Thyroid hormone is required for the phenotype transitions induced by the pharmacological inhibition of calcineurin in adult soleus muscle of rats

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1 Am J Physiol Endocrinol Metab 294: E69 E77, First published October 30, 2007; doi: /ajpendo Thyroid hormone is required for the phenotype transitions induced by the pharmacological inhibition of calcineurin in adult soleus muscle of rats Nathalie Koulmann, 1 Lahoucine Bahi, 2 Florence Ribera, 1 Hervé Sanchez, 1 Bernard Serrurier, 1 Rachel Chapot, 1 André Peinnequin, 1 Renée Ventura-Clapier, 2 and Xavier Bigard 1 1 Centre de Recherches du Service de Santé des Armées, La Tronche, Cedex; and 2 Inserm U769, Université Paris-Sud, Châtenay-Malabry, France Submitted 16 March 2007; accepted in final form 16 October 2007 Koulmann N, Bahi L, Ribera F, Sanchez H, Serrurier B, Chapot R, Peinnequin A, Ventura-Clapier R, Bigard X. Thyroid hormone is required for the phenotype transitions induced by the pharmacological inhibition of calcineurin in adult soleus muscle of rats. Am J Physiol Endocrinol Metab 294: E69 E77, First published October 30, 2007; /ajpendo The present experiment was designed to examine the effects of hypothyroidism and calcineurin inhibition induced by cyclosporin A (CsA) administration on both contractile and metabolic soleus muscle phenotypes, with a novel approach to the signaling pathway controlling mitochondrial biogenesis. Twenty-eight rats were randomly assigned to four groups, normothyroid, hypothyroid, and orally treated with either CsA (25 mg/kg, N-CsA and H-CsA) or vehicle (N-Vh and H-Vh), for 3 wk. Muscle phenotype was estimated by the MHC profile and activities of oxidative and glycolytic enzymes. We measured mrna levels of the peroxisome proliferator-activated receptor- coactivator-1 (PGC-1 ), the major regulator of mitochondrial content. We also studied the expression of the catalytic A-subunit of calcineurin (CnA) both at protein and transcript levels and mrna levels of modulatory calcineurin inhibitor proteins (MCIP)-1 and -2, which are differentially regulated by calcineurin activity and thyroid hormone, respectively. CsA-administration induced a slow-to-fast MHC transition limited to the type IIA isoform, which is associated with increased oxidative capacities. Hypothyroidism strongly decreased both the expression of fast MHC isoforms and oxidative capacities. Effects of CsA administration on muscle phenotype were blocked in conditions of thyroid hormone deficiency. Changes in the oxidative profile were strongly related to PGC-1 changes and associated with phosphorylation of p38 MAPK. Calcineurin and MCIPs mrna levels were decreased by both hypothyroidism and CsA without additive effects. Taken together, these results suggest that adult muscle phenotype is primarily under the control of thyroid state. Physiological levels of thyroid hormone are required for the effects of calcineurin inhibition on slow oxidative muscle phenotype. muscle phenotype; hypothyroidism; myosin heavy chain; oxidative capacities ADULT SKELETAL MUSCLE comprises distinct fiber types, each of which possesses a unique combination of functional, biochemical, and metabolic properties (27). Myofiber differences in contractile phenotype result mainly from the different isoforms of the myosin heavy chain (MHC) family, whereas metabolic characteristics depend on mitochondrial density and function as well as relative activities of oxidative vs. glycolytic enzymes. Commonly, slow type I fibers express higher levels of oxidative enzymes and lower levels of glycolytic enzymes than Address for reprint requests and other correspondence: N. Koulmann, Département des facteurs humains, Centre de recherches du service de santé des armées, BP La Tronche Cedex, France ( those found in fast type II fibers. However, in rodents, whereas type IIB and IIX fibers have low oxidative capacity, type IIA fibers exhibit higher oxidative and glycolytic capacity than type I fibers (30). Mammalian skeletal muscle is a dynamic system that has a remarkable capacity to adapt to changes imposed by muscle use and environment. Repeated thyroid hormone administration leads to a slow-to-fast shift in MHC isoforms, whereas increased muscle contractile activity has opposite effects (reviewed in Ref. 27). The relative importance and the combined effects of alterations in thyroid hormone and muscular functional loading on MHC expression have been studied extensively, and it has been shown that hypothyroidism predominates over the loading state in the determination of MHC distribution in slow muscle (reviewed in Ref. 4). Muscle loading state is modulated mainly by nerve activity and calcineurin activity, a calcium/calmodulin-dependent serine/ threonine protein phosphatase that is one of the main downstream effectors of the pattern of motor nerve activity (8). A role for calcineurin in the regulation of muscle fiber type was first suggested by the finding that constitutively active calcineurin selectively upregulates slow fiber-specific gene promoters in cultured muscle cells and administration of the pharmacological inhibitor cyclosporin A (CsA) promotes slowto-fast fiber transformation (8). However, the use of pharmacological inhibitors, especially CsA, sometimes led to conflicting results about the role of calcineurin on the control of muscle phenotype. As previously emphasized, discrepancies might depend on the dose of the drug, the length of treatment, the species investigated, and the type of muscle (28). Subsequent studies, and particularly those using a genetic approach to induce changes in calcineurin activity (5, 20), have confirmed that calcineurin activity regulates the slow fiber program. In parallel with MHC isoforms, calcineurin activity and thyroid hormones affect the specific activity of enzymes involved in energy metabolism, such as MM-creatine kinase isozyme (MM-CK) and the M-subunit of lactate dehydrogenase (M-LDH) (7), as well as the expression of a large subset of genes representative of the mitochondrial content within soleus muscle (SOL) (29, 35). Previous studies (3, 13) showed that the mitochondrial biogenesis induced by increased contractile activity and thyroid hormone treatment is mediated via the peroxisome proliferator-activated receptor- coactivator-1 (PGC-1 ), which has been suggested to also control type I MHC expression (17). 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 /08 $8.00 Copyright 2008 the American Physiological Society E69

2 E70 It is now well known that sustained elevations in intracellular calcium, such as those induced by tonic motor nerve activity, have long-term effects on the control of gene expression in myofiber through activation of calcineurin (22). Calmodulin complexed with Ca 2 is the only known activator of calcineurin. However, a wide range of protein inhibitors of calcineurin has been described, including cain/cabin and members of the modulatory calcineurin-interacting protein (MCIP) family (24). Two main MCIP isoforms, MCIP-1 and MCIP-2, are known to interact with calcineurin and to be subjected to distinctive mechanisms of regulation. In human and rodents, MCIP-1 is expressed primarily in heart and skeletal muscle, and transcription of the mcip-1 gene is enhanced by calcineurin activation, thereby establishing a negative feedback (34). In contrast, MCIP-2 transcript levels are increased by thyroid hormone (34). Calcineurin activity and thyroid hormone are thus expected to interact on downstream targets involved in the control of muscle phenotype through the level of MCIP-1 and MCIP-2 transcription, respectively. One of the best means to study the interaction between various regulators of the control of muscle phenotype is to examine their degree of cooperativity or competition on specific biological targets (9, 16). To further examine the interaction between thyroid hormone and calcineurin signaling pathways on muscle phenotype, we contrasted two competing influences known to exert opposite effects on muscle phenotype, i.e., calcineurin inhibition by cyclosporin (CsA) administration, which drives a slow-to-fast IIA transition with increased oxidative and glycolytic activities, and thyroid deficiency, which leads to a slower phenotype with decreased metabolic capacities. Studying both the respective and competing effects of CsA administration and hypothyroidism on different markers and regulators of contractile and metabolic properties would help to highlight the mechanisms by which these signaling pathways control muscle phenotype. The present experiment was then designed to further examine the effects of calcineurin inhibition induced by CsA administration and hypothyroidism on both contractile and metabolic phenotype, with a novel approach to the signaling pathway controlling mitochondrial biogenesis. To address this issue, we studied the MHC expression at both the protein and mrna levels and activities of several metabolic enzymes in SOL of normothyroid and hypothyroid rats treated by either CsA or its vehicle daily for 3 wk. Moreover, the expected alterations in muscle oxidative capacity were studied in relation to changes in PGC-1 mrna levels and phosphorylated state of the p38 mitogen-activated protein kinase (MAPK), one of the major regulators of PGC-1 expression (2, 12). We also examined the effects of CsA treatment and hypothyroidism on the calcineurin mrna and protein levels and on the transcript levels of two calcineurin inhibitors, MCIP-1 and MCIP-2. MATERIALS AND METHODS Animals. Twenty-eight male Wistar rats initially weighing g were purchased from IFFA Credo (L Arbresle, France). All animals were maintained on a standard diet with water ad libitum. They were housed four per cage in a room with regulated temperature (22 2 C) on a 12:12-h photo period. This investigation was carried out in accordance with the Helsinki Accords for Humane Treatment of Animals during Experimentation and was approved by the Institutional Review Committee at Centre de Recherches du Service de Santé des Armées, La Tronche, France. Experimental design. The animals were randomly assigned to four experimental groups (n 7 for each group) for 3 wk: normothyroid, hypothyroid, orally treated with a daily dose of 25 mg/kg CsA as Sandimmun (N-CsA and H-CsA groups), or orally treated with an equivalent dose of vehicle (N-Vh and H-Vh groups). Hypothyroidism was induced by thyroidectomy and administration of propylthiouracyl to drinking water (0.02% wt/vol). The complete vehicle of 25 mg/kg Sandimmun was reconstituted from two-thirds cremophor EL (BASF) and one-third alcohol (vol/vol). Doses of Sandimmun and vehicle were adjusted according to the weight gain as previously described (7). Tissue processing. Following 3 wk of treatment, animals were anesthetized with pentobarbital sodium (90 mg/kg body wt) administered intraperiteonally. SOLs were excised, cleaned of adipose and connective tissue, and weighed. Muscles were immediately frozen in liquid nitrogen for histochemical and biochemical determinations. All samples were stored at 80 C until analyses were performed. Blood cyclosporin level. Blood samples were taken from the abdominal aorta of rats 20 h after the last dose of CsA at the time of death. Levels were assayed by using a whole blood fluorescence polymerization immunoassay (Aventis Behring, Paris, France). Concentration of free triiodothyronine in serum. After blood sampling at the time of death, an aliquot of 200 l of serum was obtained by the usual methods and frozen at 20 C until assay. The concentration of free triiodothyronine (FT 3) in serum was determined in duplicate by radioimmunological assay (RIA-gnost FT 3; CIS Bio International, Gif-sur-Yvette, France). Analysis of MHC protein content by Western blotting. Samples from soleus muscles were homogenized in 50 volumes of the same extraction buffer as described above. The protein content was determined by using Bradford method. Equal amounts of muscle protein were then separated by SDS-PAGE. A standardized amount of protein prepared from intact SOL was also applied on each gel to serve as an internal standard for comparison across blots. Proteins were then transferred onto nitrocellulose sheets [Hybond enhanced chemiluminescence (ECL) Western; Amersham Pharmacia Biotech, Orsay, France]. The blots were blocked in 5% nonfat dry milk diluted in 0.1% Tween-20 in Tris-buffered saline (ph 8; Sigma, Saint- Quentin Fallavier, France) and then incubated with the primary mouse antibody against either slow type I MHC (1:200 dilution, NCL- MHCS; Novocastra, Newcastle upon Tyne, UK) or all fast type II MHC (1:1,000 dilution, MY-32; Sigma, St. Louis, MO). After being washed the blots were then incubated with horseradish peroxidaseconjugated goat anti-mouse IgG (1:2,000 dilution, sc-2005; Santa Cruz Biotechnology, Heidelberg, Germany). Washed blots were subjected to the ECL Western blotting detection reagent kit (Amersham Pharmacia Biotech) and then exposed to X-ray film (Hyperfilm ECL; Amersham Pharmacia Biotech). The relative MHC expression was determined by the ratio of sample band intensity/internal standard band intensity, using a densitometer system equipped with an integrator (GS-700; Bio-Rad, Marne-la-Coquette, France). Analysis of the distribution of MHC isoforms. Muscles were subjected to the analysis of MHC isoforms as described previously (31). Myosin was extracted from small sections of muscles in seven volumes of buffer solution (0.3 M NaCl, 0.1 M NaH 2PO 4, 0.05 M Na 2HPO 4,0.01MNa 4P 2O 7,1mMMgC 12-6H 2O, 10 mm EDTA, 1.4 mm 2 -mercaptoethanol, ph 6.5). Electrophoresis was performed using a Mini Protean II system (Bio-Rad, Marne-la-Coquette, France) with 8 and 4% acrylamide-bis (50:1) separating and stacking gels, respectively, containing 0.4% sodium dodecyl sulfate (SDS). Myofibril samples were denatured with the SDS incubation medium according to the method of Laemmli. Gels were run at constant voltage (72 V) for 31 h and then silver stained (1). The MHC protein isoform bands were scanned and quantified using the densitometer system described above.

3 E71 Analysis of MHC, MCIP-1, MCIP-2, calcineurin, and PGC-1 mrna. mrna was extracted from 10 mg of frozen muscle samples disrupted using two tungsten carbide beads in 300 l of lysis buffer RLT (Qiagen, Courtaboeuf, France). Then the extraction was carried out with MagNA Pure (Roche Diagnostics, Meylan, France) using the MagNA Pure LC mrna Isolation Kit II (tissues) procedure, following the manufacturer s protocol. mrna samples were eluted in 50 l final volume and then stored at 80 C until subsequent analyses. All oligonucleotide primers used in this study were synthesized by Eurogentec (Seraing, Belgium) and designed with MacVector software (Accelrys, San Diego, CA). MHC isoform primers have been described elsewhere (25). Other primers are described in Table 1. Reverse transcription was carried out using the Reverse Transcription Core Kit (Eurogentec, Saraing, Belgium) with 2.5 M oligo(dt) 15 primer according to the manufacturer s instructions. Quantitative PCR was made by using the LC Fast-Start DNA Master SYBR Green kit with LightCycler (Roche Applied Science, Meylan, France). The threshold cycle value was calculated from LightCycler software. Quantification was achieved using a pool of cdna samples as calibrator according to the comparative threshold cycle method (18). Accurate normalization was carried out by geometric averaging of two endogenous housekeeping genes, hypoxanthine-guanine phosphoribosyltransferase (HPRT) I and cyclophilin A (32), after verification that their mrna do not vary inside this experiment. Relative mrna values were calculated with RealQuant software (Roche Applied Science, Mannheim, Germany). Activities of metabolic enzymes. Frozen tissue samples were weighed and placed into an ice-cold homogenization buffer (30 mg wet wt/ml) containing 5 mm HEPES (ph 8.7), 1 mm EGTA, 1 mm dithiothreitol, 5 mm MgCl 2, and 0.1% Triton. Samples were homogenized using a microglass hand homogenizer and were incubated for 60 min at 0 C to ensure complete enzyme extraction. Citrate synthase (CS), creatine kinase (CK), and lactate dehydrogenase (LDH) activities were measured at 30 C (ph 7.5) using coupled enzyme systems as previously described (6). CK isoenzymes were separated using agarose (1%) gel electrophoresis performed at 200 V for 90 min. To avoid saturation of the various CK isoforms, three dilutions were used for each sample. Individual isoenzymes were resolved by incubating the gel with a paper soaked with staining solution containing 22 mm MES (ph 7.4), 50 mm magnesium acetate, 70 mm glucose, 120 mm n-acetyl cysteine, 9 mm ADP, 120 mm phosphocreatine, 9 mm NADP, 0.1 mm P1, P5-di (adenosine-59) pentaphosphate (to inhibit adenylate kinase), 9 IU/ml hexokinase, and 6 IU/ml glucose-6-phosphate dehydrogenase. Isoenzyme bands were visualized and quantified using an image analysis system (Bio-Rad). The LDH isoenzyme profile was determined using agarose gel electrophoresis (LDH reagent kit; Sigma) at 200 V for 90 min followed by image analysis. Western blot analysis for calcineurin and p38 MAPK. Samples from SOL were homogenized in ice-cold lysis buffer containing either 1% Nonidet P-40, 0.1% (wt/vol) sodium dodecyl sulphate, 0.5% sodium deoxycholate, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 M leupeptin, and pepstatin or 20 mm HEPES, ph 7.4, 2 mm EGTA, 50 mm -glycerophosphate, 1 mm dithiothreitol, 1 mm Na 3VO 4, 1% Triton X-100, 10% glycerol, and 1 l/ml protease inhibitor cocktail set III (Calbiochem, Fontenay-sous-bois, France) for calcineurin and p38 MAPK, respectively. Samples were rotated for 1 h at 4 C and centrifuged (15,000 g, 15 min, 4 C). Protein content was determined using the bicinchoninic acid method (Roche/Hitachi 912 instrument; Roche Diagnostics, Mannheim, Germany). Equal amounts of muscle protein were then separated by SDS-PAGE. A standardized amount of protein prepared from intact SOL was also applied on each gel to serve as an internal standard for comparison across blots. Proteins were then transferred onto nitrocellulose sheets (Hybond ECL Western; Amersham Pharmacia Biotech). The blots were blocked in 2% BSA and 2% nonfat dry milk diluted in 0.1% Tween-20 in Tris-buffered saline (ph 8; Sigma) and then incubated either with the primary rabbit antibody against PP2B-A (1:400 dilution, sc-9070; Santa Cruz Biotechnology, Heidelberg, Germany) or with anti-phospho-p38 MAP kinase (Thr 180 / 182 ) antibody (Cell Signaling Technology, Beverly, MA). After being washed the blots were then incubated with horseradish peroxidase-conjugated goat antirabbit IgG (1:2,500 dilution, sc-2004; Santa Cruz Biotechnology, Heidelberg, Germany). Washed blots were subjected to the ECL Western blotting detection reagent kit (Amersham Pharmacia Biotech) and then exposed to X-ray film (Hyperfilm ECL; Amersham Pharmacia Biotech). The relative calcineurin or phosphorylated p38 (P-p38) expression was determined by the ratio of sample band intensity/internal standard band intensity using the densitometer system GS-700 (Bio-Rad, Marnes-la-Coquette, France). Membranes previously conjugated with P-p38 were then stripped in a solution containing 60 mm Tris HCl, ph 7.4, 5% SDS, and 0.07% -mercaptoethanol for 30 min at 50 C, washed with Tris-buffered saline- Tween-20, and incubated overnight with anti p38 MAPK antibody (Cell Signaling Technology) and treated as described above. The bands obtained were quantified using the densitometer system de- Table 1. Nucleotide sequences for rat Primers Sequence Length, pb Gene Bank Accession No. Calcineurin 5 -TGGTCTTTGCCTTTTGTTGGAG-3 5 -TGGCTCCTTCCTCTTCGACTAAG NM_ MCIP-1 5 -GACTTTAACTACAATTTTAGCTCCCTGAT-3 5 -TTGGCCCTGGTCTCACTTTC-3 80 NM_ MCIP-2 5 -GAGACCCAGTTCAGAGGGAAGAAG-3 5 -TGAAGGGGGTGAGATGAGGAAC NM_ PGC-1 5 -ACGCAGGTCGAATGAAACTGAC-3 5 -TGGTGGAAGCAGGGTCAAAATC NM_ HPRT 5 -CTCATGGACTGATTATGGACAGGAC-3 5 -GCAGGTCAGCAAAGAACTTATAGCC NM_ CycA 5 -AGCATGTGGTCTTTGGGAAGGTG-3 5 -CTTCTTGCTGGTCTTGCCATTCC-3 92 NM_ MCIP, modulatory calcineurin-interacting protein; PGC-1, peroxisome proliferator-activated receptor- coactivator-1 ; HPRT, hypoxanthine-guanine phosphoribosyltransferase; CycA; cyclophilin A.

4 E72 Table 2. Morphological data from N and H animals treated with either Vh or CsA (N-Vh, N-CsA, H-Vh, and H-CsA, respectively) N-Vh N-CsA H-Vh H-CsA CsA effect Hypothyroidism effect Interaction FT 3 levels, pg/ml * P 0.05 P P 0.05 Body weight, g *** ** P P NS SOL absolute weight, mg *** P NS (P 0.08) P 0.01 SOL relative weight, mg/g NS P P 0.05 Values are means SE. N, normothyroid; H, hypothyroid; Vh, vehicle; CsA, cyclosporin A; FT 3, free triiodothyronine; SOL, soleus muscle; NS, nonsignificant. *P 0.05, **P 0.01, and ***P 0.001, significant difference between corresponding Vh and CsA groups (i.e., N-Vh vs. N-CsA and H-Vh vs. H-CsA groups). P 0.05 and P 0.001, significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups). ANOVA scribed above. The state of phosphorylation of p38 for each muscle sample was expressed as the ratio of P-p38 to p38 of the sample. Statistical analysis. All data are presented as means SE. Statistical changes in body weight of animals were examined using Student s t-test. CsA treatment and hypothyroidism effects were determined by a two-way analysis of variance. When appropriate, differences between groups were tested with a Newman-Keuls post hoc test. Statistical significance was accepted at P MHC-IIB, and MHC-emb transcript levels were affected by neither thyroid status nor CsA treatment, whereas MHC-neo transcript levels remained almost undetectable. MHC protein levels. No effects of hypothyroidism or CsA treatment were shown on the protein levels of MHC-I (Fig. 2A). On the contrary, there was a significant effect of both RESULTS Blood cyclosporin levels. The mean cyclosporin level in blood of animals treated with CsA was 2, and 2, ng/ml in N-CsA and H-CsA group, respectively. Then the bioavailability of the drug was in the same range as previously reported (7) and similar in both normothyroid and hypothyroid animals. Plasma FT 3 levels. Both hypothyroidism (P 0.001) and CsA treatment (P 0.05) decreased plasma FT 3 levels, with an interaction between these factors (P 0.05; Table 2). Repeated CsA administration decreased mean FT 3 levels in normothyroid rats (26%, P 0.01). As expected, hypothyroidism dramatically decreased the plasma FT 3 concentration, with no difference between CsA-treated and Vh-treated animals. Body and muscle weights. Both CsA treatment and hypothyroidism decreased the body weight of animals (P 0.001; Table 2), with additive effects in H-CsA group (36% compared with N-Vh group; 22%, P 0.01, compared with N-CsA; and 12%, P 0.05, compared with H-Vh). There was a global decrease in SOL absolute weight after CsA treatment (P 0.001; Table 2), with a significant interaction between CsA administration and hypothyroidism (P 0.01). Hence, CsA treatment decreased the SOL absolute weight only in normothyroid rats (26%, P 0.01), whereas there was no difference between H-Vh and H-CsA groups. When normalized to body weight, the relative weight of SOL increased by hypothyroidism (P 0.001), but only in CsA-treated animals (interaction P 0.05). MHC mrna levels. No effects of hypothyroidism or CsA treatment were shown on the transcript levels of MHC-I (Fig. 1A). By contrast, MHC-IIA transcript levels were markedly affected both by hypothyroidism and CsA treatment (P 0.001), with a significant interaction between these two factors (P 0.001) (Fig. 1B). MHC-IIA mrna levels decreased with hypothyroidism and increased with CsA administration, but only in normothyroid rats (P 0.001). Consistent with changes in relative protein levels, this CsA-induced increase was completely abolished in hypothyroid rats. MHC-IIX, Fig. 1. Transcript levels of myosin heavy chain (MHC) isoforms. Total RNA obtained from soleus muscles of normothyroid (N) and hypothyroid (H) rats treated by either cyclosporin A (CsA) or vehicle (Vh) was reverse transcribed and amplified using real-time quantitative PCR. Normalization of MHC mrna expression was done to the geometric average of cyclophilin A and hypoxanthine-guanine phosphoribosyltransferase (HPRT) I mrna levels determined by real-time PCR. MHC-I mrna (A) were expressed as levels higher than MHC-II mrna (B). Significant difference between corresponding Vh and CsA groups (i.e., N-Vh vs. N-CsA and H-Vh vs. H-CsA groups), *P Significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups), P

5 Fig. 2. MHC protein levels. Protein levels of MHC-I (A) and MHC-II (B) were estimated by Western blotting using antibodies against either slow-type I MHC (1:200 dilution, NCL-MHCS; Novocastra, Newcastle upon Tyne, UK) or all fast type II MHC (1:1,000 dilution, MY-32; Sigma, St. Louis, MO). Significant difference between corresponding Vh and CsA groups (i.e., N-Vh vs. N-CsA and H-Vh vs. H-CsA groups), *P Significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups), P hypothyroidism (P 0.001) and CsA treatment (P 0.05) on the protein levels of MHC-II (Fig. 2B). MHC-II protein levels increased with CsA administration only in normothyroid rats (P 0.05) and decreased in hypothyroid rats with lower levels in H-CsA rats than in N-CsA (P 0.01). MHC distribution using SDS-PAGE. Only MHC-I and MHC-IIA isoforms were detected using SDS-PAGE analysis. The relative amount of MHC-I and MHC-IIA was affected both by CsA treatment (P 0.001) and thyroid status (P 0.001; Table 3). Moreover, a strong interaction between these two factors was shown (P 0.001). CsA treatment decreased the relative amount of MHC-I and increased the relative amount of MHC-IIA only in normothyroid rats (P 0.001); no detectable change occurred in hypothyroid animals. Biochemical determinations. Enzyme activities in SOL are shown in Table 4 and Fig. 3. The activity of citrate synthase (CS), an enzyme of the tricarboxylic acid cycle, was influenced Table 3. MHC distribution in SOL muscles assessed by SDS-PAGE and silver staining by both CsA treatment (P 0.01) and thyroid status (P 0.001), with an interaction between these factors (P 0.01) (Fig. 3). CsA treatment increased CS activity only in normothyroid rats (P 0.01), whereas hypothyroidism decreased CS activity both in Vh- and CsA-treated rats (P 0.001). Total CK activity, the main phosphotransfer enzyme, was affected only by thyroid status (P 0.001), with decreased levels reported in both Vh- and CsA-treated groups (34 and 37%, respectively, P 0.001) (Table 4). The specific activity of CK isoforms also decreased by hypothyroidism (P 0.001). Hypothyroidism decreased the cytosolic muscle CK isoform, MM-CK, by 34 and 36% in Vh and CsA groups, respectively (P 0.001). The mitochondrial CK isoform, mi-ck, was affected both by CsA treatment (P 0.05) and thyroid status (P 0.001). Mi-CK activity decreased in SOL muscles of hypothyroid rats after either Vh or CsA administration (32%, P 0.05, and 57%, P 0.001, respectively). Moreover, hypothyroidism decreased the total LDH activity by 16 and 23% in Vh and CsA groups, respectively (P 0.05). The specific activity of LDH subunits was also affected by thyroid status (P 0.01). Hypothyroidism decreased the specific activity of the M-subunit in Vh- and in CsA-treated rats (21%, P 0.01, and 29%, P 0.001, respectively). Moreover, CsA treatment affected only the activity of the M-subunit, and only in normothyroid animals (22%, P 0.05). In the present study, CsA administration failed to affect the H-subunit activity. As for contractile phenotype, metabolic changes induced by CsA in SOL muscle were reported only in normothyroid rats and nearly abolished in hypothyroid animals. Analysis of MCIP-1 and MCIP-2 mrna levels. Both hypothyroidism and CsA treatment significantly decreased MCIP-1 and MCIP-2 mrna levels (P 0.01 and P 0.05 for MCIP-1 and MCIP-2, respectively), with an interaction between these factors (P 0.01 and P 0.05 for MCIP-1 and MCIP-2, respectively) (Fig. 4). CsA treatment decreased both MCIP-1 and MCIP-2 mrna levels in normothyroid rats (P 0.01). Moreover, MCIP-1 and MCIP-2 mrna levels were strongly decreased in hypothyroid rats (P and P 0.05 for MCIP-1 and MCIP-2, respectively), with no difference between H-Vh and H-CsA groups. Catalytic A-subunit of calcineurin mrna and protein levels. Both CsA treatment and hypothyroidism decreased catalytic A-subunit of calcineurin (CnA) mrna levels (P 0.05), whereas only CsA treatment decreased calcineurin protein levels (P 0.05; Fig. 5). There was a strong interaction between these factors (P 0.01). CsA administration halfdecreased CnA mrna and protein in normothyroid rats (P 0.01 and P 0.05 for mrna and protein, respectively). Moreover, hypothyroidism reduced calcineurin mrna levels, but only in Vh-treated groups (P 0.01). There were no ANOVA E73 N-Vh N-CsA H-Vh H-CsA CsA effect Hypothyroidism effect Interaction Type I MHC, % * P P P Type IIA MHC, % * P P P Values are means SE. Myosin heavy chain (MHC) isoforms are expressed as the relative proportion of total MHC. *P 0.001, different from corresponding Vh-treated animals. P 0.001, different from corresponding normothyroid animals.

6 E74 Table 4. Energy metabolism in SOL ANOVA N-Vh N-CsA H-Vh H-CsA CsA effect Hypothyroidism effect Interaction Total CK , NS P NS MM-CK , NS P NS mi-ck P 0.05 P NS Total LDH NS P NS Activity H-subunit NS P 0.01 NS Activity M-subunit * P 0.05 P 0.01 NS Values are means SE. CK, creatine kinase; MM-CK, cytosolic muscle CK isoform; mi-ck, mitochondrial CK isoform; LDH, lactate dehydrogenase. Enzyme activities are expressed as units of activity per gram of tissue wet weight (IU/g). *P 0.01, significant difference from corresponding Vh-treated animals. P 0.05 and P 0.001, significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups). significant differences in CnA mrna and protein levels between H-CsA group and either N-CsA or H-Vh group. Analysis of PGC-1 mrna levels. PGC-1 transcript levels were significantly increased by CsA treatment only in normothyroid rats (56%, P 0.05; Fig. 6A). There was no response from PGC-1 transcript levels to CsA treatment in hypothyroid rats. Hypothyroidism strongly decreased PGC-1 transcript levels both in Vh- (61%, P 0.01) and CsA-treated animals (69%, P 0.01). p38 MAPK phosphorylation. There was no significant effect of either hypothyroidism or CsA treatment on the total level of p38 MAPK protein (data not shown). Both hypothyroidism (P 0.001) and CsA treatment (P 0.05) affected the p38 MAPK pathway activity, as assessed by the ratio of P-p38 to total p38 protein, with a strong interaction between these factors (P 0.001; Fig. 6B). Activity of the p38 MAPK pathway increased in normothyroid animals (P 0.05), whereas hypothyroidism strongly decreased it in Vh- and CsA-treated rats (P 0.001). hypothyroidism were similar in soleus muscle of rats treated or not by CsA); 2) changes in the oxidative profile were strongly related to alterations in PGC-1 mrna, which were associated with changes in the phosphorylation state of p38 MAPK; 3) thyroid deficiency per se decreased MCIP-1 mrna abundance, a marker of calcineurin activity; and 4) both calcineurin inhibition and thyroid deficiency strongly decreased MCIP-2 mrna, without additive effects. Taken together, present results show that thyroid hormone is required for CsA effects on DISCUSSION The present experiment was designed to examine both the respective and combined effects of calcineurin inhibition induced by CsA administration and hypothyroidism on MHC transition and metabolic profile of soleus muscle in rats. The main results were the following: 1) the specific effects of CsA administration on MHC and metabolic profile were blocked when thyroid hormone was decreased, whereas effects of Fig. 3. Activity of the citrate synthase, an enzyme of the tricarboxylic acid cycle used as a marker of oxidative capacities, in soleus muscles of N and H rats treated with either CsA or Vh. Significant difference between corresponding Vh and CsA groups (i.e., N-Vh vs. N-CsA and H-Vh vs. H-CsA groups), *P Significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups), P Fig. 4. Transcript levels of the modulatory calcineurin-interacting protein-1 (MCIP-1; A) and MCIP-2 (B) in soleus muscles of N and H rats treated with either CsA or Vh. Total RNA obtained from soleus muscles was reverse transcribed and amplified using real-time quantitative PCR. Normalization of MCIP mrna expression was done to the geometric average of cyclophilin A and HPRT mrna levels determined by real-time PCR. Significant difference between corresponding Vh and CsA groups (i.e., N-Vh vs. N-CsA and H-Vh vs. H-CsA groups), *P 0.05 and **P Significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups), P 0.05 and P

7 E75 to changes in MHC-IIA mrna, variations in MHC-IIA protein can be considered as due mainly to pretranslational events. Moreover, because CsA administration had no effect on MHC-I expression, whereas MHC-IIA transcript and protein levels increased, it can be suggested that calcineurin activity mainly repressed MHC-IIA expression. CsA administration was associated with an increase in the specific activity of M-LDH, the major isoform in glycolytic muscle, and a slight, albeit nonsignificant, increase in total and MM-CK activities (9 and 10%, respectively). Thus CsA administration induced changes in the contractile and glycolytic profile, consistent with a slow-to-fast phenotype transition (7). Moreover, we (7, 26) also found an increase in citrate synthase activity in the soleus muscle of CsA-treated rats, representative of an increase in the mitochondrial content. The PGC-1 protein coactivates transcription factors responsible for mediating the coordinated expression of mitochondrial proteins and mitochondrial biogenesis (15, 23). Interestingly, we show for the first time that CsA administration increased PGC-1 transcript levels in muscle, consistent with the increased oxidative capacity and CS expression in soleus muscle (7, 26) and the CsA-induced increase in MHC-IIA expression (30). This result is at first Fig. 5. Calcineurin transcript (A) and protein levels (B) in soleus muscles of N and H rats treated with either CsA or Vh. Total RNA obtained from soleus muscles was reverse transcribed and amplified using real-time quantitative PCR. Normalization of calcineurin mrna expression was done to the geometric average of cyclophilin A and HPRT mrna levels determined by real-time PCR. Calcineurin protein levels were estimated by Western blotting using the sc-9070 antibody (Santa Cruz Biotechnology). Significant difference between corresponding Vh and CsA groups (i.e., N-Vh vs. N-CsA and H-Vh vs. H-CsA groups), *P 0.05 and **P Significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups), P soleus muscle phenotype and plays a regulatory role on calcineurin activity by controlling MCIPs mrna abundance. As previously shown in several studies, both thyroid deficiency and CsA administration slowed down the body weight gain of animal models. CsA consistently depressed the body weight growth rate together with a parallel decrease in skeletal muscle mass (6, 26). Using untreated rats fed with the same amount of food ingested by CsA-treated rats, we previously showed that the CsA-induced depression in growth rate was attributed mainly to a decrease in food consumption (body weight of CsA-treated and nontreated pair-fed rats, and g, respectively; Koulmann N and Bigard X, unpublished data). Although this issue needs to be carefully addressed in the future, the CsA-induced alterations in body weight and muscle mass seem to be related mainly to a central depression of food intake rather than to peripheral toxic effects. Consistent with previous studies, calcineurin inhibition by CsA administration induced a transition in the MHC profile of soleus muscle restricted to MHC-I toward MHC-IIA (7, 8), due to an increase in MHC-II protein levels, specifically in MHC-IIA, without detectable change in MHC-I protein levels. Considering that MHC-II protein levels vary accordingly Fig. 6. Peroxisome proliferator-activated receptor- coactivator-1 (PGC-1 ) transcript levels (A) and phosphorylated state of p38 MAPK (B). Total RNA obtained from soleus muscles of N and H rats treated either by CsA or Vh was reverse transcribed and amplified using real-time quantitative PCR. Normalization of PGC-1 mrna expression was done to the geometric average of cyclophilin A and HPRT mrna levels determined by real-time PCR. The state of phosphorylation of p38 (P-p38) for each muscle sample was expressed as the ratio of P-p38 to p38 of the sample. P-p38 and total p38 levels were estimated by Western blotting using anti-phospho-p38 MAPK (Thr 180/182 ) and p38 MAPK antibodies (Cell Signaling Technology). Significant difference between corresponding Vh and CsA groups (i.e., N-Vh vs. N-CsA and H-Vh vs. H-CsA groups), *P Significant difference between corresponding N and H groups (i.e., N-Vh vs. H-Vh and N-CsA vs. H-CsA groups), P 0.01 and P

8 E76 surprising because calcineurin has been reported (17) to activate PGC-1 expression. However, such a discrepancy was recently reported (11) following exercise training in triceps muscle. This finding clearly suggests that different signaling pathways are involved in regulating PGC-1 expression in skeletal muscle. Since it has been shown (2) that upregulation of PGC-1 transcript level is strongly dependent upon the phosphorylation of the p38 MAPK, at least during exercise, we decided to verify the level of phosphorylation of p38 under CsA treatment. We found that CsA administration for 3 wk induced an increase in the phosphorylated state of p38, which may explain the increase in PGC-1 mrna and then the increase in oxidative capacities observed in soleus muscles of CsA-treated rats. Whether the effect of CsA on the phosphorylation of p38 is a direct effect of this pharmacological compound or due to the inhibition of the phosphatase calcineurin is not known yet. However, it has been established that calcineurin and MAPK pathways are interconnected and that, in particular, calcineurin is able to dephosphorylate p38 MAPK (19), which could explain the CsA-induced increase in p38 MAPK phosphorylation. Hypothyroidism markedly decreased the expression of both transcript and protein levels of fast MHC isoforms without any changes in MHC-I expression. This finding provides additional support to the hypothesis that thyroid hormone is necessary for the expression of fast MHC isoforms (14, 16). Hypothyroidism induced a concomitant decrease in all the activities, either oxidative or glycolytic, of metabolic enzymes. Although the decrease in glycolytic enzymes is consistent with the decrease in fast MHC isoforms, the decrease in oxidative capacities without detectable change in MHC-I expression underlines the dissociated regulation of contractile and metabolic phenotype under altered thyroid state. It has been previously shown that PGC-1 responds to thyroid hormone treatment (13, 33). We show here that thyroid deficiency dramatically decreased PGC-1, at least partly in response to decreased levels of phosphorylated p38 and/or calcineurin activity, and that this decrease led to decreased levels of oxidative enzyme activities. This result further supports the hypothesis that PGC-1 may mediate some of the thyroid hormone actions on mitochondrial biogenesis and metabolic rate in skeletal muscle (3). In the present study, two interventions known to have opposite effects on muscle phenotype were competed. The results demonstrate that thyroid deficiency neutralized the effects of pharmacological inhibition of calcineurin activity. Calcineurin dephosphorylates the nuclear factors of activated T cells (NFAT), allowing them to translocate to the nucleus and to activate the transcription of genes involved in muscle phenotype. To understand the alteration of calcineurin activity in hypothyroid state, we measured transcript levels of MCIP-1, which contains 15 repeats of the NFAT binding site and thus has been shown (34) to be the most sensitive indicator of calcineurin transcriptional activity. CsA treatment strongly decreased both MCIP-1 and MCIP-2 mrna levels. It has been shown previously (34) that only MCIP-1 mrna increased with enhanced calcineurin activity, and only MCIP-2 mrna levels increased in hearts of thyrotoxic rodents, with unchanged MCIP-1 transcript levels. Surprisingly in soleus muscle, we found a strong decrease in MCIP-1 mrna not only after CsA treatment, but also by thyroid deficiency. Moreover, we also observed that MCIP-2 was decreased not only by hypothyroidism, but also by CsA administration. Then it seems that, in soleus muscle, MCIP-1 and MCIP-2 respond equally to conditions of thyroid deficiency and decreased calcineurin activity. Decreased calcineurin activity by CsA administration led to a decrease in CnA mrna and protein levels. Such a finding is consistent with the role played by NFAT on the CnA mrna and protein expression. The regulation of the several calcineurin subunits has previously been examined in cardiac muscle (21). Calcineurin activity, through NFAT- and GATAdependent mechanisms, was shown to activate the promoter of CnA, one of the main CnA subunits. This study clearly suggests that CnA protein expression is subject to transcriptional regulation, especially by NFAT, one of the biological targets of calcineurin. Our results are fully consistent with this finding and suggest that, in the present study, CnA gene transcription decreased through the CsA decrease in calcineurin activity and associated drop in dephosphorylated NFAT. Then the question arises whether calcineurin and thyroid hormone interact together within muscle cells to control muscle phenotype. Analyzing the combined effects of CsA treatment and hypothyroidism on the different variables studied in muscle, we did not find additive effects of those factors, which suggests that they act within a similar pathway. Considering that pharmacological inhibition of calcineurin by CsA treatment only slightly decreased the level of thyroid hormone, whereas hypothyroidism dramatically decreased calcineurin activity estimated by the level of MCIP-1 mrna, we hypothesize that thyroid hormone acts upstream of calcineurin activity. Indeed, the dramatic decrease in MCIP mrna abundance in hypothyroid soleus muscle shows that calcineurin activity is inhibited in the absence of thyroid hormone, explaining the blunted effects of CsA in hypothyroid state. This suggests that, by increasing calcineurin activity in soleus muscle, thyroid hormone could facilitate the setting of the slow muscle phenotype despite its own effects on fast MHC expression. Interestingly, plantaris muscle that is poorly responsive to thyroid hormone (3) is also much less responsive to calcineurin inhibition (7). This strong interplay between thyroid state and calcineurin could thus be specific to soleus muscle that comprises a high amount of slow fibers. Further studies, especially careful examination of the respective and combined effects of hyperthyroidism and changes in calcineurin activity, are needed to establish the exact molecular link between thyroid hormone receptors, calcineurin activation, and the regulation of MCIP genes. In conclusion, the present experiments strongly suggest that the slow oxidative muscle phenotype is primarily under the control of thyroid state. In conditions of thyroid hormone deficiency, there is no response to the pharmacological inhibition of calcineurin, usually known to induce a slow-to-fast IIA transition associated with an enhancement of mitochondrial biogenesis in normothyroid rats. On the contrary, hypothyroidism effects on soleus muscle phenotype occur in rats with either basal or inhibited levels of calcineurin activity. Moreover, both thyroid deficiency and calcineurin inhibition decreased MCIP-1 and MCIP-2 mrna levels and then control calcineurin activity, suggesting that thyroid hormone and calcineurin pathways are interconnected.

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