The calcineurin-NFAT pathway and muscle fiber-type gene expression

Steven J. Swoap1, R. Bridge Hunter2, Eric J. Stevenson2, Heather Mitchell Felton2, Nilesh V. Kansagra1, Joshua M. Lang3, Karyn A. Esser3, and Susan C. Kandarian2

1 Department of Biology, Williams College, Williamstown 01267; 2 Department of Health Sciences, Boston University, Boston, Massachusetts 02215; and 3 Department of Kinesiology, University of Illinois at Chicago, Chicago, Illinois 60608


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To test for a role of the calcineurin-NFAT (nuclear factor of activated T cells) pathway in the regulation of fiber type-specific gene expression, slow and fast muscle-specific promoters were examined in C2C12 myotubes and in slow and fast muscle in the presence of calcineurin or NFAT2 expression plasmids. Overexpression of active calcineurin in myotubes induced both fast and slow muscle-specific promoters but not non-muscle-specific reporters. Overexpression of NFAT2 in myotubes did not activate muscle-specific promoters, although it strongly activated an NFAT reporter. Thus overexpression of active calcineurin activates transcription of muscle-specific promoters in vitro but likely not via the NFAT2 transcription factor. Slow myosin light chain 2 (MLC2) and fast sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1) reporter genes injected into rat soleus (slow) and extensor digitorum longus (EDL) (fast) muscles were not activated by coinjection of activated calcineurin or NFAT2 expression plasmids. However, an NFAT reporter was strongly activated by overexpression of NFAT2 in both muscle types. Calcineurin and NFAT protein expression and binding activity to NFAT oligonucleotides were different in slow vs. fast muscle. Taken together, these results indicate that neither calcineurin nor NFAT appear to have dominant roles in the induction and/or maintenance of slow or fast fiber type in adult skeletal muscle. Furthermore, different pathways may be involved in muscle-specific gene expression in vitro vs. in vivo.

SERCA1; myosin heavy chain; myosin light chain; fast-twitch muscle; slow-twitch muscle; nuclear factor of activated T cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MUSCLE FIBER TYPE DIVERSITY in striated muscle is a result of a complex pattern of gene expression (for review see Ref. 6). For example, the myosin heavy chain (MHC) and myosin light chain (MLC) gene families have multiple isoforms that have different functional properties related to contraction velocity. Similarly, proteins involved with calcium handling, such as the sarcoplasmic reticulum Ca2+-ATPase gene, are also part of a gene family whose differential expression results in altered contractile kinetics. Muscle fiber types also differ in substrate preference for metabolism, as reflected by differential expression of glycolytic or oxidative genes that influence fatigability.

The transcription factors and signaling pathways involved in regulating muscle fiber type are poorly understood. Involvement of members of the myogenic regulatory factor (MRF) gene family has been suggested, but the evidence so far is correlative rather than causal. Myogenic determination gene D (MyoD) and myogenin are differentially expressed in fast- and slow-twitch muscle, respectively (19, 20, 37). MyoD expression patterns correlate with fiber type patterns in many (20, 22, 38) but not all (17, 22) interventions known to alter fiber type. Manipulation of MRF family members in the mouse genome shows differing effects on muscle fiber type. MyoD-null mice show normal fiber type distribution as determined by histochemical analysis in some but not all muscles examined (19). Overexpression of myogenin in fast-twitch muscle leads to an increase in oxidative metabolism resembling that of slow muscle (18). However, the number of genes expressed in a fiber type-specific pattern that do not have functional binding sites for MRFs suggests that they alone are not likely responsible for regulation of fiber type-specific gene expression.

Another pathway recently postulated as having a regulatory role in fiber type-specific gene expression is the calcineurin-NFAT (nuclear factor of activated T cells) pathway (8). The most well-characterized role of calcineurin (CaN) is in the transduction of immune cell activation via nuclear translocation of NFAT transcription factors (reviewed in Ref. 31). Elevated intracellular calcium levels activate CaN, the only known Ca2+/calmodulin-dependent protein phosphatase, which then dephosphorylates any number of NFAT family members, causing their nuclear translocation. Once in the nucleus, NFAT presumably binds DNA in a sequence-specific fashion, resulting in altered transcription of several different genes. In skeletal muscle, Chin et al. (8) proposed that the CaN-NFAT pathway is important for slow fiber-type specificity. The conceptual basis for this model is that slow motor units are characterized by chronic recruitment patterns; thus the CaN-NFAT pathway might induce the activation of slow fiber-specific genes via higher chronic intracellular Ca2+ levels. In contrast, fast motor units, which are characterized by a phasic recruitment pattern, would not have chronically elevated Ca2+ levels and hence would not activate slow genes. Evidence to support this hypothesis is that treatment of rats with cyclosporin A, an inhibitor of CaN, increases the number of fast fibers within the slow soleus muscle as determined by histochemical analysis (8). However, promoter analysis of the slow troponin I gene demonstrated that the NFAT site in the SURE (slow upstream regulatory element) region was not required for fiber type-specific transcription in transgenic mice (7). Therefore, it is unclear what role, if any, the CaN-NFAT pathway has in the maintenance or acquisition of fiber type-specific gene expression in adult skeletal muscle.

The purpose of the present study was to test the hypothesis that the CaN-NFAT pathway has a regulatory role in fiber type-specific gene expression using in vivo transfection into rat fast (extensor digitorum longus) and slow (soleus) muscles. Expression of either slow or fast muscle-specific promoter constructs was not enhanced by cotransfection with either activated CaN or NFAT2 expression plasmids. Although overexpression of active CaN did enhance transcription of muscle promoters in C2C12 myotubes, this effect was not specific to fast or slow fiber promoters and did not appear to act via nuclear NFAT2. Thus the findings from this study do not support a dominant role for either CaN or NFAT proteins in regulating fiber type-specific gene expression in skeletal muscle.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Reporter plasmids. NFAT-GL3 (also called NFATx3) contains the distal NFAT site from the human interleukin-2 (IL-2) gene inserted as a trimer within the IL-2 minimal promoter (11) driving expression of luciferase in pGL3-basic (gift from Dr. S. Ho). Sarco(endo)plasmic reticulum Ca2+-ATPase [SERCA1] (-3636)-pGL3 was made as previously described (27). SERCA1 (-1373)-pGL3 was isolated using Erase-a-Base (Promega) performed according to the directions of the manufacturer.

The MLC2slow promoter contains 270 bp in pGL3-basic (12). The MHC IIB plasmid contains 2,560 bp of promoter upstream of the luciferase gene in pGL3-basic (33). The type I or beta -MHC promoter containing 3,538 bp of the 5' flank was a gift from Dr. K. Baldwin. pGL3-PGAM-M (muscle-specific isoform of phosphoglycerate mutase) contains 441 bp of the rat PGAM-M 5' flank (21). pGL2-skeletal alpha -actin was a gift from Dr. R. Wade (9). The c-jun promoter plasmid was a gift from Dr. R. Prywes (15), and cytochrome c was from Dr. F. Booth (41). The pRL-cytomegalovirus (pRL-CMV) and pRL-SV40 were purchased from Promega.

A 2.5-kb region of the perinatal MHC gene promoter/enhancer was isolated using genome walking (Clontech). It includes 973 bp of 5' flank, exon 1, intron 1, and a portion of exon 2 (containing the translation start site). From this clone PCR techniques were used to subclone the -973 to +1 region of the human perinatal MHC gene upstream of the luciferase cDNA (pGL3basic) for promoter analysis (GenBank accession no. AF067143). Using in vivo DNA injection into regenerating soleus and extensor digitorum longus (EDL) muscles, this portion of the perinatal MHC promoter was sufficient to direct expression in regenerating fast and slow skeletal muscles (K. Esser and A. Tidhar, unpublished data).

Overexpression plasmids. The NFAT2 vectors were gifts from Dr. C. Beals. The vector SH160mSRR contains mutations in the serine-rich region of NFAT, creating a constitutively nuclear NFAT2 overexpression plasmid. In the vector SH160Delta 418, a COOH-terminal deletion creates a dominant negative (2). The NFAT2-negative control used was SH-null, in which SH160mSRR was digested with EcoR I, the NFAT2 cDNA was removed, and the vector was recircularized. CaN overexpressors used during the study were pCIneo-CaN and pcDNAI-CaN. An active mutant of subunit A of CaN (30) was cloned into the EcoR I site of pCI-neo, downstream of a CMV promoter, and was a gift of Dr. R. Bassel-Duby (8). To create the controls, pCI-neo-null and pcDNAI-null plasmids were digested with EcoR I, CaN was removed, and plasmids were recircularized. In vivo plasmid injections of CaN in pcDNAI were not different from CaN in pCI-neo.

Transfection in mammalian cell culture. Approximately 3 × 105 C2C12 myoblasts were seeded into gelatin-coated wells of a six-well plate in growth media (20% fetal bovine serum, 25 µg/ml gentamicin, in DMEM). Transfections were performed the next day using Lipofectamine (GIBCO) with 1 µg of a firefly luciferase reporter plasmid, 76 ng of pRL-CMV, and 0.25 µg of expression vector. After 5 h of transfection, growth medium was added to each well for incubation overnight. The following morning, growth medium was replaced with a low-serum medium (2% horse serum, 10 µg/ml insulin, 10 µg/ml transferrin, 50 mM HEPES, pH 7.5, and 25 µg/ml gentamicin, in DMEM) to induce differentiation. After 48 h, protein extracts were made from the cells using a passive lysis buffer (Promega).

Luciferase assays. All muscle samples, cell culture samples, and reagents were brought to room temperature before the assay was performed. Firefly and Renilla luciferase activities were determined using a dual luciferase kit (Promega) in a Turner Designs Luminometer (model TD 20/20). In the whole muscle luciferase activity from MLC2slow injections, a Berthold luminometer was used.

Somatic gene transfer using intramuscular DNA injection. Plasmid DNA injections into rat skeletal muscle have been previously detailed (12, 27, 33). Plasmid DNA was prepared using Endo-free Maxi and Mega Prep Kits (Qiagen). Appropriate amounts of the reporter and overexpression plasmids were mixed, ethanol-precipitated overnight, and resuspended at 4°C overnight in 25% sucrose-1× PBS. Soleus and EDL muscles were injected with 75 µg of SERCA1 or MLC2slow reporter and 50 µg of the expression vectors in a volume of 50 µl. Muscles were removed for analysis of luciferase activity 7 days after injection. For regenerating muscle, muscles were injected with 5 µg MLC2 reporter and 3 µg expression vector in a volume of 40 µl, 3 days after Marcaine injection (12).

RNA isolation and Northern blotting. RNA was isolated, size fractionated, and transferred to nylon membrane as described previously (33). Membranes were prehybridized in 6× SSPE (0.9 M NaCl, 31 mM Na2HPO4 · H2O, and 10 mM EDTA), 10× Denhardt solution, 0.1% SDS, and 150 µg/ml salmon sperm DNA at 42°C for 1 h. 32P end-labeled oligonucleotides specific for beta -MHC (5'-GGTCTCAGGGCTTCACAGGC-3') and 28S (5'-GGTCTAAACCCAGCTCACGTTC-3') were added to 6× SSPE + 0.1% SDS and hybridized to the membrane overnight at 42°C. For random-primed probes, CaN (30) and SERCA1 (40), membranes were prehybridized in 50% formamide, 5× SSPE, 5× Denhardt solution, 0.1 mg/ml salmon sperm DNA, and 0.1% SDS for 4 h at 42°C. Labeled cDNA was added to freshly made prehybridization buffer and hybridized at 42°C overnight. The membranes were washed extensively, with a final wash in 0.1× SSC + 0.1% SDS at 42°C for 30 min. Blots were exposed to a phosphorimaging screen.

Nuclear and cytosolic extract preparation from whole muscle. Nuclear extracts were prepared from rat skeletal muscle according to Blough et al. (5). The cytosolic extract was obtained from the first supernatant of the nuclear extract preparation. The supernatant was added to Millipore Ultrafree-4 centrifugal columns, prewet with 0.5 ml of dilution buffer [20 mM HEPES, 40 mM KCl, 10% glycerol, 0.2 mM EDTA, and 1 mM dithiothreitol (DTT)], and centrifuged (7,500 g) at 4°C for 30 min. A 0.8-ml volume of dilution buffer was added to the column, and the 30 min spin was repeated. Protein concentration was determined using the Bio-Rad Bradford Protein Assay for the nuclear samples and the Detergent Compatible Assay for the cytosolic samples.

Western analysis of CaN and NFAT. Forty micrograms of nuclear and cytosolic extracts from rat soleus and EDL muscles was dissolved in SDS loading buffer, boiled for 3 min, centrifuged (10,000 g) for 5 min (to remove insoluble material), and separated on 4-15% or 4-10% SDS-polyacrylamide gels. Protein was transferred onto Hybond ECL (Amersham) nitrocellulose membrane. Membranes were blocked in 5% nonfat milk diluted in TBS-Tween for 1 h and then incubated for 1-2 h with either anti-calcineurin A (Sigma, clone CN-A1), anti-NFAT1 (Affinity Bioreagents, clone 25A10.D6.D2), anti-NFAT2 (Affinity Bioreagents, clone 7A6), anti-NFAT4 (Santa Cruz Biotechnology, clone F1-X), or a broadly reactive NFAT polyclonal antibody (Santa Cruz Biotechnology, clone K-18), diluted according to the manufacturer. Horseradish peroxidase-conjugated secondary antibodies (Vector Labs) and a chemiluminescence detection system (Amersham) were used for visualization. Nuclear extracts used for positive controls were obtained from manufacturers (Jurkat T cells, Geneka; CTL cells, Upstate Biotechnology). Cerebellar microsomal extracts were isolated from rat brain for a positive control for the CaN antibody.

Electrophoretic mobility shift assays. Binding reactions were performed with labeled probe and 10 µg of crude nuclear extract from soleus or EDL muscle in 25 mM Tris · HCl, pH 7.5, 50 mM KCl, 1 mM DTT, and 25% (vol/vol) glycerol in a total volume of 20 µl. Probes were end labeled with either [gamma -32P]ATP (New England Nuclear) using T4 polynucleotide kinase (New England Biolabs). The DNA-protein binding reaction was carried out at room temperature for 30 min with 50,000 cpm (0.1-0.2 ng) of double-stranded SERCA1 oligonucleotide containing the NFAT cis-element found at -1305 (5'-CCTTGGTGGAAATCAAAAGA-3'). For comparison, some shifts were performed using an NFATc consensus oligonucleotide from Santa Cruz Biotechnology (5'-CGCCCAAAGAGGAAAATTTGTTTCATA-3'). Supershifts were performed by the addition of 2 µl of the respective antibodies and incubated at room temperature for 15 min prior to the addition of the labeled probe. The binding reaction mixture was loaded onto a 7% or 5% (supershifts) native polyacrylamide gel (high ionic strength) and run at 180 V for 3 h (13). Cold competition was performed using double-stranded NFAT and Sp1 (5'-CTCTGGGGCCCCGCCCACATGACTGCC-3') oligonucleotides (data not shown). One nanogram of nonspecific DNA (dI · dC:dI · dC) was added to all reactions. Autoradiography was performed by exposing dried gels to X-ray film for ~40 h.

Statistics. Data for the variables studied are reported as means ± SE. Statistically significant differences were determined using the Dunn test following an ANOVA. The 0.05 level of confidence was accepted for statistical significance.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activated CaN induced muscle-specific but not non-muscle-specific genes in C2C12 cells. A series of in vitro transfection experiments were initially performed to expand on the original findings of Chin et al. (8). To determine whether CaN could activate different fast, slow, or ubiquitously expressed genes, C2C12 cells were cotransfected with activated CaN and one of several different muscle-specific or non-muscle-specific reporter plasmids. As a positive control, a multimerized NFAT site upstream of the IL-2 minimal promoter (NFATx3) was strongly induced by the activated CaN plasmid (Fig. 1A). Overexpression of active CaN significantly induced expression of the fast-twitch PGAM-M, SERCA1, and MHC IIB promoters, the slow-twitch MLC2slow and beta -MHC promoters, the perinatal MHC promoter, and the non-fiber-type-specific skeletal alpha -actin promoter (Fig. 1A). Although all of these muscle-specific promoters tested were activated by CaN, the non-muscle-specific promoters (c-jun, SV40, and cytochrome c) were not (Fig. 1A). Several of the muscle-specific promoters were then tested for inhibition by cyclosporin A, a classic inhibitor of CaN activity. The activation by CaN in the four muscle-specific promoters tested (PGAM-M, type IIB MHC, beta -MHC, skeletal alpha -actin) was blunted by the addition of cyclosporin A (Fig. 1B).


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Fig. 1.   Activated calcineurin (CaN) induced all muscle-specific promoters tested but not non-muscle-specific promoters in C2C12 cells. A: C2C12 myoblasts were cotransfected with activated CaN or pCI-null, and 1 of 11 firefly luciferase reporter plasmids: NFATx3, phosphoglycerate mutase (PGAM), skeletal muscle sarco(endo)plasmic reticulum calcium-ATPase (SERCA1), myosin heavy chain IIB (IIB MHC), myosin light chain 2 slow (MLC2slow), beta -myosin heavy chain (beta -MHC), perinatal MHC, skeletal alpha -actin, c-Jun, SV40, and cytochrome c (Cyt-C). A Renilla luciferase plasmid was used for transfection efficiency, except for pRL-SV40 transfections, which were cotransfected with pGL3-CMV. Firefly luciferase of C2C12s was examined 3 days after transfection and normalized to Renilla luciferase. B: C2C12 myoblasts were cotransfected with activated CaN or pCI-null, and one of four reporter plasmids. After transfection, cells were treated with 125 nM cyclosporin A, an inhibitor of CaN, or vehicle. Data are relative to transfection with pCI-null and are means ± SE of 4 separate sets of transfections, each done in duplicate or triplicate.

Activated CaN overexpression induces endogenous SERCA1 and beta -MHC mRNA expression in C2C12 cells. To verify that the changes seen with the reporter constructs were indicative of endogenous gene expression, Northern analysis of RNA isolated from C2C12 cells transfected with either activated CaN or a null vector was performed. Overexpression of active CaN upregulated fast SERCA1 (2.5 ± 0.5-fold) and slow beta -MHC (5.6 ± 1.3-fold) mRNA (Fig. 2). Hence, the changes detected in reporter gene activity reflect the changes in expression of the endogenous genes.


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Fig. 2.   Reporter gene activity in C2C12 cells reflected endogenous gene expression with overexpression of activated CaN. C2C12 myoblasts were transfected with either activated CaN or with pCI-null. After 3 days, RNA was isolated and probed for beta -MHC mRNA and SERCA1 mRNA. The RNA gel was stained showing 28S and 18S rRNA.

NFAT2 overexpression does not induce muscle reporters in C2C12 cells. One of the well-characterized targets of active CaN is NFAT, and this pathway has been suggested to be important for fiber type specificity (8). Interestingly, although the NFAT reporter (NFATx3) was strongly activated by overexpression of wild-type or constitutively nuclear NFAT2 in C2C12 cells, neither the SERCA1 or type IIB MHC promoters were activated by overexpression of constitutively nuclear or wild-type NFAT2 (Fig. 3). It is important to note that both the SERCA1 (27) and type IIB MHC (34) promoters used in these experiments contain multiple NFAT consensus sequences.


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Fig. 3.   C2C12s were transfected with NFAT2 (wt, wild type; cn, constitutively nuclear) or null plus either SERCA1 or type IIB MHC. The cells were harvested 3 days after transfection, and firefly and Renilla luciferase activity was measured. Data are relative to transfection with pCI-null and are means ± SE for 3 sets of transfections, each done in triplicate.

Effects of CaN or NFAT2 overexpression on the Renilla luciferase control plasmid. Since the Renilla luciferase vector was used to control for the efficiency of plasmid uptake by C2C12 cells, it was possible that overexpression of CaN or NFAT2 could affect CMV-driven Renilla expression. However, Renilla luciferase activity did not vary more than 10% and not in a consistent direction with any one treatment: CaN, NFAT2, or cyclosporin A.

Overexpression of activated CaN or constitutively nuclear NFAT2 does not induce a fast fiber-specific reporter (SERCA1) in slow or in fast muscle. In vivo plasmid injection into rodent skeletal muscle has been successfully used to functionally overexpress proteins (12, 28). Therefore, we used this approach to test for a role of CaN or NFAT2 in the activation of a fast muscle gene (SERCA1) in adult fast and slow muscle. The SERCA1 promoter (-1373 to +172) driving luciferase was coinjected with either a plasmid encoding active CaN or constitutively nuclear NFAT2 protein. The SERCA1 promoter contains three consensus NFAT binding sites (sense strands -1367, -1305, -1239; Ref. 27). In contrast to results in vitro, overexpression of constitutively active CaN was not sufficient to transactivate the NFATx3 (NFAT-pGL3) promoter in either soleus or EDL muscles (Fig. 4A), nor was it sufficient to activate the SERCA1 promoter in soleus or EDL muscles (Fig. 4B). In fact, activated CaN appeared to decrease reporter expression in soleus muscles. Reporter activity of NFATx3 increased 100-fold when nuclear NFAT2 was overexpressed in both soleus and EDL muscles (Fig. 4C), demonstrating the fidelity of the coinjection methodology. However, SERCA1 reporter gene activity was either unchanged or repressed by NFAT2 overexpression (Fig. 4D). It is important to note that previous work has established that soleus muscles take up plasmid better than EDL muscles using direct injection (27), thus a quantitative comparison of luciferase values between the two muscles is not appropriate.


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Fig. 4.   In vivo injection of soleus and extensor digitorum longus (EDL) muscles with reporter and overexpression plasmids analyzed 7 days postinjection for firefly luciferase activity. RLU, relative light units. A: soleus and EDL muscles were coinjected with NFATx3 and activated CaN or empty vector (null). B: coinjection of muscles, same as in A except the reporter plasmid is SERCA1 5' flank from -1373 to +172. C: muscles coinjected with NFATx3 and constitutively nuclear NFAT2 or empty vector (null). NFATx3 luciferase activity increased 100-fold in soleus and EDL muscles when nuclear NFAT2 was overexpressed. D: coinjection of muscles, same as in C except the reporter plasmid is SERCA1 5' flank from -1373 to +172 bp. Data are means ± SE; n = 8 muscles for each group. Note: comparison of activity between soleus and EDL muscles is not valid (see text).

Overexpression of CaN or NFAT2 does not induce an MLC2slow reporter gene in control or regenerating slow or fast muscle. The next experiment was to determine whether the MLC2slow promoter was sensitive to overexpression of CaN or NFAT2 in vivo. This slow muscle-specific promoter confers fiber type-specific expression (12) but has no consensus NFAT sites. However, the model proposed by Chin et al. (8) predicts that NFAT should activate a slow specific promoter, if not via an NFAT site, then potentially via its myocyte specific enhancer factor 2 (MEF2) site (8). As was seen with the SERCA1 promoter, overexpression of active CaN inhibited rather than induced expression of the MLC2slow promoter in control (Fig. 5A) or in regenerating (Fig. 5B) soleus muscles and had no effect in EDL muscles. Interestingly, overexpression of nuclear NFAT2 also had an inhibitory effect on MLC2slow promoter in both control (Fig. 5C) and regenerating (Fig. 5D) soleus muscles. This is consistent with the same inhibitory effect when active CaN is cotransfected with MLC2slow in soleus muscles. In control or regenerating EDL muscle, NFAT2 overexpression did not significantly change MLC2slow reporter activity (Fig. 5, C and D).


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Fig. 5.   In vivo injection of MLC2slow reporter and overexpression plasmids into regenerating and nonregenerating soleus and EDL muscles. Muscles were analyzed 7 days postplasmid injection for firefly luciferase activity. A: coinjection of MLC2slow and active CaN or null plasmid into control (i.e., nonregenerating) soleus and EDL muscle. B: same as in A except injection was into regenerating muscles. Regeneration was induced by direct injection of Marcaine 3 days before plasmid injection. C: control soleus and EDL muscles were injected with MLC2slow and either constitutively nuclear NFAT2 or null plasmid. D: DNA injection into regenerating muscles conducted as in B except MLC2slow reporter and either wild-type (wt) NFAT2, constitutively nuclear (cn) NFAT2, dominant-negative (dn) NFAT2, or null plasmid was injected. Data are means ± SE; n = 4-8 muscles per group. A different luminometer was used for MLC2slow vs. SERCA1.

CaN expression in fast and slow whole muscle. Although the role of the CaN-NFAT pathway has become a popular area of research in muscle, there is still very little known regarding the relative expression patterns of both CaN and NFAT isoforms in adult skeletal muscle. Thus experiments were performed to better characterize the fiber type expression of components of the CaN-NFAT pathway. Northern analysis of total RNA from the tibialis anterior, plantaris, EDL, and soleus muscles demonstrated that the slow soleus muscle contains significantly less mRNA of the catalytically active subunit of CaN (subunit A) compared with three different fast-twitch muscles (Fig. 6A). Western analysis using a monoclonal antibody against the catalytically active subunit of CaN (subunit A) detected a band in both nuclear and cytosolic fractions from soleus and EDL muscles with a molecular mass of ~61 kDa (Fig. 6B). Within a given blot, the cytosolic fraction always gave greater signal intensity than the nuclear fraction in both muscle types. As seen with the mRNA distribution, the relative protein expression of CaN A was consistently greater in the nuclear and cytosolic fractions from EDL compared with soleus muscles.


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Fig. 6.   CaN A mRNA and protein expression is greater in fast vs. slow muscle. A: CaN mRNA using Northern analysis of the tibialis anterior (TA), soleus (Sol), plantaris (Plan), and EDL muscles (n = 6 for each muscle type). Blot was stripped and reprobed for 28S rRNA for quantification. Expression of CaN mRNA was significantly lower in the slow soleus than in other fast muscles. B: CaN protein expression measured using CaN A-specific antibody. Positive control for CaN A was a crude membrane (M) preparation from rat brain (B). EDL (E) cytosolic (C) and nuclear (N) extracts had greater expression than soleus (S) extracts (n = 8 per extract). Blots were quantified by densitometric analysis. Data are means ± SE.

NFAT isoform expression in slow and fast muscle. Characterization of NFAT1, NFAT2, and NFAT4 protein expression in skeletal muscle was performed using previously characterized antibodies. With use of an NFAT2-specific antibody (36), four bands ranging from 80 to 115 kDa were found in the nuclear fraction of the soleus (Fig. 7A). This banding pattern is consistent with what has been detected previously using this antibody in B cells (24, 36) and in C2C12 myotubes (1, 32). Three NFAT2 splice variants are known to exist, and their sizes may vary based on phosphorylation state (36). However, only one prominent band was detected in nuclear extracts from EDL muscles. The cytosolic fractions of both the soleus and EDL revealed slower migrating bands, from 100 to 140 kDa, consistent with more phosphorylated forms of cytosolic NFAT2 (24, 36). At least four NFAT1 immunoreactive bands were detected in soleus, EDL, and Jurkat cell nuclear extracts (Fig. 7B) of molecular masses ranging from 125 to 190 kDa. Three NFAT1 splice variants are known, and their sizes may also vary based on phosphorylation state (31). Stronger banding intensity was consistently detected in extracts from soleus compared with EDL muscles. We could not detect NFAT1 in cytosolic fractions of either muscle type (not shown). Anti-NFAT4 did not detect NFAT4 (~190 kDa) in nuclear or cytosolic muscle fractions (not shown). When a broadly reactive NFAT antibody was used, the NFAT2 immunoreactive bands were prominent, whereas the NFAT1 bands were barely detectable. These observations may suggest that NFAT2 is the most abundant NFAT isoform in skeletal muscle, consistent with that seen in L6 myotubes (29).


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Fig. 7.   NFAT protein expression differs in slow vs. fast muscles: Western blot analysis using anti-NFAT2 (A) and anti-NFAT1 (B) from nuclear (Nuc) and cytosolic (Cyto) extracts (40 µg) from soleus (S) and EDL (E) muscles. Gels of 4-15% were used except for left panel in B, which is a 4-10% gel, so bands are more separated. Jurkat (J) T-cell lysates were used as a positive control for NFAT1 and NFAT2.

Protein complexes containing CaN and NFAT2 bind to an NFAT oligonucleotide in nuclear extracts from slow and fast muscle. Because NFAT1, NFAT2, and CaN A were differentially expressed in nuclear extracts from slow vs. fast skeletal muscle, we went on to determine whether there were differences in their interaction with a conserved NFAT binding site found in the 5' regulatory sequence of the SERCA1 gene. Electrophoretic mobility shift assays were performed using nuclear extracts from four separate soleus and EDL muscles and an oligonucleotide probe spanning the SERCA1 NFAT site. Incubation of the double-stranded probe with skeletal muscle nuclear extract revealed two bands referred to as complex A and B (Fig. 8A). Cold competition with excess cold NFAT oligonucleotide (data not shown) was more successful than with nonspecific DNA as previously shown with the use of immune cell nuclear extracts (25, 36). The complexes always gave greater band intensity in soleus vs. EDL nuclear fractions, and this is consistent with the greater intensity of NFAT bands found in the soleus nuclear extracts with immunoblotting. Results were the same when a commercially available consensus NFATc oligonucleotide was used (data not shown). Incubation with antibodies to NFAT2 supershifted complex B in both soleus and EDL extracts, suggesting that NFAT2 is a component of the complex (Fig. 8B). Antibodies against NFAT1 or NFAT4 did not alter the binding complexes. Incubation with the antibody directed against CaN supershifted complex A (Fig. 8C), suggesting that CaN is also part of the complex at this NFAT site. The observation that one of these complexes contains immunologically detectable CaN is consistent with the recent finding that CaN may be bound in a complex with NFAT during transcriptional activation (42).


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Fig. 8.   Electrophoretic mobility shift assays performed with 10 µg of soleus or EDL nuclear extract incubated with 0.1 ng of 5'-end labeled SERCA1 NFAT oligonucleotide. A: binding of soleus vs. EDL extracts on 7% gel. Multiple lanes of soleus and EDL represent extracts isolated from separate rats. B and C: supershifts (5% gels) were performed by preincubation of antibodies specific for NFAT1, NFAT2, NFAT4, or CaN plus labeled probe. Shifts were observed using antibodies specific for NFAT2 and CaN, but not NFAT1 or NFAT4. Jurkat (J) nuclear extracts were used as positive control for NFAT binding. Arrows labeled "A" or "B" represent specific binding complexes. Unlabeled arrows indicate supershifted bands.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In the present work, overexpression of activated CaN in cultured C2C12 cells strongly induced all (fast and slow) muscle-specific, but not non-muscle-specific, promoters tested. Chin et al. (8) also showed strong induction of slow muscle genes by activated CaN in C2C12 cells. However, because CaN overexpression did not activate the muscle creatine kinase gene in that study, it was interpreted to indicate that only slow muscle genes are induced by active CaN. In contrast, we found that several fast muscle genes are induced by activated CaN including SERCA1, PGAM-M, and type IIB MHC. Genes that are expressed in muscle but are not muscle specific, such as cytochrome c and c-jun, were not induced by CaN. Importantly, we also showed that NFAT2 overexpression did not activate the muscle-specific promoters tested, SERCA1 or type IIB MHC in C2C12s, even though they have consensus NFAT binding sites. These data indicate that the activating effect of CaN on muscle-specific promoters in vitro is not likely acting through NFAT sites. This conclusion is consistent with the observation that CaN can activate viral promoters via MEF2 sites (23) or via p53 and NF-kappa B sites (14). In T cells, CaN has been implicated in the regulation of the Nur77 steroid receptor promoter through an MEF2 rather than NFAT sites (39).

In adult soleus muscle, active CaN overexpression inhibited rather than activated SERCA1 (fast) and MLC2slow reporters, whereas in EDL muscles reporter activity was unchanged. In regenerating soleus muscles, characterized by satellite cell-mediated myogenesis, there was also an inhibitory effect of active CaN on MLC2slow reporter activity. This suggests that in adult muscle and during muscle fiber formation and/or repair, active CaN may be inhibitory for slow gene activation. This inhibitory role of CaN would not be unusual, as previous studies have shown that active CaN inhibits the transcription factors Elk-1 (35) and CREB (4) through dephosphorylation. We interpret these in vivo observations as indicating that CaN does not play a dominant regulatory role in fiber type acquisition or maintenance in the adult animal. The contrasting results between in vitro and in vivo transfection studies also indicate that a different factor(s) exists or that distinct pathways are activated by CaN in cell culture vs. whole muscle.

The observations reported herein do not support the hypothesis that the CaN-NFAT pathway is involved with slow-muscle-specific gene expression. The strongest evidence to date to support this hypothesis is that animals treated with cyclosporin A demonstrate an increase in the number of fast fibers within a slow muscle (8) and a blunted fast-to-slow fiber transition in functionally overloaded rat fast muscle (10). However, Biring et al. (3) found no change in either fiber type or cross-sectional area following 4 wk of cyclosporin A treatment. It is also important to note that Mercier et al. (26) and Hokanson et al. (16) demonstrated that muscle mitochondrial respiration was decreased due to suppression of mitochondrial electron chain capacity following 2 wk of cyclosporin A administration. Thus changes in muscle gene expression in animals treated with cyclosporin may be a secondary result of its side effects on mitochondrial respiration and not associated with specific effects involving CaN.

Overexpression of the NFAT2 transcription factor also does not appear necessary for fiber type-specific gene expression. This is evidenced by the lack of activation of MLC2slow and SERCA1 reporter genes by NFAT2 in either the slow soleus or fast EDL muscles; however, the NFAT2 overexpressor did activate an NFAT reporter in both muscle types, suggesting that the lack of effect on muscle-specific reporters is not due to a limitation of the direct plasmid injection methodology. These data are consistent with the report of Calvo et al. (7), who found that the NFAT site located in the region of the troponin I slow gene that confers slow specificity was not necessary for slow specific expression in transgenic mice.

Endogenous expression of CaN and NFAT were different in fast vs. slow whole muscle. CaN was expressed at higher levels in both nuclear and cytosolic fractions in the EDL muscle. In contrast, NFAT nuclear localization and DNA binding activity was higher in soleus vs. EDL muscles, consistent with the model proposed by Chin et al. (8); however, the CaN expression data in slow vs. fast muscle is in contrast to what would be predicted from this model, where nuclear CaN and NFAT levels might parallel each other, and both would be higher in slow soleus compared with fast EDL muscles. However, since CaN has many roles in the cell and our reporter overexpression data do not support a role for CaN in regulating fiber type specificity, the functional significance of the differential expression is unclear. Although the results from the present study do not define a specific transcriptional function for either CaN or NFAT in skeletal muscle, they do suggest that these factors have more complex functions in vivo.

In summary, results from this study demonstrate that active CaN is not sufficient to differentially regulate fiber type-specific gene expression in whole muscle or in cell culture. Although CaN induced both fast and slow muscle promoters in C2C12 myotubes, it did not induce these promoters in fast or slow whole muscle. Moreover NFAT2, a downstream target of CaN, did not induce muscle-specific promoters, but it strongly activated transcription from a multimerized NFAT reporter in both fast and slow muscle and in C2C12s. Thus, in C2C12 myotubes, CaN induces transcription via alternative transcription factors, and a role for transactivation of muscle-specific genes in whole muscle was not supported.


    ACKNOWLEDGEMENTS

This work was supported by National Science Foundation Grant IBN-9723351 (to S. J. Swoap) and by National Institutes of Health Grants AR-41705 (to S. C. Kandarian) and AR-43349 to (K. A. Esser). S. C. Kandarian is an Established Investigator of the American Heart Association.


    FOOTNOTES

Address for reprint requests and other correspondence: S. C. Kandarian, Dept. of Health Sciences, Boston Univ., 635 Commonwealth Ave., Boston, MA 02215 (skandar{at}bu.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.

Received 11 February 2000; accepted in final form 11 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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