Functional overload increases beta -MHC promoter activity in rodent fast muscle via the proximal MCAT (beta e3) site

Julia M. Giger, Fadia Haddad, Anqi X. Qin, and Kenneth M. Baldwin

Department of Physiology and Biophysics, University of California, Irvine, California 92697


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Functional overload (OL) of the rat plantaris muscle by the removal of synergistic muscles induces a shift in the myosin heavy chain (MHC) isoform expression profile from the fast isoforms toward the slow type I, or, beta -MHC isoform. Different length rat beta -MHC promoters were linked to a firefly luciferase reporter gene and injected in control and OL plantaris muscles. Reporter activities of -3,500, -914, -408, and -215 bp promoters increased in response to 1 wk of OL. The smallest -171 bp promoter was not responsive to OL. Mutation analyses of putative regulatory elements within the -171 and -408 bp region were performed. The -408 bp promoters containing mutations of the beta e1, distal muscle CAT (MCAT; beta e2), CACC, or A/T-rich (GATA), were still responsive to OL. Only the proximal MCAT (beta e3) mutation abolished the OL response. Gel mobility shift assays revealed a significantly higher level of complex formation of the beta e3 probe with nuclear protein from OL plantaris compared with control plantaris. These results suggest that the beta e3 site functions as a putative OL-responsive element in the rat beta -MHC gene promoter.

gel mobility shift assay; plantaris muscle; direct gene transfer; dual luciferase; beta -myosin heavy chain


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SKELETAL MUSCLE FIBERS have the potential to adapt their phenotypic properties to meet different functional demands. The beta -myosin heavy chain (MHC) isoform is typically expressed in "slow" muscles, which are characterized by a relatively slow speed of shortening but exhibit fatigue resistance and thus are able to maintain contractile activity for long periods of time. They typically function as weight-bearing and postural muscles. In the soleus, a slow muscle in the hindlimb, ~90% of fibers express the beta -form of MHC (13, 16). The relationship between weight-bearing activity and the expression of beta -MHC in soleus muscle is demonstrated by removing the factor of load in experimental animal models. This is achieved by subjecting the animal to either the microgravity environment of spaceflight (14) or through the ground-based model of hindlimb suspension (4) such that the leg extensor muscles bear little or no weight. When these manipulations are imposed, the expression of beta -MHC is significantly reduced along with a shift toward increased expression of faster MHC isoforms (4, 14).

At the other extreme, functional overload induces the expression of beta -MHC in the plantaris, a fast-twitch ankle extensor muscle that normally expresses the fast MHC isoforms, IIb and IIx, which account for >80% of the MHC pool (28). In this model, the overload stress is induced by the removal of synergistic muscles along with normal weight-bearing activity, which collectively causes compensatory hypertrophy of muscle fibers (1, 26). Adaptation to the overload stimulus also prompts a shift in the fiber-type profile toward a slow fiber-type pattern, characterized by a lower myofibrillar ATPase activity, reduced glycogenolytic enzyme activities, decreased expression of fast MHC isoforms IIx and IIb, and increased expression of slow myosin light chain isoforms and slower MHC isoforms beta  (type I) and IIa (2, 28, 30, 34).

We are interested in determining the molecular mechanisms responsible for the increase in the slow, beta -MHC gene expression in overloaded plantaris. It is believed that the phenotypic adaptations are mediated in some way by the interaction of transcriptional regulatory proteins with specific cis elements within the beta -MHC gene's promoter sequence. It may be that fast and slow fibers express a different pool of transcriptional regulatory proteins, or transcription factors, that interact with the cis elements that are present only in the genes of "fast" or slow isoforms. Thus we tested the hypothesis that the upregulation of beta -MHC expression in response to overload is regulated through specific elements of the beta -MHC promoter.

The proximal region of the beta -MHC promoter has been shown to be required for muscle-specific expression in cardiac and skeletal cell culture (7, 29) and transgenic mice models (20, 34). Several highly conserved positive and negative regulatory elements within the proximal region (-408 bp) promoter have been identified (7, 20, 29, 34). A putative repressor element, beta e1 (-321/-297), appears to play a distinct role in beta -MHC expression in skeletal and cardiac muscle cells (7, 9, 10) and has been implicated in conferring the decreased expression of beta -MHC that occurs in response to soleus unloading (23). It has been suggested that four positive elements [beta e2 or muscle CAT (MCAT)-binding site (-285/-269); an A/T-rich site (-267/-259); the CCAC box (-245/-233); and the beta e3 element (-214/-190)] are essential for muscle-specific gene activation (20, 29, 34). In fact, the region containing these positive motifs appears to be required for the increased expression of the human beta -MHC gene after plantaris overload in transgenic mice (34).

Unlike previous studies (20, 30, 32), the present report involves a comprehensive mutation analysis of all of the putative regulatory elements in the rat beta -MHC proximal promoter to resolve their role in the load responsiveness of the beta -MHC gene in vivo. Herein, we describe a direct gene transfer approach whereby deleted or mutated promoters are linked to a firefly luciferase reporter gene and injected directly in rat plantaris muscles. After injection, the synergistic gastrocnemius and soleus muscles were removed from the left leg to induce plantaris overload, and the right leg was left intact to act as the control muscle. After 7 days, the plantaris muscles were harvested, and luciferase activity was measured. We have successfully used this gene transfer technique to examine the relevant promoter sequences for maintaining high levels of beta -MHC activity in normal slow-twitch soleus muscles and after unloading (11).


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Animal Procedures

Female Sprague-Dawley rats weighing 110-125 g were anesthetized (10 mg ketamine and/or 20 mg acepromazine/100 g) for all aseptic surgical/injection procedures.

DNA injection. A skin incision was made to expose the muscle of interest. PBS (20 µl) containing a mixture of two supercoiled DNA plasmids [beta -MHC test plasmid (molar equivalent to 10 µg of -3,500 bp MHC-Fluc) and a control skeletal alpha -actin-Rluc reporter plasmid (molar equivalent to 5 µg of -3,500 bp MHC-Fluc)] was injected in the muscles using a 29-gauge needle attached to a 0.5-ml insulin syringe.

Electroporation. To improve plasmid uptake by the muscle, electropermeabilization was induced on the muscles immediately after plasmid injection. This procedure, termed electroporation, was performed according to the method described by Mir et al. (24). Two gold-plated stainless steel electrodes set 4 mm apart were placed in contact with the muscle at the injection site. Using a Grass electrical stimulator, four pulses (200 V/cm, lasting 50 ms each, 1 Hz) were delivered, and then four additional pulses of opposite polarity were applied.

Unilateral functional overload. Plantaris overload was performed via removal of the synergistic muscles, gastrocnemius, and soleus in one leg only, as described in detail previously (2). The contralateral leg remained intact, and the contralateral plantaris represented the control. The overload surgical procedure was carried out immediately after plasmid injections/electroporation. All experiments were for a duration of 7 days. After 7 days, plantaris muscle tissues were excised after pentobarbital sodium (100 mg/kg) anesthesia and were quick-frozen and stored at -80°C. All animals in the study were allowed food and water ad libitum, and all procedures were approved by the institutional animal care and use committee.

Reporter Plasmid Constructs and Expression Assays

The plasmids -3300right-arrow+34 and -215right-arrow+34 rat beta -MHC-CAT were a kind gift from Dr. P. C. Simpson (University of California San Francisco; see Ref. 18) and the -3500right-arrow+462 beta -MHC-Luc was kindly provided by Dr. K. Ojamaa (North Shore Univ. Hospital; see Ref. 25). The -3500 and -215 sequences were subcloned into a firefly luciferase expression vector (pGL3 basic; Promega; see Ref. 35). Deletion mutations of the 5'-end were derived from this long fragment using mapped sites for restriction endonucleases and were subcloned by standard procedures in the reporter plasmid; all beta -MHC sequences terminated at +34 from the transcription start site. Site-directed mutagenesis employed the MORPH mutagenesis kit (5primeright-arrow3prime) to introduce base substitutions in the promoter sequences using the wild-type promoters as templates (see Table 1).

                              
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Table 1.  

Frozen muscle tissues were homogenized in ice-cold passive lysis buffer from Promega, using a glass homogenizer. The homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was reserved for the luciferase activity assay using Promega's dual luciferase assay kit, which is designed for sensitive detection of both firefly and renilla luciferase activities in a single extract aliquot. Activities were measured as total light output (as measured by a Monolight 2010-C luminometer) per muscle per second and were expressed as relative light units. Background levels, based on luciferase activities of noninjected tissue, were subtracted from the activities of test samples. These experiments were predicated on the assumption that the level of luciferase activity is proportional to the degree of promoter activity.

Justification for the Use of Skeletal alpha -Actin as a Reference Promoter

To correct for variation in gene transfer efficiency in the direct injection technique, a reference vector linked to a different reporter gene is coinjected with the test plasmid. Typically, a plasmid containing a viral promoter, such as the cytomegalovirus (CMV) promoter, is selected as the reference vector because the viral promoter is ubiquitously and constitutively expressed in transfected cells. The activity of the reference promoter is used as an independent internal control for the variability of gene transfer efficiency. However, in our hands, the CMV or the Simian virus (SV)-40 viral promoters subcloned into a renilla luciferase expression vector (CMV-Rluc, SV40-Rluc; Promega) proved unsatisfactory as control vectors. As we have reported previously (11), CMV-RLuc activity was persistently variable, often significantly lower than that of the test promoter-firefly luciferase (-3500 bp beta -MHC-Fluc) and thus did not reflect transfection efficiency. Therefore, we chose instead to use a 2-kb promoter sequence of the human skeletal alpha -actin (gift from S. Swoap, Williams College) linked to a renilla luciferase reporter as the reference vector. Both MHC and skeletal alpha -actin are integral sarcomeric muscle proteins, but, unlike the MHC, skeletal alpha -actin is the only actin isoform expressed in adult skeletal muscle and therefore is not fiber-type specific. Any change in alpha -actin expression reflects global changes in general transcription activity of sarcomeric proteins resulting from hypertrophic or atrophic stimuli. Unlike a viral promoter, the alpha -actin promoter will not be expressed in surrounding nonmuscle tissue, which compromises its function in the normalization of plasmid uptake in muscle cells. We have previously shown (11) that the level of firefly luciferase activity (beta -MHC-Fluc) correlated well with the level of renilla luciferase (alpha -actin-Rluc) activity in control soleus muscles, demonstrating the effectiveness of alpha -actin reference plasmid as a control for transfection efficiency.

Ideally, the reference promoter activity should not change in response to experimental manipulation. However, as a sarcomeric muscle protein, we expect alpha -actin to be upregulated in response to the hypertrophic stimuli of plantaris overload. Indeed, we know that alpha -actin promoter is responsive to functional overload in chicken anterior latissimus dorsi muscles (6). Yet, because the overload procedure by synergist ablation is the same in all experiments, we expect the degree of overload to be equivalent in each subject as demonstrated by the similar level of plantaris enlargement (see RESULTS). Therefore, although the activity of alpha -actin promoter increases in response to overload, the degree of increase should remain relatively constant from one experiment to another. Indeed, in the present paper, the increase of alpha -actin activity was not significantly different among the experiments, with an average increase of 135% above control. The consistency of the alpha -actin promoter activity validates its function as a reference vector and thus allows comparisons between the following test promoters: 1) different length promoter fragments and 2) wild-type vs. mutated sequences.

As an additional control, plasmid uptake can be determined by directly measuring the amount of plasmid DNA in the tissue using PCR. One set of experiments was sampled using this method to assess whether plasmid uptake was different in overloaded and normal control plantaris. No difference was detected in either the firefly luciferase test plasmid (beta -MHC-pGL3) or the reference alpha -actin renilla luciferase plasmid between normal control and overloaded groups. Given this fact, normalizing the firefly luciferase activity to plasmid DNA would exaggerate the beta -MHC response to overload because it would not reflect the overall increase in global transcriptional and posttranscriptional activity that likely occurs with hypertrophic stimuli. Therefore, we feel that dividing the beta -MHC promoter-driven firefly activity by alpha -actin-driven renilla activity is a better conceived method of normalization because it factors out this generalized increase in transcriptional activity. Thus any increase in the beta -MHC-Fluc/alpha -actin-Rluc reporter activity in response to overload is a result of the specific increase in beta -MHC isoform promoter activity.

Gel Mobility Shift Assay

Nuclear extraction of skeletal muscle tissue. Nuclear protein was extracted from skeletal muscle according to the method described by Blough et al. (3). Briefly, frozen plantaris muscle tissue (400-700 mg) was homogenized in 35 ml of buffer 1 [10 mM HEPES (pH 7.5), 10 mM MgCl2, 5 mM KCl, 0.1 mM EDTA (pH 8.0), 0.1% Triton X-100, 0.2 mM phenylmethylsulfonyl fluoride (PMSF), 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 1 mM dithiothreitol (DTT)]. Homogenates were centrifuged for 5 min at 3,000 g at 4°C. The pellets were resuspended in 500-1,000 µl of buffer 2 [20 mM HEPES (pH 7.5), 500 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA (pH 8.0), 25% glycerol, 0.2 mM PMSF, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 0.5 mM DTT]. The suspension was incubated on ice with intermittent mixing for 30 min and was centrifuged for 5 min at 4,000 g at 4°C. The supernatant was transferred to an Amicon 2-ml Centricon filter unit (YM-10; Millipore, Bedford, MA). An equal volume of binding buffer [20 mM HEPES (pH 7.9), 40 mM KCl, 2 mM MgCl2, 10% glycerol, 0.2 mM PMSF, 2.5 µg/ml aprotinin, 2.5 µg/ml leupeptin, and 0.5 mM DTT] was added to the filter unit and centrifuged for 30 min at 4,500 g at 4°C. Another 500-1,000 µl were added and centrifuged again for 3 min; centrifugation was repeated until the concentrate achieved the desired volume (1 ml). The protein concentration of the nuclear extract was determined using the Bio-Rad protein assay with BSA as a standard. Nuclear extract samples were stored at -80°C.

Gel mobility shift assay. Gel mobility shift assays (GMSAs) were used to examine and quantitate binding of nuclear extract protein to the beta e3 cis-regulatory element of the rat type I MHC gene promoter (29). This approach allows one to detect changes in transacting factor content and/or changes in the pattern of shifted bands resulting from binding of one or more factors. The beta e3 oligonucleotide sequences were purchased from GIBCO, and the sense strand was of the following sequence: beta -MHC beta e3: 5'-CATGCCATACCACAACAATGACGC-3' (bases that were mutated are underlined). After strand annealing, the double-stranded probe was end labeled with [gamma -32P]ATP (6,000 Ci/mmol) using T4 polynucleotide kinase (Promega). For each binding reaction, 20-µg nuclear extract were preincubated for 10 min at room temperature with 225 ng poly(dI-dC) homopolymer, which was used as a nonspecific competitor, in a binding buffer containing 40 mM KCl, 1 mM DTT, 1 mM EDTA, 1 mM MgCl2, 7.5% glycerol, 0.05% BSA, and 20 mM HEPES (pH 7.9) in a total volume of 20 µl. For competition studies, the preincubation was carried out in the presence of 150× molar excess of either cold beta e3 wild type (specific), a mutated cold beta e3, or an A/T-rich oligonucleotide (nonspecific). At the end of the preincubation, 100,000 counts/min of labeled beta e3 were added and incubated for 30 min at room temperature. At the end of the reaction, 2 µl loading buffer (20% ficoll, 0.2% bromphenol blue, and 0.2% xylene cyanol) were added, and the reaction mixtures were loaded on a 6% polyacrylamide gel, which was preelectrophoresed at 20 mA/gel for 2 h. Electrophoresis was carried out in 0.5× Tris-borate-EDTA buffer at constant current (30 mA) at room temperature for 3 h. After electrophoresis, the gels were dried and visualized by phosphorimaging (Molecular Dynamics, Sunnyvale, CA). The intensity of the shifted bands was quantified by scanning densitometry (Molecular Dynamics) using volume integration on the shifted bands (Image Quant; Molecular Dynamics).

Statistical Analysis

Statistical analysis was performed using the Graphpad Prism 2.0 statistical software package. Values are means ± SE. Differences among groups were determined by ANOVA followed by the Newman-Keuls posttest. Differences between the means of two experimental groups were assessed by an unpaired, two-tailed t-test. P < 0.05 was taken as the level of statistical significance.


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Body/Muscle Weights

Compensatory growth of the plantaris muscle of the left leg was observed after 1 wk of unilateral functional overload. The contralateral right leg remained intact, and the plantaris muscle of this leg served as the normal control muscle in the experiments. Previously, we have demonstrated that the unilateral functional overload model does not alter MHC gene expression in the contralateral control (unpublished observations). The average body weight of female Sprague-Dawley rats after 1 wk of overload was 147.1 ± 0.96 g (n = 100). Muscle wet weight of normal control plantaris muscle was 138 ± 1.6 mg (muscle wt/body wt = 0.94 ± 0.01 mg/g). Overloaded plantaris muscle was 316 ± 4.9 mg (muscle wt/body wt = 2.16 ± 0.03 mg/g), which corresponds to a significant 129% increase in muscle wet weight (Students t-test, P < 0.0001). Thus the overload stimulus provides a robust stimulus of muscle enlargement in these young female rats.

Deletion Analysis of the beta -MHC Promoter in Normal Control Plantaris Muscle

The in vivo approach of direct gene injection was employed to ascertain which segments of the beta -MHC promoter sequence are essential for the upregulation of the slow beta -MHC gene in the fast-twitch plantaris muscle in response to functional overload. Four fragments (-914, -408, -215, and -171) of the -3500-bp beta -MHC parent promoter were examined in both normal control and overloaded plantaris muscles. In the control plantaris, all of the deletion fragments were sufficient to activate reporter activity (Fig. 1A) and were at least 10-fold above the mean reporter activity of the promoterless pGL3 basic plasmid (basic-FLuc/actin-Rluc = 0.0003 ± 0.00012; n = 6). Of interest to note is that the beta -MHC promoter activity of the fast-twitch plantaris was an order of magnitude lower than that of the slow-twitch soleus (Fig. 1B), which demonstrates muscle fiber-type specificity concerning beta -MHC gene expression.


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Fig. 1.   A: deletion analysis of beta -myosin heavy chain (MHC) promoter in normal control muscles. Deletion analyses in plantaris muscle of normal control rats. Derived from the parent promoter, -3500 bp (n = 14), deletion fragments -914 (n = 11), -408 (n = 15), -215 (n = 20), and -171 (n = 7) were subcloned into the firefly luciferase (Fluc) reporter plasmid. Each construct was injected in an equimolar amount relative to the parent plasmid construct of 10 µg and was coinjected with the alpha -actin-renilla luciferase (Rluc) control plasmid (relative to one-half equimolar amount to -3500, i.e., 3.3 µg). Luciferase activity is expressed as beta -MHC-to-skeletal alpha -actin ratios. Values are means ± SE. *P < 0.05 vs. all, as determined by ANOVA. B: comparison of slow-twitch soleus and fast-twitch plantaris muscles. Luciferase activity ratio values of plantaris samples are the same samples depicted in A but were doubled to normalize to the amount of alpha -actin-Rluc (6.6 µg) injected in soleus. With the exception of the -171 construct, deletion analyses of the parent promoter had no effect on activity levels in normal control (NC) plantaris, but a significant difference was observed between the deletions -3500 (n = 23), -914 (n = 7), -408 (n = 14), -215 (n = 14), and -171 (n = 11) in soleus muscles. *P < 0.05 vs. all (ANOVA). ^P < 0.05 vs. -215 (t-test); ** From Ref. 11.

Surprisingly, with the exception of the -171 construct, deletion analyses of the parent promoter had no significant effect on activity levels in normal control plantaris. This is worth noting because, in soleus muscle, the activity is very responsive to deletion mutations (Fig. 1B and Ref. 11). Also in soleus muscle, beta -MHC-Fluc reporter activity was highest using the -3500 promoter, whereas in plantaris the smallest deletion fragment, -171, showed the highest level of activity. The very different pattern of promoter activity in response to deletion analysis between the soleus and plantaris muscles suggests that distinct mechanisms are functioning in the regulation of this gene in these two different muscles of contrasting fiber type.

Deletion Analysis in Overloaded Plantaris

Deletion analysis revealed that the -3500, -914, -408, and -215 reporter constructs were all responsive to overload (Fig. 2A). There was no significant increase in activity resulting from overload using the -171 construct despite the higher level of reporter activity in both control and overloaded plantaris. The highest level of the overload response was observed using the -408 promoter, which responded with more than two times the reporter activity measured in normal control plantaris (Fig. 2B). The relative level of upregulation in response to overload dropped significantly from 128% with the -408 construct to only 52 and 36% with the -215 and -171 constructs, respectively. The decrease in response that resulted from further deletion of the -408 promoter suggests that certain regulatory elements within this proximal region are required for activation of the beta -MHC promoter in response to overload.


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Fig. 2.   A: deletion analysis of beta -MHC promoter in control and overloaded (OL) plantaris. Deletion analysis in OL plantaris of the left leg and normal control (NC) plantaris of the right leg. After the injection of the beta -MHC-Fluc and sk alpha -RLuc plasmid mixture, the functional OL procedure was performed on the left hindlimb. Reporter activities of deletion fragments [OL: -3500 (n = 18), -914 (n = 17), -408 (n = 17), -215 (n = 18), and -171 (n = 10)] were compared in NC and OL plantaris. All values are means ± SE. *P < 0.05, NC vs. OL (t-test). B: bar graph of percent increase of promoter activity in OL plantaris over control values. Data were derived from A, showing that the relative level of upregulation was highest with the -408 promoter. ^P < 0.05 vs. -408 (ANOVA).

Mutation Analysis of -408 beta -MHC Promoter

The proximal region of the beta -MHC promoter sequence between -408 and -171 contains numerous putative regulatory elements, including beta e1 (-321/-297); beta e2 or MCAT-binding site (-285/-269); A/T rich (-267/-259); CCAC box (-245/-233); and a beta e3 element (-214/-190). All of these elements have been implicated to play a role in muscle-specific gene activation in transgenic mice (20,34) and in cardiomyocyte cell culture (29) experiments. To determine which cis element(s) confer the response to overload in plantaris muscle, mutation analysis was performed on these putative elements (Fig. 3). Comparison of -408 promoters that contain single mutations of the beta e1, beta e2, or CACC elements showed that the promoter activity was still responsive to overload despite these individual mutations. The overload response of each of the three mutants was not significantly different from that of the wild-type construct (Fig. 3B), suggesting that these elements do not confer the activation of the beta -MHC promoter in response to overload. A triple mutation construct harboring the simultaneous mutations of beta e2, beta e3, and A/T-rich elements showed only a small upregulation with overload (53%), which was significantly lower than the wild-type response (128%). Therefore, one of these mutated elements or the interaction of two or more of these elements may be responsible for the upregulation of the beta -MHC promoter in response to overload. Given that the beta e2 single mutation had already been tested and found to respond to overload similarly to the wild type (Fig. 3), we excluded this construct from further analysis and concentrated on only testing the A/T-rich and beta e3 single mutations.


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Fig. 3.   A: mutation analysis of -408 beta -MHC promoter in control and OL plantaris. Three -408 bp promoter constructs harboring single mutations of beta e1 (n = 6 NC; 7 OL), beta e2 (n = 8; 9), and CACC (n = 8; 8) regulatory elements; one "triple" construct (n = 12, 21) harboring three mutations (beta e2, beta e3, and A/T-rich sites) simultaneously; and -408 wt (from Fig. 2) were examined in NC and OL plantaris. Location of regulatory elements in -408 promoter are shown. See Table 1 for exact sequences. *P < 0.05, NC vs. OL (t-test). B: bar graph of the percent increase of promoter activity in OL plantaris over control values. Data were derived from A. The triple mutant was the only mutant that was significantly different compared with the wild type (wt). ^P < 0.05 vs. -408 wild type (ANOVA).

A -408-A/T-rich mutant was tested in overloaded and normal control plantaris muscles (Fig. 4). Evidence supporting the A/T-rich mutant (or GATA element) as a load-responsive element stems from a report by Vyas et al. (33) that demonstrated increased binding of the A/T-rich element by nuclear extracts from overloaded plantaris compared with normal control plantaris.


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Fig. 4.   A: beta e3 mutant abolishes OL effect. Two -408 bp promoter constructs harboring single mutations of A/T-rich (n = 9 NC and 14 OL) and beta e3 (n = 17) vs. -408 wild type (from Fig. 2) were examined in NC and OL plantaris. See Table 1 for exact sequences. The OL response was abolished in beta e3 mutant. *P < 0.05, NC vs. OL (t-test). B: bar graph of the percent increase of promoter activity in OL plantaris over control values. Data were derived from A. Despite higher levels of activity, A/T-rich mutant has similar OL response as the wild type. The beta e3 mutant was the only mutant that was significantly different compared with the wild type. ^P < 0.05 vs. -408 wild type (ANOVA).

Compared with the -408 wild-type promoter, the A/T-rich mutant exhibited a large increase in reporter activity in both normal control and overloaded muscle, a pattern that suggests that a negative/repressor element may have been altered. However, despite this upregulation compared with the wild type, the mutant was still highly responsive to overload, with the reporter activity increasing up to 160% over control activity levels (Fig. 4B).

The last element to be tested that could be responsible for the diminished activity of the triple mutant was the beta e3 site. The -408 beta e3 mutation eliminated the overload response, indicating that the beta e3 element may be a critical site by which the beta -MHC gene is transcriptionally activated in plantaris muscle after overload (Fig. 4). Initially, the potential role of the beta e3 element in the overload response was not expected, given that 1) this element is located within the -215 promoter fragment, and 2) the majority of the regulation appears to be taking place upstream of the -215 deletion fragment (within the -408 fragment). However, as seen in the deletion analysis (Fig. 2A), although the -215 promoter exhibited an overload response, the response was significantly smaller than that of the -408 construct. In contrast, the -171 construct, which lacks the beta e3 element, did not respond to the overload stimulus. Of all the -408 mutations tested in overloaded plantaris, only the triple mutant (i.e.. beta e2, beta e3, and A/T rich) and the beta e3 single mutant were significantly different than the response of the -408 wild type.

Gel Mobility Shift Analysis

Further evidence supporting a role for the beta e3 element in the overload response was derived from a GMSA comparing nuclear extracts from normal control and overloaded rat plantaris. A 24-bp oligonucleotide corresponding to the beta e3 sequence (-214 to -191) of the rat beta -MHC promoter sequence was used as a probe, and several DNA-protein complex bands were detected (Fig. 5A). When challenged with unlabeled competitors, complexes labeled A and B represented specific high-affinity complexes because they were disrupted by competition with unlabeled beta e3 but were not competed by nonself oligonucleotides (beta e3 mut or A/T rich). There was only a 3-bp difference between the competitors beta e3 and beta e3 mut, and yet complexes A and B remained intact with beta e3 mut competition. Therefore, the protein factor(s) forming complexes A and B most likely bind the site containing these 3 bp. Significantly, the level of binding of overloaded nuclear extracts with complexes A and B was 280 and 150%, respectively, higher than with normal control extracts (Fig. 5B). The same overload and normal control extract samples were also compared using an A/T-rich probe (Fig. 5C). Several complexes were resolved but showed no differences in the level of binding of overloaded and normal control nuclear proteins (quantitative measurements are not shown). The similarity of shifted bands between normal control and overload extracts indicates that the small amount of binding of normal control extract to the beta e3 probe observed in Fig. 5A is not the result of a smaller amount of nuclear protein in the sample.


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Fig. 5.   Representative gel mobility shift assays (GMSAs) assessing the interaction of radiolabeled oligonucleotide probes with 20-µg nuclear protein from 6 OL and 3 NC plantaris muscles samples. A: with the use of rat beta -MHC beta e3 as a probe, several binding complexes were resolved. Binding specificity was analyzed through competition assays by preincubating extracts with 150-fold molar excess of unlabeled oligonucleotides: beta e3 (self), mutated beta e3 (mut), and A/T rich (nonself). Only complexes A and B denote specific high-affinity complexes because they were disrupted by competition with self but were not competed by nonself oligonucleotides. NS, nonspecific binding. B: quantification of band intensities demonstrates highly enriched binding activity of complexes A and B with OL plantaris nuclear extracts (n = 10) compared with NC extracts (n = 7). Values are means ± SE. *P < 0.05, NC vs. OL (t-test). C: GMSA using A/T-rich probe comparing same samples as in A shows no difference between binding of OL and NC plantaris extracts. Binding specificity was analyzed through competition assay by preincubating extracts with 150-fold molar excess of unlabeled oligonucleotide [A/T rich (self)]. Oligonucleotide probes/competitors were double stranded. Sequences were as follows: rat beta -MHC beta e3 WT: 5'-CATGCCATACCACAACAATGACGC-3'; rat beta -MHC beta e3 mut: 5'-CATGCCATcggACAACAATGACGC-3'; and rat beta -MHC A/T rich: 5'-AATGTAAGGGATATTTTTGCTTCA-3'.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

There is a relationship between the functional characteristics of a muscle and the types of MHC isoforms that are expressed in the muscle fibers. In the hindlimb, for example, the chronically weight-bearing soleus muscle expresses primarily the slow, beta -MHC isoform (15), whereas its fast-twitch synergist, the plantaris muscle, expresses a predominance of the fast MHC isoforms, IIx and IIb (14). Muscle fibers have the unique ability to undergo changes in MHC phenotype as an adaptive response to altered loading states; however, the regulatory mechanisms involved in these phenotype transitions are largely unknown. Our interest lies in further understanding how beta -MHC isoform gene expression is regulated in fast and slow muscles in response to various loading conditions. Deletion analysis revealed that beta -MHC promoter activity responds uniquely in slow-twitch soleus compared with the fast-twitch plantaris muscle, suggesting that distinct mechanisms are involved in the regulation of this gene in these two different muscle types. Previously, we have noted that, in soleus muscle (11) and cardiac muscle (35), the largest level of beta -MHC promoter activity occurs in the parent promoter (-3500), and promoter activity decreases with successive deletions. Of further interest, herein we report that, in the normal control plantaris muscle, a muscle that normally does not abundantly express the beta -MHC gene (28), no such promoter length dependence was demonstrated (Fig. 1). The only difference in activity was seen with the smallest deletion construct (-171), which unexpectedly had the highest level of activity. In fact, aside from the A/T-rich mutant, even when specific regulatory elements were mutated, activity levels in normal control plantaris were not changed. It is conceivable that a pool of inhibitory transcription factors bind the beta -MHC promoter to maintain low levels of expression that are typical of normal fast-twitch plantaris, and only after the binding sites are eliminated by severe deletion are the inhibitory factors removed. Support for this concept stems from the findings of Lamph et al. (21), which suggest that the cAMP-responsive element-binding protein (CREB) in an unphosphorylated state can act as a repressor of transcriptional activity of the c-jun promoter. The sequence between -215 and -171 contains, among others, a putative CREB binding site. Perhaps CREB may be acting as a repressor in the beta -MHC promoter in normal plantaris muscle. Consequently, with the creation of the -171 construct, this "repressor" is removed.

Mechanical overload of the plantaris results in marked increases in expression of beta -MHC isoform mRNA and protein (28) and an increase in beta -MHC promoter activity (Fig. 2). However, the mechanism by which loading stimuli affect beta -MHC gene activation is unclear. Based on the findings by Vyas et al. (33), we tested a mutant -408-A/T-rich rat promoter in our gene transfer model to determine if an intact A/T-rich element was required for the overload response. Despite an upregulation in activity with the mutant -408-A/T-rich promoter, the overload response was similar to that of the wild type. Therefore, factors binding to the A/T-rich element do not appear to be relevant in conferring the overload-stimulated activity of the rodent beta -MHC promoter. Perhaps our results differ from Vyas et al. (33) because their GMSA experiments used the human beta -MHC A/T-rich sequence, which contains about 10 extra base pairs that flank the 5'-A/T-rich region, whereas we used a rat sequence that did not contain these flanking base pairs.

In the present study, a triple mutant (beta e2, beta e3, and A/T rich) and the beta e3 single mutant were the only mutants that had a significantly lower overload response than the -408 wild-type construct. Interestingly, only the beta e3 single mutant completely abolished the overload response. One curious finding was that the activity of the triple mutant in overloaded plantaris was higher than the beta e3 mutant alone, even though beta e3 was one of the three mutations (see Figs. 3 and 4). Perhaps the reason for this unexpected result is that one of the other mutations within the triple mutant was the A/T-rich element, which exhibited a large upregulation in activity when it alone was mutated. In other words, the A/T-rich mutation may have compensated for the loss of activity brought about by the beta e3 mutation.

To our knowledge, we are the first to identify the beta e3 site as a putative overload-responsive element in the rat beta -MHC gene promoter. We base this conclusion on three findings. First, in the normal control and overloaded plantaris gene transfer studies, the -171 construct, which lacks the beta e3 element, did not respond to the overload stimulus (Fig. 2A). Second, the overload response was completely abolished when 3 bp of the beta e3 element were mutated (Fig. 4A). Finally, in the GMSA experiment (Fig. 5) a significant increase in the level of specific binding of the beta e3 probe was detected with the extracts from overloaded plantaris muscles compared with normal control plantaris extracts. However, this conclusion conflicts with a transgenic mouse plantaris overload study using a mouse beta -MHC promoter-CAT reporter transgene that harbored simultaneous mutations of beta e2, CACC, and beta e3 elements (30). That report found that, despite the mutations, reporter activity increased in response to 8 wk of bilateral plantaris overload similar to that of the wild type. These findings may differ between the two studies because of the different model systems employed. Not only were different species and different mutated sequences used, but we used a direct gene transfer technique to introduce the promoter-reporter construct, whereas the other investigators used a transgenic mouse model. Moreover, the time period of the overload stimulus differed greatly between the two studies. Our 1-wk model explored the regulatory processes that are involved during the adaptive phase of the overload response. In contrast, their 8-wk study (30) assessed the promoter activity at the steady-state level of the overload stimulus. It is possible that the mechanisms regulating promoter activity are different during the steady state compared with the adaptive phase such that, after such a long period of overload perturbation, the involvement of other elements may override the beta e3 mutation and induce an increase in promoter activity.

In the GMSA experiment (Fig. 5), mutant competition suggests that the protein(s) comprising complexes A and B bind at the site including the 3 bp ACC of the MCAT sequence. The MCAT motif has been shown to be required for muscle-specific expression in genes of sarcomeric proteins, such as alpha -skeletal actin (19), alpha -MHC (12), cardiac tropin T (22), and beta -MHC (9). Therefore, our results indicate that the overload stimulus triggers an as yet unknown signaling pathway whereby transcription factor(s) bind the MCAT core of the beta e3 element, thus initiating the transactivation of the beta -MHC promoter.

Although we have not yet examined which transcription factors may be binding the MCAT motif of beta e3 in our skeletal muscle overload model, evidence from cardiomyocyte culture studies indicate that the transcriptional enhancer factor (TEF) 1 (or MCAT binding factor; see Ref. 8) may be involved. In fact, two members of the TEF family, TEF1 and TEF-related (TEFR) 1, have been shown to regulate transactivation of the beta -MHC and other muscle genes such as alpha -MHC (12) and skeletal alpha -actin (19) through an MCAT site. For example, sequential mutation analysis in GMSA assays determined that both phenylephrine and protein kinase C (PKC)-beta stimulation of cardiomyocytes activated the -215 beta -MHC promoter through the MCAT core of the beta e3 element (18) and the protein factor binding the beta e3 sequence immunoreacted with a TEF1 antibody (17). Stewart et al. (27), on the other hand, concluded that, although basal levels of beta -MHC activity require an intact MCAT for TEF1 and TEFR1 activation, the pathway whereby phenylephrine potentiates -215 beta -MHC expression does not involve the TEF family of factors or the MCAT box. The MCAT-TEF1-TEFR1 signaling pathway may also play a role in the regulation of skeletal alpha -actin, another fetal gene activated in response to alpha 1-adrenergic stimulation. For example, Karns et al. (19) maintain that PKC-phosphorylated TEF1 binds to an MCAT site within the skeletal alpha -actin proximal promoter, whereas Ueyma et al. (31) contend that it is the mitogen-activated protein kinase phosphorylation of serine-322 in TEFR1, a site not present in TEF1, that transactivates the skeletal alpha -actin promoter. Stewart et al. (27) noted that alpha 1-adrenergic stimulation of skeletal alpha -actin is via TEFR1, yet not through an MCAT site. Given the differences of opinion about the role of TEF1/TEFR1 factors and signaling pathways in beta -MHC and skeletal alpha -actin gene regulation in cardiomyocyte cultures, both TEF1 and TEFR1 factors need to be tested in our model.

More relevant to our in vivo model are findings by Carson et al. (5, 6) that indicate a role of TEF1 in the regulation of the skeletal alpha -actin gene in hypertrophied skeletal muscle. With the use of a stretch-overload model of the chicken anterior latissimus dorsi muscle, skeletal alpha -actin promoter activity increased significantly in response to stretch overload (6). Also, 3 days of stretch increased TEF-1 binding to the skeletal alpha -actin promoter, as demonstrated by GMSA. These results support the hypothesis that the TEF1 family of transcription factors may be involved in conferring the overload-stimulated increase in beta -MHC gene activity in skeletal muscle.

Finally, it is important to note that we cannot completely disregard the fact that other elements upstream of beta e3/-215 may play some role in our model given the fact that the -408 construct has a significantly higher level of overload responsiveness than that of the -215 promoter. In conclusion, the major findings derived from this study indicate that the proximal region (-408) of the rat beta -MHC promoter is essential for maximal promoter activity in response to plantaris overload, and an intact MCAT sequence within the beta e3 element is required for the overload responsiveness.


    ACKNOWLEDGEMENTS

This work was performed during the tenure of a fellowship for J. M. Giger from the American Heart Association, Western States Affiliate, and was supported by National Institute of Arthritis and Musculoskeletal Diseases Grant AR-30346.


    FOOTNOTES

Address for reprint requests and other correspondence: K. M. Baldwin, Dept. of Physiology and Biophysics, Univ. of California, Irvine, Irvine, CA 92697 (E-mail: kmbaldwi{at}uci.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.

10.1152/ajpcell.00444.2001

Received 17 September 2001; accepted in final form 31 October 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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