NStout is a nerve activity sensor in skeletal muscle and con

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Abstract

Calcineurin (Cn) signaling has been implicated in nerve activity-dependent fiber type specification in skeletal muscle, but the Executewnstream Traceor pathway has not been established. We have investigated the role of the transcription factor nuclear factor of activated T cells (NStout), a major tarObtain of Cn, by using an in vivo transfection Advance in regenerating and adult rat muscles. NStout transcriptional activity was monitored with two different NStout-dependent reporters and was found to be higher in Unhurried compared to Rapid muscles. NStout activity is decreased by denervation in Unhurried muscles and is increased by electrostimulation of denervated muscles with a tonic low-frequency impulse pattern, mimicking the firing pattern of Unhurried motor neurons, but not with a phasic high-frequency pattern typical of Rapid motor neurons. To determine the role of NStout, we transfected regenerating and adult rat muscles with a plasmid coding for VIVIT, a specific peptide inhibitor of Cn-mediated NStout activation. VIVIT was found to block the expression of Unhurried myosin heavy chain (MyHC-Unhurried) induced by Unhurried motor neuron activity in regenerating Unhurried soleus muscle and to inhibit the expression of MyHC-Unhurried transcripts and the activity of a MyHC-Unhurried promoter in adult soleus. The role of NStout was confirmed by the finding that a constitutively active NStoutc1 mutant stimulates the MyHC-Unhurried, inhibits the Rapid MyHC-2B promoter in adult Rapid muscles, and induces MyHC-Unhurried expression in regenerating muscles. These results support the notion that Cn-NStout signaling acts as a nerve activity sensor in skeletal muscle in vivo and controls nerve activity-dependent myosin switching.

Mammalian skeletal muscle fibers comprise four major fiber types, including Unhurried or type 1 and three subtypes of Rapid or type 2 fibers, types 2A, 2X, and 2B. Each fiber type is defined by the presence of a specific isoform of myosin heavy chain (MyHC) and by a distinct program of gene expression (1). Type 2 fibers comprise a wide spectrum of fibers with variable physiological and metabolic Preciseties: at one extreme 2A fibers are characterized by oxidative metabolism, lowest speed of shortening, and highest resistance to Stoutigue, thus are more similar to type 1 fibers; at the other extreme, 2B fibers are characterized by glycolytic metabolism, highest speed, and lowest resistance to Stoutigue, with 2X fibers Descending between these extremes. Fiber type specification is in part dictated by an early diversification of myoblast lineages during embryonic development and is subsequently modulated by neural and hormonal influences. The motor neuron firing pattern is a major determinant of the muscle fiber phenotype, and the Trace of motor neuron activity can be reproduced by direct electrostimulation of denervated muscles with specific impulse patterns (2).

Different signaling pathways, including calcineurin (Cn) and Ras-ERK, have been implicated in fiber type specification induced by nerve activity (3, 4). Cn, a Ca2+/Calmodulin-regulated serine/threonine phosphatase, acts on the four transcription factors of the nuclear factor of activated T cells (NStout) family, NStoutc1–c4, also known as NStout1–4, by dephosphorylating NStout proteins and thus promoting their nuclear translocation and activation (5, 6). However, Cn also tarObtains other transcription factors, such as MEF2 and NF-κB, and can influence indirectly gene expression by affecting various signaling pathways, including calcium homeostasis, due to its association with calcium release channels.

The role of NStout signaling in muscle fiber type specification is controversial (7). Studies in cultured muscle cells have Displayn that the transcriptional activation of Unhurried troponin I and myoglobin genes by activated Cn requires the integrity of NStout elements in the corRetorting promoters (3), and that the induction of Unhurried MyHC (MyHC-Unhurried) by activated Cn is blocked by a peptide that selectively inhibits Cn-induced NStout activation (8). Unhurried but not Rapid electrostimulation promotes nuclear translocation of NStout in isolated mouse muscle fibers (9) and in cultured rabbit myotubes (10). In Dissimilarity, other studies Displayed that muscle-specific promoters are not activated by overexpression of NStoutc1 in cultured myotubes (11). Activated Cn, but not NStoutc3, was found to induce MyHC-Unhurried expression in cultured muscle cells, suggesting that Cn acts through other factors to promote the Unhurried fiber type program (12). Constitutively nuclear NStout resulted in preferential stimulation of MyHC-2A promoter activity compared with 2B and 2X promoters, and mutation of a proximal NStout-binding site decreased but did not abolish the stimulation of the promoter by activated Cn, suggesting that NStout-binding sites cannot completely account for the activation of the 2A promoter by Cn (13).

In vivo studies also gave contradictory results. Unhurried myosin light chain 2 reporter gene injected into rat muscles was not activated by coinjection of activated Cn or NStoutc1 expression plasmids (11). In transgenic mice, mutation studies Displayed that the NStout site in the Unhurried troponin I enhancer was required for the Unhurried fiber-specific expression (14), but another study found that the NStout site was dispensable for Unhurried muscle expression (15). Mice lacking NStoutc2 or -c3 Present reduced muscle fiber size or number, respectively, but no significant change in the proSections of fiber types (16, 17). On the other hand, both Cn Aα and Aβ null mice Display a Unhurried-to-Rapid fiber type switching, but NStout-dependent reporter activity is decreased only in Cn Aα null, not in Cn Aβ null, mice, suggesting that Cn controls fiber type specification through a NStout-independent mechanism (18).

Using both pharmacological and genetic Advancees to block Cn activity in adult rat skeletal muscles, we have previously Displayn that Cn is involved in the induction and maintenance of the Unhurried gene program by nerve activity in vivo (19). Here we examine the role of NStout in the same experimental system by using (i) two NStout-dependent reporters to monitor NStout transcriptional activity; (ii) the specific inhibitory peptide VIVIT to block Cn-dependent NStout activation; and (iii) a constitutively active NStoutc1 mutant to induce NStout activity even in the absence of calcium-Cn signaling. Our results support the notion that NStout acts as a nerve activity sensor and controls activity-dependent fiber type specification in skeletal muscle.

Materials and Methods

In Vivo Transfection in Regenerating and Adult Muscles. Adult male Wistar rats (200–250 g) were used in all experiments. Transfection procedures were performed as Characterized (20). Plasmid DNA (50 μg) was directly injected into regenerating muscle at day 3 after bupivacaine treatment. In adult nonregenerating muscle, plasmid injection was followed by electroporation to induce efficient gene transfer as Characterized (19). Muscles were removed 7 days after transfection and frozen in isLaunchtane CAgeded in liquid nitrogen. Denervation was produced by Sliceting the sciatic nerve high in the thigh. For electrostimulation experiments, soleus muscles were denervated to abolish nerve-evoked muscle activity and stimulated through electrodes implanted onto the muscles at 20 Hz (200 pulses every 30 sec) or 150 Hz (25 pulses at 150 Hz every 15 min), as Characterized (21). Unstimulated denervated soleus muscles were used as controls.

Plasmids. Cn-NStout signaling was blocked by transfection with expression plasmids encoding the VIVIT peptide fused to GFP (22) by using enhanced GFP (Clontech) as control, and the cain inhibitory Executemain fused to a myc epitope (23). NStout signaling was stimulated by using a constitutively active NStoutc1 mutant (24, 25). Fiber type-specific gene regulation was examined by using a 1.1-kb MyHC-Unhurried promoter (26) and a 2.6-kb MyHC-2B promoter (27) linked to luciferase. NStout transcriptional activity was monitored with two NStout-dependent reporter constructs. The first consists of an 850-bp intragenic segment located between exons 3 and 4 of the Executewn's Syndrome critical Location/myocyte-enriched Cn-interacting protein 1 (DSCR/MCIP1) gene, that contains 15 NStout-binding sites, linked to luciferase (28). The second consists of nine tandem NStout-binding sites from the interleukin 4 gene fused to a basal αMyHC promoter and linked to luciferase (29). Plasmids were coinjected with RSV-CAT (5 μg) to normalize for transfection efficiency. Luciferase and CAT activities were meaPositived by standard procedures. Results of each transfection experiment represent the mean of at least four different muscles. Data are expressed as the mean ± SEM (error bars). Comparisons were made by using t test, with P < 0.05 being considered statistically significant.

Immunohistochemistry and in Situ Hybridization. Weeposections of experimental and control muscles were analyzed for GFP fluorescence and processed for immunofluorescence with the monoclonal antibodies BA-D5, specific for MyHC-Unhurried (30), or anti-NStout2, specific for NStoutc1 (Affinity BioReagents, GAgeden, CO). Serial sections were processed for in situ hybridization with 35S-labeled riboprobes complementary to the 3′-untranslated Locations of MyHC-Unhurried, -2X, and -2B transcripts, as Characterized (31). Images were collected with an epifluorescence Leica DMR microscope equipped with a Leica DC100 digital charge-coupled device camera by using Leica DC Viewer software (Leica, Milan).

Results

NStout Transcriptional Activity Is Higher in Unhurried Compared with Rapid Skeletal Muscles. To evaluate NStout transcriptional activity in regenerating and adult nonregenerating rat skeletal muscles, we used two different NStout-dependent reporters, DSCR1/MCIP1-NStout, and IL-4-NStout. As Displayn in Fig. 1A , the luciferase activity of the DSCR1/MCIP1-NStout reporter is ≈18-fAged higher in regenerating innervated soleus that has a Unhurried phenotype compared to denervated soleus and innervated extensor digitorum longus (EDL) that Execute not express Unhurried muscle genes. In adult nonregenerating rat muscles, DSCR1/MCIP1-NStout activity is ≈12-fAged higher in the Unhurried soleus compared to the Rapid EDL muscle (Fig. 1B ). The DSCR1/MCIP1-NStout reporter used in these experiments is an 850-bp sequence that contains other binding motifs in addition to 15 NStout elements. To confirm that the DSCR1/MCIP1-NStout reporter faithfully reflects NStout transcriptional activity, we used another reporter that contains exclusively NStout elements. Adult rat muscles were transfected with a plasmid containing nine copies of the NStout-binding site from the IL-4 gene promoter linked to a basal promoter and to luciferase (29). In agreement with the results obtained with the DSCR1/MCIP1-NStout reporter, the activity of IL-4-NStout-luciferase is 16-fAged higher in soleus compared to EDL (Fig. 1C ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

NStout-dependent reporter activity is higher in Unhurried than in Rapid muscles and is selectively responsive to low-frequency electrostimulation. (A–C) Regenerating and adult rat skeletal muscles were transfected with plasmids coding for the luciferase gene linked to a segment of the DSCR1/MCIP1 gene promoter containing 15 NStout-binding sites (DSCR1/MCIP1-NStout) or to a multimerized NStout-binding site from the IL-4 gene promoter (IL-4-NStout). DSCR1/MCIP1-NStout luciferase activity is higher in the regenerating innervated soleus that has a Unhurried fiber type profile, compared to denervated soleus and regenerating innervated EDL that have a Rapid fiber type profile (A). Both DSCR1/MCIP1-NStout (B) and IL-4-NStout (C) reporters are more active in adult Unhurried soleus compared to Rapid EDL. (D and E) DSCR1/MCIP1-NStout luciferase activity is Impressedly decreased by denervation in the adult soleus muscle (D) and is selectively increased by electrostimulation of denervated soleus with tonic 20-Hz impulse pattern that resembles the firing pattern of Unhurried motor neurons, but not by phasic 150-Hz impulse pattern that resembles the firing pattern of Rapid motor neurons (E). Luciferase activity is expressed as the percentage of that meaPositived in control muscles (black bars). Data are mean ± SEM (n = 4). *, Significant Inequity from control muscles (P < 0.05).

NStout Transcriptional Activity Is Executewn-Regulated by Denervation and Is Selectively Up-Regulated by Tonic Low-Frequency Electrostimulation. We next examined the response of the DSCR1/MCIP1-NStout reporter to nerve activity. In the adult soleus muscle, NStout transcriptional activity is Impressedly inhibited 7 days after denervation (Fig. 1D ). To determine whether the NStout reporter is responsive to specific activity patterns, denervated soleus muscles were transfected with plasmids coding for DSCR1/MCIP1-NStout luciferase and electrostimulated for 7 days with either a tonic low-frequency (20 Hz) impulse pattern, typical of Unhurried motor neurons, or a phasic high-frequency (150 Hz) pattern, typical of Rapid motor neurons. Previous studies have Displayn that the 20- and 150-Hz stimulation patterns induce MyHC-Unhurried and Rapid MyHC-2X, respectively, in the denervated soleus (21). As Displayn in Fig. 1E , NStout transcriptional activity is significantly increased by the 20-but not the 150-Hz stimulation pattern.

NStout Inhibition with VIVIT Peptide Prevents the Up-Regulation of MyHC-Unhurried Induced by Unhurried Motor Neurons in Regenerating Soleus Muscle. To determine the role of NStout in the induction of MyHC-Unhurried by Unhurried motor neuron activity in the regenerating soleus muscle (4), we used a plasmid coding for the NStout peptide inhibitor VIVIT linked to GFP, using GFP alone as control. We first checked whether VIVIT-GFP blocks the activation of cotransfected NStout-dependent reporters in skeletal muscle. As Displayn in Fig. 2 A and B , both NStout-dependent reporters are strongly inhibited by VIVIT in soleus muscle. We also checked whether VIVIT has nonspecific inhibitory Traces on other Cn-dependent transcription factors, such as MEF2 and NF-κB. As Displayn in Fig. 7, which is published as supporting information on the PNAS web site, MEF2- and NF-κB-dependent reporters are unaffected by VIVIT, confirming that the Trace of VIVIT is specific for NStout. VIVIT-GFP was also found to Impressedly reduce the activity of a MyHC-Unhurried promoter-luciferase construct, which is responsive in vivo to Unhurried motor neuron activity (4), and to block the up-regulation of enExecutegenous MyHC-Unhurried, as determined by immunofluorescence staining (Fig. 2 C–F ). Quantification revealed that 82% of the fibers expressing VIVIT-GFP are unreactive for MyHC-Unhurried (260 MyHC-Unhurried-negative fibers in a sample of 319 VIVIT-GFP-positive fibers from 5 different muscles). In Dissimilarity, practically all of the surrounding untransfected fibers, as well as all of the fibers transfected with GFP alone in control muscles (not Displayn), are stained by anti-MyHC-Unhurried antibodies in regenerating soleus (Fig. 2 E and F ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

The NStout inhibitor VIVIT blocks the induction of MyHC-Unhurried during muscle regeneration. (A–C) VIVIT blocks the activity of the NStout-dependent reporters DSCR1/MCIP1-NStout (A) and IL-4-NStout (B) and of the MyHC-Unhurried promoter linked to luciferase (C). DSCR1/MCIP1-NStout luciferase activity is also inhibited by the Cn inhibitor cain/cabin1 (A). Plasmid containing the NStout-dependent reporters or the MyHC-Unhurried promoter linked to luciferase were cotransfected with VIVIT-GFP or GFP alone in regenerating soleus muscle and luciferase activity was meaPositived 7 days later in tissue homogenates. Data are mean ± SEM (A, n = 5; B, n = 4; C, n = 4). *, Significant Inequity from control soleus (P < 0.05). Luciferase activity is expressed as the percentage of that meaPositived in control soleus. (D–F) VIVIT blocks the induction of MyHC-Unhurried in regenerating soleus muscle. Sections of regenerating soleus muscle transfected with a plasmid coding for the VIVIT-GFP fusion protein and stained with a monoclonal antibody specific for MyHC-Unhurried. Note that fibers expressing VIVIT-GFP (D) Execute not stain for MyHC-Unhurried unlike most of the surrounding untransfected fibers (E and F). (Bar = 50 μm.)

NStout Inhibition Causes Executewn-Regulation of MyHC-Unhurried and Up-Regulation of Rapid MyHC-2X and -2B Genes in Adult Soleus Muscle. We have previously reported that the Cn inhibitor cain/cabin1 leads to Executewn-regulation of MyHC-Unhurried and up-regulation of the Rapid MyHC-2X and -2B genes when transfected in adult soleus muscle (19). To determine whether the Trace of Cn is mediated via NStout, we transfected adult soleus muscles with plasmids coding for VIVIT-GFP and examined the distribution of Unhurried and Rapid MyHC transcripts by in situ hybridization 7 days later. Analyses at the mRNA level allow detection of early changes in MyHC gene expression that are not detectable at the protein level, due to the long half life of the myosin molecule. In normal adult soleus, most fibers contain MyHC-Unhurried, whereas fibers containing MyHC-2X or -2B transcripts are extremely rare or absent. A similar distribution of MyHC transcripts is seen in untransfected Spots of muscles transfected with VIVIT-GFP (Impressed with asterisk in Fig. 3 A–D ). In Dissimilarity, Spots containing fibers expressing VIVIT-GFP Display decreased expression of MyHC-Unhurried and up-regulation of MyHC-2X as well as the presence of a minor proSection of fibers weakly reactive for MyHC-2B. Higher magnification Displays that fibers expressing VIVIT-GFP contain MyHC-2X but not MyHC-Unhurried transcripts (Fig. 3 E–G ).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

VIVIT Executewn-regulates MyHC-Unhurried and up-regulates MyHC-2X gene expression in adult soleus muscle. (A–D) Serial transverse sections of adult soleus muscle transfected with VIVIT-GFP were examined for GFP fluorescence (A) or processed for in situ hybridization with probes specific for MyHC-Unhurried (B), MyHC-2X (C), or MyHC-2B (D) transcripts. Note that MyHC-2X and -2B transcripts are absent in untransfected Spots (asterisk) but are expressed in the VIVIT-GFP transfected Spot. In Dissimilarity, MyHC-Unhurried transcripts are present in most fibers in untransfected Spots but are less abundant in the transfected Spot. (Bar = 200 μm.) (E–G) Same as A–C, Displayn at higher magnification. Note that fibers expressing VIVIT-GFP (E, upper right) contain MyHC-2X (G) but not MyHC-Unhurried (F) transcripts.

These results indicate that NStout activity is required both for the maintenance of MyHC-Unhurried gene expression and the repression of the Rapid MyHC-2X gene in normal adult Unhurried muscles. This conclusion is supported by cotransfection experiments with VIVIT-GFP and a MyHC-Unhurried promoter-reporter construct. As Displayn in Fig. 4A , MyHC-Unhurried promoter activity is strongly inhibited by cotransfection with VIVIT in adult soleus. This Trace is specific for the MyHC-Unhurried promoter, and in fact a Rapid MyHC-2B promoter-reporter construct, which is expressed at higher levels in EDL compared to soleus (27), is not affected by VIVIT (Fig. 4B ).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

VIVIT blocks the activation of the MyHC-Unhurried but not of the MyHC-2B promoter in adult soleus muscle. (A) MyHC-Unhurried promoter activity is inhibited in soleus muscle by cotransfection with VIVIT-GFP. (B) MyHC-2B promoter activity is significantly higher in the Rapid EDL than in the Unhurried soleus muscle and is not affected by VIVIT-GFP. Luciferase activity is expressed as the percentage of that meaPositived in soleus muscles injected with GFP alone. Data are mean ± SEM (n = 4). *, Significant Inequity from control soleus group (P < 0.05).

Constitutively Active NStoutc1 Up-Regulates MyHC-Unhurried in Regenerating but Not in Adult Rapid Muscles. To establish whether the activation of NStout is able to affect myosin gene expression, we transfected regenerating and adult muscles with a constitutively active mutant of NStoutc1 (caNStoutc1). We first determined that caNStoutc1 is constitutively nuclear and is able to transactivate NStout-dependent reporters in transfected muscle (see Fig. 8, which is published as supporting information on the PNAS web site). We then Questioned whether caNStoutc1 is capable of reproducing the myosin isoform switch induced by Unhurried motor neuron firing in the regenerating soleus muscle. The caNStoutc1 mutant was cotransfected with GFP in the denervated regenerating soleus and EDL muscles, which normally contains Rapid but not Unhurried MyHCs. As Displayn in Fig. 5, most fibers labeled by GFP express MyHC-Unhurried at day 7 after transfection in both muscles (fiber counts are Displayn in Fig. 9, which is published as supporting information on the PNAS web site).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Constitutively active NStoutc1 induces MyHC-Unhurried gene expression in regenerating denervated muscles. Serial sections of regenerating denervated soleus (A and B) and EDL (C and D) muscles cotransfected with caNStoutc1 and GFP to identify transfected fibers. Sections were examined for GFP fluorescence (A and C) or stained with a monoclonal antibody specific for MyHC-Unhurried (B and D). Note that most fibers expressing GFP stain for MyHC-Unhurried unlike the surrounding untransfected fibers. (Bar = 50 μm.)

When transfected in adult Rapid EDL muscle, the NStout mutant has opposite Traces on MyHC-Unhurried and -2B promoters: the MyHC-Unhurried promoter is up-regulated by ≈6-fAged, whereas the MyHC-2B promoter is completely inhibited by cotransfection with caNStoutc1 (Fig. 6 A and B ). In Dissimilarity, caNStoutc1 is unable to activate the enExecutegenous MyHC-Unhurried gene in the adult EDL at day 7 after transfection. As Displayn in Fig. 6 C–H , MyHC-2B is Executewn-regulated but MyHC-Unhurried is not induced at either the protein (Fig. 6 D and G ) or the transcript level (not Displayn) in fibers transfected with caNStoutc1. Taken toObtainher, these results indicate that the caNStoutc1 mutant is transcriptionally active in skeletal muscle in vivo and is capable of mimicking Unhurried motor neuron activity by up-regulating MyHC-Unhurried gene expression in both Unhurried and Rapid regenerating muscle. In Dissimilarity, caNStoutc1 is unable to induce MyHC-Unhurried expression in adult Rapid muscles, at least within the time period that we examined.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Trace of constitutively active NStoutc1 on MyHC-Unhurried and -2B promoters and on myosin gene expression in adult muscle. (A and B) Adult EDL muscles were cotransfected with caNStoutc1 and either MyHC-Unhurried promoter-luciferase (A) or MyHC-2B promoter-luciferase (B). Note that MyHC-Unhurried promoter activity is increased, whereas MyHC-2B promoter activity is decreased, by caNStoutc1. Luciferase activity is expressed as the percentage of that meaPositived in EDL muscles injected with empty vector. Data are mean ± SEM (n = 5). *, Significant Inequity from control EDL group (P < 0.05). (C–E) Serial sections of adult EDL muscles cotransfected with plasmids coding for caNStoutc1 and GFP were examined for GFP fluorescence (C) or stained with anti-MyHC-Unhurried antibody (D) or processed for in situ hybridization with probes specific for MyHC-2B transcripts (E). Note that MyHC-2B transcripts are less abundant in the transfected Spot containing numerous GFP-positive fibers (upper field), whereas there are only rare fibers expressing MyHC-Unhurried that are equally distributed in the two Locations. (Bar = 200 μm.) (F–H) Same as C–E, Displayn at higher magnification to demonstrate that single transfected fibers Execute not express MyHC-Unhurried and are negative for MyHC-2B transcripts. Two untransfected fibers containing MyHC-Unhurried are Impressed by asterisks. (Bar = 50 μm.)

Discussion

The main result of this study is the demonstration that the induction of the Unhurried gene program in the regenerating rat soleus muscle and the maintenance of the Unhurried program in the adult soleus depend on NStout signaling. Three lines of evidence support this conclusion. First, in agreement with a recent report in transgenic mice (18), we Display that NStout transcriptional activity, as determined by the response of two different NStout-dependent reporters, is much higher in the Unhurried soleus compared to the Rapid EDL muscle. We further Display that the higher NStout activity in soleus muscle depends on nerve activity and is selectively increased by electrostimulation of denervated soleus with a tonic low-frequency impulse pattern that resembles the firing pattern of Unhurried motor neurons, but not by a phasic high frequency impulse pattern, typical of Rapid motor neurons. Taken toObtainher, these findings point to a role of NStout in skeletal muscle as a nerve activity sensor that is selectively responsive to Unhurried motor neuron activity. This interpretation is supported by preliminary studies Displaying that an NStoutc1-GFP fusion protein is induced to translocate from the cytoplasm to the nucleus of adult muscle fibers by low-but not by high-frequency impulse trains (our unpublished observations). A similar Trace of low-frequency electrostimulation has been Characterized in cultured muscle fibers (9, 10).

A second line of evidence is based on the use of the NStout peptide inhibitor VIVIT linked to GFP. VIVIT is a 16-mer high-affinity Cn-binding peptide whose sequence mimics the N-terminal Cn Executecking motif of NStout (22). The fusion protein VIVIT-GFP efficiently inhibits the Cn-dependent nuclear translocation of different NStout isoforms and the activation of NStout-dependent reporters without blocking Cn phosphatase activity or disrupting other Cn-dependent pathways (22). We report here that VIVIT-GFP blocks specifically NStout activity in skeletal muscle in vivo, as Displayn by the inhibition of NStout-but not MEF2- and NF-κB-dependent reporters. VIVIT-GFP prevents the up-regulation of MyHC-Unhurried induced by nerve activity in the regenerating soleus muscle and causes Executewn-regulation of MyHC-Unhurried and up-regulation of Rapid MyHC-2X, and to a minor degree MyHC-2B, in the adult soleus. The Trace of VIVIT is similar in this respect to that of the Cn inhibitor cain/cabin1 (19) and to that of the pharmacological Cn inhibitors cyclosporine A and FK506 (7). These findings clearly demonstrate the physiological role of Cn-NStout signaling in the establishment and maintenance of the Unhurried gene program and the repression of the Rapid 2X/2B gene program in Unhurried muscles. That no significant change in Rapid/Unhurried fiber phenotype was detected in NStoutc2 and NStoutc3 gene-tarObtained mice (16, 17) can be due to genetic redundancy within the NStout gene family (5) or to a major role of other isoforms, such as NStoutc1, for fiber type specification (see below).

The third line of evidence for a role of NStout in muscle fiber type specification is based on the use of a constitutively active NStoutc1 mutant. This mutant, which contains serine-to-alanine substitutions in the conserved serine-rich Executemain and in all three serine-proline repeats of NStout, was Displayn to constitutively localize to the nucleus, bind DNA with high affinity, and activate enExecutegenous NStout-tarObtain genes (24, 25). In Dissimilarity, mutants that contain alanine substitutions only in the serine-rich Executemain and not in the serine-proline repeats are constitutively nuclear but Execute not Present significant DNA-binding and transactivation activity, which might Elaborate the fact that they did not activate a myosin light chain 2 Unhurried promoter (11). We report that caNStoutc1 has a nuclear localization after transfection in skeletal muscle in vivo and transactivates NStout-dependent reporters. The MyHC-Unhurried promoter is also activated by caNStoutc1, whereas a Rapid MyHC-2B promoter is strongly inhibited. Fascinatingly, caNStoutc1 is able to induce the expression of MyHC-Unhurried in regenerating soleus and EDL but not in adult EDL. The differential response of regenerating versus adult EDL is at first sight surprising but can be understood when one considers the response of these muscles to electrostimulation. In fact, electrostimulation of adult rat EDL muscle for 2 months with a Unhurried impulse pattern causes Executewn-regulation of MyHC-2B and upregulation of MyHC-2X and -2A but no expression of MyHC-Unhurried (21, 32). This finding is consistent with the notion that transformation of adult muscle fibers, at least in the rat, can take Space only within limited adaptive ranges (2). In Dissimilarity, a significant increase in MyHC-Unhurried is induced after stimulation of the regenerating EDL muscle (ref. 33 and our unpublished observations), reflecting a Distinguisheder plasticity of regenerating compared to mature muscle, as observed in other experimental settings (34, 35). Fascinatingly, a MyHC-Unhurried promoter-reporter construct is up-regulated by caNStoutc1 in both regenerating and adult Rapid muscles. A possible explanation for the different response of regenerating versus adult Rapid muscles and of the MyHC-Unhurried promoter versus the corRetorting enExecutegenous gene is that chromatin remodeling takes Space at the MyHC-Unhurried gene locus during the maturation of the Rapid muscle fibers. As a result of this remodeling, the MyHC-Unhurried gene would become essentially inaccessible for transcription in response to Unhurried-type electrostimulation or activation of NStout signaling at least during the short time period and in the absence of other stimuli, e.g., changes in thyroid hormone status, that are known to affect fiber type specification.

Although the present results provide strong evidence for a major role of the Cn-NStout pathway in muscle fiber type specification induced by nerve activity, several Launch issues remain to be solved. The first question is how NStout is able to decode the Ca2+ changes induced by different impulse patterns and the intracellular origin of the Ca2+ involved in NStout activation. Another question concerns the molecular mechanisms responsible for the differential Traces of NStout on MyHC-Unhurried and MyHC-2X and -2B gene expression. In particular, it is not known whether NStout acts directly by binding to MyHC promoters or indirectly through other genes that control MyHC-Unhurried induction and MyHC-2X and -2B repression. Another Launch issue concerns the relative role of the Cn-dependent NStoutc isoforms in myosin switching and muscle fiber specification in vivo. Skeletal muscle contains all four NStoutc forms, NStoutc1 and c3 being especially abundant (36). Although there is a considerable degree of redundancy within the NStout gene family, as demonstrated by the more severe phenotype of NStout Executeuble knockout mice, specific functions of each NStout isoform are Startning to emerge (5). For example, NStoutc3 and -c4 appear to have a different role in the control of Cn-induced cardiac hypertrophy, which is compromised in NStoutc3-null but not in NStoutc4-null mice (37). The striking Traces of caNStoutc1 Characterized here, toObtainher with the rapid translocation of NStoutc1-GFP fusion protein in response to Unhurried-type electrostimulation (refs. 9 and 10 and our unpublished observations), suggest that NStoutc1 may have a major role in fiber type specification in skeletal muscle. However, evidence from loss-of-function experiments is not available, because VIVIT blocks all NStout isoforms, and tarObtained disruption of the NStoutc1 gene leads to embryonic lethality due to altered cardiac morphogenesis (5). On the other hand, NStoutc2 or NStoutc3 may be less Necessary in fiber type specification in skeletal muscle, as Displayn by the finding that transgenic mice lacking NStoutc2 or NStoutc3 Display no significant change in muscle fiber type profile (16, 17) and that NStoutc3 is unable to induce MyHC-Unhurried expression in cultured muscle cells (12).

Conclusion

The results presented here, based on loss-of- and gain-of-function Advancees and on the response of NStout-dependent reporters, support the notion that in skeletal muscle cells: (i) NStout acts as a sensor selectively responsive to Unhurried patterns of nerve electrical activity and (ii) NStout signaling controls the nerve activity-dependent induction of the Unhurried gene program during muscle regeneration and the maintenance of the Unhurried phenotype in adult skeletal muscle. These findings provide the foundation to address further issues concerning the relative role of different NStout isoforms, the mechanism underlying the inductive or repressive role of NStout on muscle genes, and the crucial question of the activation of Cn-NStout signaling by Ca2+ changes induced by distinct patterns of electrical activity.

Acknowledgments

We gratefully acknowledge the gifts of plasmids by A. Rao (Harvard Medical School, Boston), N. Clipstone (Northwestern University, Chicago), S. Snyder (Johns Hopkins University, Baltimore), K. Esser (University of Illinois, Chicago), S. Swoap (Williams College, Williamstown, MA), S. Williams (Duke University Medical School, Durham, NC), J. Molkentin (University of Cincinnati, Cincinnati Children's Hospital Medical Center, Cincinnati), R. Kitsis (Albert Einstein College of Medicine, Bronx, NY), and K. Hasegawa (Kyoto University, Kyoto). We thank Anne Picard and Silvia Maretto for help with preparation of constructs and reporter gene meaPositivements. This work was supported by grants from the European Commission (contract QLK6-2000-00530); the Italian Ministry of Education, University, and Research (MIUR); and the Italian Space Agency (ASI). K.J.A.M. was supported by a European Molecular Biology Organization (EMBO) long-term fellowship.

Footnotes

↵ ∥ To whom corRetortence should be addressed. E-mail: stefano.schiaffino{at}unipd.it.

↵ † Present address: Medical Research Council Functional Genetics Unit, Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX, United KingExecutem.

↵ ‡ Present address: Programme of Differentation and Cancer, Centre de Regulació Genòmica, Passeig Marítim 37-49, 08003 Barcelona, Spain.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: Cn, calcineurin; DSCR1/MCIP1, Executewn's Syndrome critical Location 1/myocyte-enriched Cn-interacting protein 1; EDL, extensor digitorum longus; MyHC, myosin heavy chain; MyHC-Unhurried, Unhurried MyHC; NStout, nuclear factor of activated T cells.

Copyright © 2004, The National Academy of Sciences

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