in the cytoplasm, suggesting that calcineurin is inactive in those muscles or there is an additional mechanism that removes NFATc1 from the nucleus. An alternative explanation is that even though calcineurin is elevated and activated in atrophying muscle, nuclear-localized NFATc1 may not be functional. Nuclear localization of NFATc1 does not necessarily mean that it is involved in transcription. DNA binding by NFATs is quite weak, and, as a consequence, the protein requires a partner for tight association with DNA (13). In atrophying muscles, it is likely that these partners are missing, because other growth signals, such as insulin-like growth factors, are decreased. However, MEF2 family proteins have been shown to cooperatively bind with NFAT (22), and their expression was also increased in hindlimb-suspended soleus muscle in this study. Therefore, the exact role of nuclear localization of NFAT in atrophied muscle remains to be determined.

It is also possible that during atrophy the increase in calcineurin expression (and possibly activity) is indeed acting as a compensatory mechanism to minimize or prevent further muscle loss and that CsA has properties that could affect the muscle independent of calcineurin. In addition to being an inhibitor of calcineurin, CsA has also been shown to act as an uncompetitive inhibitor of proteasome activity and to prevent nuclear factor-kappa B (NF-kappa B) activation (31). Muscle atrophy induced by hindlimb suspension is correlated with an increase in protein degradation (47). Thus, during hindlimb suspension, CsA may act to decrease degradation by inhibiting the proteasome, effectively balancing the detrimental result of its actions on calcineurin. In addition, NF-kappa B has been shown to mediate protein loss in muscle cells (23, 28); therefore, blocking activation of NF-kappa B by CsA during atrophy may counteract the loss of the beneficial effects of calcineurin due to CsA administration.

On the other hand, calcineurin in hindlimb-suspended muscles may not be associated with the control of muscle mass per se, but rather with apoptosis. Calcineurin has been shown to induce apoptosis in different neuronal cells (4, 48), thymocytes (26), fibroblasts (44), and cardiomyocytes (41). The proapoptotic effects of calcineurin seem to be NFAT independent and do not require new protein synthesis (44). Therefore, an increase in calcineurin activity could result in apoptosis, contributing to muscle atrophy. Calcineurin has been shown to be able to induce or suppress apoptosis in the same cells, depending on the cellular context (29); therefore, different intracellular pathways in muscles undergoing atrophy may be influencing calcineurin in opposing ways. We and others have shown that atrophy in soleus muscle is associated with an increase in apoptotic nuclei and that muscle mass-maintaining interventions decrease this nuclear death (2, 19). The fact that NFATc1 was not localized to nuclei in soleus muscles from rats that were hindlimb suspended and intermittently reloaded indicates that calcineurin may be inactive in these muscles, and therefore apoptosis may be suppressed with intermittent reloading.

Our results contradict a recent report showing that calcineurin was decreased on atrophy due to spinal cord transection in the vastus intermedius muscle (45). We suggest that the discrepancy between these two sets of data can be explained by the fact that muscle mass is regulated differently in muscles with dissimilar fiber type compositions. The rat soleus muscle is composed of mainly type I fibers, while the vastus intermedius muscle is of mixed fiber type (45). We found that plantaris muscles (composed of mainly type II muscle fibers) atrophied without a decrease in myofiber nuclear number, whereas in soleus muscle, myonuclear number decreases with atrophy, most likely due to apoptotic nuclear loss (19). Therefore, calcineurin may promote apoptosis and muscle atrophy in specific muscles, such as the soleus. Indeed, we found that calcineurin protein levels were not different between plantaris control and hindlimb-suspended rats (data not shown).

In summary, results presented in this study indicate that CsA treatment does not inhibit the beneficial effects of muscle-maintaining interventions during atrophy, suggesting that the calcineurin pathway is likely not involved in the control of normal muscle mass or in the maintenance of muscle mass during atrophy. However, because calcineurin levels were elevated with atrophy, it may have opposing roles in the regulation of muscle mass under these circumstances. Depending on the cellular and physiological context of the muscle, calcineurin may mediate hypertrophic effects or apoptosis.


    ACKNOWLEDGEMENTS

This research was supported by a research grant from the American Federation for Aging Research to E. E. Dupont-Versteegden and by National Institute of Neurological Disorders and Stroke Grant NS-40008 (to J. D. Houlé and C. A. Peterson).


    FOOTNOTES

Address for reprint requests and other correspondence: E. E. Dupont-Versteegden, Reynolds Dept. of Geriatrics, 4301 West Markham, Slot 807, Little Rock, AR 72205 (E-mail: dupontesthere{at}uams.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published January 30, 2002;10.1152/ajpcell.00424.2001

Received 4 September 2001; accepted in final form 29 January 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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