Transcriptional regulation of the type I myosin heavy chain promoter in inactive rat soleus

K. A. Huey1, R. R. Roy3, F. Haddad1, V. R. Edgerton2,3, and K. M. Baldwin1

1 Department of Physiology and Biophysics, University of California, Irvine, Irvine 92697; and 2 Department of Physiological Science, and 3 Brain Research Institute, University of California, Los Angeles, Los Angeles, California 90095-1761


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chronic muscle inactivity with spinal cord isolation (SI) decreases expression of slow type I myosin heavy chain (MHC) while increasing expression of the faster MHC isoforms, primarily IIx. The purpose of this study was to determine whether type I MHC downregulation in the soleus muscle of SI rats is regulated transcriptionally and to identify cis-acting elements or regions of the rat type I MHC gene promoter involved in this response. One week of SI significantly decreased in vivo activity of the -3500-, -408-, -299-, -215-, and -171-bp type I MHC promoters. The activity of all tested deletions of the type I MHC promoter, relative to the human skeletal alpha -actin promoter, were significantly reduced in the SI soleus, except activity of the -171-bp promoter, which increased. Mutation of the beta e3 element (-214/-190 bp) in the -215- and -408-bp promoters and deletion of this element (-171-bp promoter) attenuated type I downregulation with SI. Gel mobility shift assays demonstrated a decrease in transcription enhancer factor-1 binding to the beta e3 element with SI, despite an increase in total binding to this region. These results demonstrate that type I MHC downregulation with SI is transcriptionally regulated and suggest that interactions between transcription enhancer factor-1 and the beta e3 element are likely involved in this response.

beta e3 DNA regulatory element; transcription enhancer factor-1; spinal cord isolation; chronic muscle inactivity


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

CHRONIC DECREASES IN NEUROMUSCULAR activity strongly influence myosin heavy chain (MHC) expression in rodent skeletal muscle (for reviews see Refs. 27 and 36). Decreased neuromuscular activation and unloading, as occurs with spinal cord transection (37), and reduced loading or weight bearing, as induced by hindlimb suspension (simplified here as unloading) (14, 23), decrease the relative expression of slow type I MHC while increasing the relative expression of the faster MHC isoforms in the normally slow-twitch rat soleus muscle. Although these models decrease overall muscle activity and load, some residual neuromuscular activation remains, which could influence muscle gene expression. Specifically, MHC expression could be altered by any remaining activation-associated factors, despite the dramatic reduction in load and activity. Although denervation results in complete inactivity, it also removes non-activity-dependent neurotrophic factors, which may be involved in regulation of MHC plasticity (32, 33). Consequently, it has been difficult to develop a suitable model to study the effects of complete inactivity on MHC gene regulation while maintaining an intact motoneuron-muscle connection, i.e., without direct interruption of potential activity-independent neurotrophic factors.

Spinal cord isolation (SI) provides a unique model in which the hindlimb muscles are silenced for prolonged periods by functionally isolating the lumbar region of the spinal cord (6, 25, 28). This model eliminates supraspinal, infraspinal, and peripheral afferent input to the isolated cord segment while maintaining motoneuron-muscle connectivity. Thus SI provides a model that removes neuromuscular activation and loading, while the motoneuron continues to exert activity-independent neurotrophic effects on the inactive muscles (6, 28).

We recently reported that SI results in a time-dependent decrease in type I MHC in the rat soleus muscle, which is primarily offset by an increase in fast type IIx MHC (16). These MHC shifts were characterized by an early loss in type I MHC, as evidenced by the rapid muscle atrophy and concomitant decreases in muscle protein and MHC content over the first 8 days of SI. The loss of nearly half of the type I MHC protein in the soleus muscle after only 8 days of inactivity is the result of negative balance between protein synthesis and degradation. This imbalance may be due to alterations in transcriptional/translational and/or posttranslational processes. In a previous study (16), analysis of the time course of changes in type I MHC protein and mRNA expression in the soleus muscle of SI rats revealed that the changes at the mRNA level preceded changes at the protein level. Furthermore, over the complete SI time course of 4-90 days, there was a tight coupling between changes at the mRNA and protein levels, suggesting that pretranslational processes dominated MHC isoform shifts in the chronically inactive soleus muscle. However, the question remains as to whether these pretranslational processes are transcriptional; therefore, the present study further characterizes the molecular mechanisms mediating downregulation of type I MHC expression in inactive slow-twitch muscle. It is hypothesized that the molecular remodeling of the soleus muscle associated with SI is regulated in part at the transcriptional level via interaction of specific transcription factors with cis-acting regulatory elements located on muscle gene promoters. Furthermore, inactivity is likely associated with changes in the nuclear milieu that render it unfavorable for transcription of slow-twitch muscle genes.

Within the MHC gene family, slow type I MHC expression has been studied most extensively in cardiac and skeletal muscle, and the gene has been isolated from several mammalian species, including human (21, 30), rat (22), mouse (11, 42), and rabbit (8). In vivo studies involving transgenic mice (26) have provided evidence that type I MHC promoter activity is muscle specific. Reporter activity was detected primarily in cardiac and slow-twitch skeletal muscles. In contrast, no reporter activity was detected in pure fast-twitch muscles (masseter) or nonmuscle tissue of transgenic animals harboring the type I MHC promoter fused to the chloramphenicol acetyltransferase reporter gene. Furthermore, in vivo studies using direct muscle gene injection demonstrated that the type I MHC promoter is 25-fold more active in the slow-twitch soleus than in the fast-twitch tibialis anterior muscle (9), which corresponds to endogenous gene expression. On the basis of transient transfection assays in cardiac and muscle cell culture (5, 7, 17, 18, 31, 38), several highly conserved positive and negative cis-acting regulatory elements have been identified within the most proximal region of the type I MHC promoter [within 350 bp of the transcription start site (TSS)]. For example, at least three positive elements that are necessary for muscle-specific expression have been identified: beta e2 (-285/-269 bp), CACC box (-245/-233 bp), and beta e3 (-210/-188 bp) (38). A negative element, beta e1 (-330/-300 bp), has also been identified (5). In addition, the upstream distal region (-3500/-2500 bp) was required for optimal promoter activity in cardiac and slow-twitch skeletal muscles (9, 45).

Although several positive and negative regulatory elements have been identified and found necessary for type I MHC promoter activity under normal conditions, their role in modulating type I MHC gene expression under altered physiological conditions is unclear. Studies have implicated the beta e1 repressor region in type I MHC downregulation in response to skeletal muscle unloading (9, 23), while all three positive elements have been implicated in the adaptation to skeletal muscle overloading (39, 43).

Although transcriptional regulation of the type I MHC gene has been demonstrated under altered loading states, regulation of this gene has never been studied under conditions of complete neuromuscular inactivity. It remains to be elucidated whether decreases in type I MHC expression with chronic inactivity in slow-twitch muscles are mediated transcriptionally and whether transcriptional downregulation would result from increased activity of a repressor element or decreased activity of an enhancer element. Consequently, SI provides an excellent model to begin to establish the functional significance of these positive and negative elements of the type I MHC promoter during periods of complete muscle inactivity. These findings will provide an excellent baseline with which to compare type I MHC regulation in other models of decreased muscle activity and/or neurotrophic influence such as hindlimb unloading and denervation.

The purpose of the present study, therefore, was to test the hypothesis that downregulation of type I MHC expression in the soleus muscle of SI rats is regulated at the transcriptional level using the in vivo direct gene transfer approach. A second goal was to identify cis-acting regulatory sequences on the rat type I MHC gene promoter necessary for its downregulation in response to chronic inactivity. We found that 1 wk of SI results in a significant decrease in type I MHC promoter activity in the inactive rat soleus muscle and that important cis-acting DNA regulatory elements mediating this response are contained within 215 bp of the proximal promoter. Within this fragment, the beta e3 element is likely involved in the type I MHC downregulation, inasmuch as deletion and mutation of this element attenuated the decrease in promoter activity associated with SI.


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INTRODUCTION
METHODS
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Experimental design and animal procedures. The experiments were conducted over a period of ~1 yr and consisted of repeating the following experimental design. For each tested promoter construct, adult female Sprague-Dawley rats (145 ± 10 g) were randomly assigned to normal control (NC) and SI groups (n = 8 animals/group). Rats were anesthetized and underwent surgery for intramuscular plasmid injection into left and right soleus muscles, as described previously (9). A skin incision was made to expose the soleus muscle, and 20 µl of phosphate-buffered saline containing plasmid DNA mixtures were injected into the muscles using a 29-gauge needle attached to a 0.5-ml insulin syringe. Plasmid mixtures consisted of the rat type I MHC test promoter (molar equivalent to 10 µg of -3.5-kb type I MHC-pGL3 promoter construct) and a fixed amount (3.29 µg) of the human skeletal alpha -actin reference promoter construct (HSA2000-pRL). After plasmid injection, the rats in the SI group were subjected to complete spinal cord transections at midthoracic and upper sacral levels, along with bilateral deafferentation between the transection sites, as described previously (10, 29). Additional rats were assigned to NC (n = 12) and SI (n = 24) groups and used to obtain soleus muscle for muscle nuclei extraction for use in the DNA gel mobility shift assays (see below). The care and maintenance of the SI rats have been described in detail previously (10, 29). All experiments were 7 days in duration. After 7 days, the animals were euthanized, and the soleus muscle was quickly removed, frozen in isopentane cooled by liquid nitrogen, and stored at -80°C until subsequent analysis. This study was approved by the Animal Use Committee at the University of California, Los Angeles, and followed the animal care guidelines of the American Physiological Society.

Plasmid constructs. The test promoter consisted of the rat type I MHC gene promoter linked to the firefly luciferase (FLuc) reporter gene in the pGL3 vector (Promega). A series of deletions of the type I MHC promoter were tested: -3500-, -408-, -299-, -215-, and -171-bp promoter fragments, all extending to position +34 at the 3' end relative to the TSS. These promoter constructs were the same as those used previously by Wright et al. (44). The -408- and -215-bp type I MHC promoter constructs with beta e3 mutation (-408-bp beta e2/beta e3 mutant) were also tested in the NC and SI soleus muscle. Mutations of the target promoter sequence consisted of changing ACC to CGG in the beta e3 element (17, 38) and GTG to TGT in the beta e2 element (38) and were designed to disrupt specific transcription factor binding sites (see Ref. 44 for details on mutagenesis). The reference promoter consisted of the human skeletal alpha -actin gene extending from -2000 to +250 bp relative to the TSS, which was linked to the Renilla luciferase (RLuc) reporter gene in the pRL vector (Promega).

In addition, a type IIb MHC promoter construct was injected into NC and SI soleus muscle to examine the promoter response of a gene upregulated in response to SI (16). This experiment would also rule out the possibility of global transcriptional downregulation with SI. The test plasmid consisted of a mouse type IIb MHC promoter fragment extending from -1400 to +13 bp relative to the TSS and linked to an FLuc reporter gene (pGL3 basic, Promega). The type IIb MHC and human skeletal alpha -actin promoter fragments were the kind gift of Dr. Steve Swoap (Williams College, Williamstown, MA), and their activity in rat muscle corresponds to relative expression of the endogenous gene (35). Plasmids were amplified in Escherichia coli cultures and purified by anion-exchange chromatography using disposable columns (Endofree Maxiprep, Qiagen). Plasmids were suspended in sterile phosphate-buffered saline, and the concentration was determined by ultraviolet absorbance at 260 nm using the conversion factor 50 µg/ml per optical density unit. Plasmid preparations were examined by ethidium bromide staining after agarose gel electrophoresis to verify their supercoiled nature and that they were free of genomic DNA and cellular RNA.

Reporter gene assay. Each soleus muscle was homogenized in 1 ml of ice-cold passive lysis buffer (Promega) supplemented with protease inhibitors [0.2 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 5 µg/ml aprotinin, 5 µg/ml leupeptin], and the homogenate was centrifuged at 10,000 g for 10 min at 4°C. The supernatant was reserved to measure reporter gene activity using the Promega dual-luciferase assay kit, which is designed for sensitive detection of FLuc and RLuc activities within a single extract aliquot. Five microliters of supernatant were used in the assay, and light output was integrated over 10 s and expressed as relative light units (RLUs) using a luminometer (Analytical Luminescence). Background activity levels, based on measurements of noninjected tissue for both luciferases, were determined and subtracted from the activities measured in plasmid-injected tissue.

Rationale for use of skeletal alpha -actin as a reference promoter. In the present study, activity of the type I MHC promoter linked to an FLuc reporter was tested in the SI soleus muscle using reporter gene assays after direct muscle gene transfer. This approach is predicated on the assumption that reporter gene expression (FLuc activity) is directly proportional to the activity of the promoter linked to the 5'-upstream region. The method of gene transfer (in vivo or in cell culture) is associated with variability in DNA uptake by the cells, which makes it difficult to interpret changes in reporter gene activity. Consequently, a reference promoter is coinjected with the test promoter and used as an internal control for variable gene transfer efficiency. The reference promoter drives expression of a different reporter gene, which allows independent measurement of its activity along with that of the test promoter. The reference gene promoter should not interact with test promoter activity and should ideally not change in response to experimental manipulations. Skeletal alpha -actin is a good reference promoter for studies utilizing muscle gene promoters for the following reasons: 1) Skeletal alpha -actin and MHC are sarcomeric genes that are intrinsically active in muscle cells; thus the transfection of exogenous promoters of these genes into muscle cells is less likely to disturb the nuclear milieu balance and cause competition for nuclear factors than transfection with strong viral promoters (simian virus 40 and cytomegalovirus). 2) Skeletal alpha -actin and MHC are muscle-specific genes that are not expressed in nonmuscle cells; thus the possibility of confounding expression in surrounding nonmuscle cells is unlikely. 3) FLuc and RLuc reporter expression can be assayed in a single aliquot with similar detection sensitivity and range (Promega dual-luciferase assay protocol). As evidence of the validity of alpha -actin for normalizing gene transfer efficiency in muscle, Giger et al. (9) reported a strong correlation between FLuc and RLuc activity after coinjection of type I MHC pGL3 and human alpha -actin pRL in the NC soleus.

Although there is strong evidence supporting the skeletal alpha -actin promoter as a reference in NC muscle, its use as reference promoter in manipulated muscle needs further justification. We present reasons that strongly validate the use of skeletal alpha -actin as a reference promoter in the soleus of SI rats, especially when comparing the responsiveness of two or more different promoters. The actin promoter has been shown to be responsive to altered loading conditions (2, 3), and on the basis of mRNA analysis, after 8 days of SI, expression of total MHC and skeletal alpha -actin mRNA relative to 18S ribosomal RNA in the soleus muscle was reduced by ~74 and 72%, respectively (unpublished observations). This is not surprising, considering the fact that MHC and actin are basic muscle structural genes, and their expression should be tightly regulated when the muscle undergoes atrophy or hypertrophy.

In the present experiments, the response of the skeletal alpha -actin promoter (SI vs. NC) was expected to be relatively constant across all experiments, because it was subjected to the same experimental manipulation (7 days of SI). For all eight experiments in this project (8 rats per group per construct), experimental procedures and personnel were identical, and soleus atrophy was homogeneous (Table 1), suggesting comparable actin responsiveness across experiments. As expected, the actin promoter response to SI was not significantly different among all experiments, with an average decrease of ~90% (data not reported).

                              
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Table 1.   Body weight, muscle weight, and muscle weight-to-body weight ratios in NC and SI groups for individual experiments in deletion and mutation analysis of type I MHC promoter

Unlike the actin promoter, which responded to SI in a predictable and comparable manner, responses of the different type I MHC promoters could vary depending on the presence or absence of regulatory elements. In gene transfer assay systems, measured reference and test reporter activities depend on plasmid uptake, and possible variations must be accounted for in the interpretation of the results. When actin and MHC promoter constructs are coinjected into the muscle, their uptake efficiency is thought to be similar. Thus normalizing type I MHC test promoter activity to actin eliminates differences in plasmid uptake across experiments utilizing different MHC promoters. With this normalization, the results are interpreted as changes in test promoter activity relative to actin, rather than absolute changes per se. Consequently, by using this approach (normalization to actin), the behavior of different test promoter constructs could be evaluated to define critical regulatory regions of the promoter.

Plasmid uptake. Plasmid DNA uptake amounts can be used to normalize reporter activity without use of a reference plasmid; however, this approach does not account for possible alterations in posttranscriptional processes induced by the experimental manipulation. During the course of this study, it was clear that SI caused a dramatic decrease in test and reference promoter activities. Consequently, the possibility existed that plasmid uptake efficiency was less in SI muscles, which would cause this decrease in test and reference promoter activity. To gain insight into plasmid uptake in this model, tissue plasmid content at the time of analysis for reporter activity was determined using PCR.

Total tissue DNA (genomic and plasmid) was extracted from the resulting tissue pellet after centrifugation of the soleus homogenate in the reporter gene assay protocol described above. DNA extraction was performed using the Qiagen QIAamp DNA Mini Kit according to manufacturer's protocol for tissue extraction. DNA was eluted in 200 µl of nuclease-free water.

Plasmid detection and quantification were based on PCR amplification. PCR primers to detect pGL3 plasmid were targeted specifically to the FLuc cDNA, and their sequence shares no identity with any of the rat genomic DNA sequence. The primers used to detect the pGL3 plasmid are CGGGCGCGGTCGGTAAAGT [5' pGL3 primer (1163-1181)] and AACAACGGCGGCGGGAAGT [3' pGL3 primer (1542-1524)], where the numbers in parentheses refer to the base position within the pGL3 basic sequence (GenBank accession no. U47295), and they generated a 380-bp PCR product. The FLuc cDNA gene is located from 88 to 1737 bp (Promega Technical Reference).

Five microliters of the DNA template were amplified using PCR (final concentrations): 1× PCR buffer, 20 µM each dNTP, 1 µM primers, 2.8 mM MgCl2, and 0.75 U of Taq polymerase. Amplification was carried out in a thermocycler (Stratagene, La Jolla, CA) and was started with an initial denaturation step at 96°C for 3 min, followed by 1 min at 96°C, 45 s at 59°C, and 50 s at 72°C for 25 cycles and a final elongation step for 3 min at 72°C. Under these PCR conditions, the signal was in the linear range of a semilogarithmic plot of the yield vs. number of PCR cycles. Samples were run in duplicate with appropriate negative and positive controls. PCR products were separated on 2% agarose gels containing ethidium bromide. Pictures of the gels were obtained under ultraviolet light with Polaroid 55 film to obtain a negative and a positive gel image. The negatives were scanned (Molecular Dynamics personal densitometer), and band intensity was determined with the Image Quant 5.0 software package (Molecular Dynamics) using volume integration.

Gel mobility shift assays. Skeletal muscle nuclei were isolated and extracted according to the method described previously by Blough et al. (1). Nuclear extract was prepared from NC and SI soleus muscles (n = 6/group, each n consisted of a pool of 4 muscles for NC and 8 muscles for SI, ~200 mg/pool) and stored at -80°C. Double-stranded oligonucleotides consist of 24 bp from the type I MHC gene promoter spanning specific regulatory elements. The beta e3 oligonucleotide sense strand sequence and beta e3 mutant sense strand sequence are given in Table 2. After strand annealing, double-stranded probes were end labeled with 32P using T4 polynucleotide kinase (Promega).

                              
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Table 2.   Oligonucleotides used as probes or competitors in gel mobility shift assay

For the binding reaction, 20 µg of nuclear protein were preincubated for 10 min on ice with 15 ng of nonspecific homologous DNA [poly (dIdC)] in a buffer containing 50 mM KCl, 20 mM HEPES, pH 7.9, 1 mM dithiothreitol, 1 mM MgCl2, 1 mM EDTA, 7.5% glycerol, and 0.025% BSA. At the end of this preincubation, 5'-end-labeled probe (~200,000 cpm) was added, and reactions (20 µl total volume) were incubated at room temperature for 30 min. For the supershift experiments, 0.5 µg of transcription enhancer factor-1 (TEF-1) antibody (BD Transduction Laboratories, Los Angeles, CA) was added to the reactions after 20 min of incubation with the probe, and the sample was incubated at room temperature for an additional 20 min. Reactions were terminated by addition of 2 µl of loading buffer (10% Ficoll, 0.01% xylene cyanol, and 0.01% bromphenol blue). The samples were immediately loaded on a 7% polyacrylamide gel and electrophoresed at 250 V for 2 h in 0.5× Tris base-boric acid-EDTA under nondenaturing conditions. Gels were dried and exposed to a PhosphorImager (Molecular Dynamics) storage screen for 24 h. Gel images were obtained by laser scanning of the storage screen with a PhosphorImager. For competition experiments, 100-fold molar excess of unlabeled probes was included in the preincubation described above (i.e., before addition of the labeled probe). Band intensity was quantified with Image Quant 5.0 software (Molecular Dynamics).

Specificity of the TEF-1 antibody used in the gel supershift assays was confirmed using Western blots and supershifts with a double-stranded DNA probe, which is unrelated to the TEF-1 binding site, such as BNP-GATA (41). In Western blots using soleus nuclear extract, along with HeLa nuclear extract and a positive control provided by the commercial source of TEF-1 antibody (BD Transduction), TEF-I antibody immunoreacted with a single specific band corresponding to 53 kDa (data not shown).

Statistical analysis. Statistical analysis was performed with Graphpad Prism 3.0 statistical software. Values are means ± SE. An unpaired, two-tailed t-test was used to detect differences between NC and SI groups. For multiple comparisons, one-way ANOVA with Newman-Keuls post hoc tests were performed. P < 0.05 was accepted as the level of statistical significance.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Body and muscle weights. Animals assigned to separate experiments were matched in initial body weight: 139 ± 1 and 140 ± 1 g for NC and SI groups, respectively. At the termination of the 7-day experiment, body weight increased to 166 ± 1 g in NC rats and decreased to 122 ± 1 g in SI rats. SI resulted in similar changes in body weight and muscle weight relative to NC across all experiments (Table 1). Average body weight was 26% less, absolute soleus muscle weight was 60% lower, and normalized soleus weight-to-body weight ratio was 46% lower in SI than in NC rats (Table 1). All these differences were highly significant (P < 0.0001).

Deletion analysis of the type I MHC promoter. The validity of the direct gene injection technique to study MHC regulation in intact skeletal muscle has been previously demonstrated in our laboratory (9). Transfected type I and IIb MHC promoter activities, based on reporter assays, correlated well with muscle fiber-type-specific expression of the corresponding endogenous genes.

In the NC soleus muscle, a "full-length" -3500-bp type I MHC promoter demonstrated the greatest activity, confirming the existence of a distal enhancer in the upstream region of the promoter (Fig. 1). In addition to the -3500-bp type I MHC promoter, four shorter promoter fragments were tested for their responsiveness to SI. All the deletion fragments (-408, -299, -215, and -171 bp) were sufficient to activate reporter activity in the soleus muscle of NC rats above background levels; however, activity was further decreased as additional upstream sequences were deleted. Activity of the -408-bp promoter was reduced 75% (P < 0.001), while -171-bp promoter activity was reduced 95% (P < 0.001), relative to the 3500-bp promoter (Fig. 1).


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Fig. 1.   Deletion analysis of type I myosin heavy chain (MHC) promoter in soleus muscle of normal control (NC) and spinal cord-isolated (SI) rats. One week of SI significantly decreased activity of full-length (-3500 bp) and several deletion fragments (-408 to -215 bp) of type I MHC promoter. Activity is expressed as the ratio of type I MHC to skeletal (Sk) alpha -actin promoter activity. Schematic illustration of type I MHC promoter-reporter depicts several DNA regulatory elements. Values are means ± SE. *P < 0.05 vs. NC. There were significant differences in promoter activity in NC soleus muscle among all constructs, except 408 vs. 299 bp and 215 vs. 171 bp (ANOVA). There were no significant differences in activity among promoters in SI soleus muscle. Inset: percent changes in type I promoter activity with SI compared with NC: (SI - NC)/NC. FLuc, firefly luciferase.

Normalized activity of the full-length -3500-bp type I promoter, as well as the -408-, -299-, and -215-bp deletion fragments, was significantly decreased in the soleus muscle of SI rats compared with NC rats (Fig. 1). The -3500-bp promoter exhibited the greatest responsiveness to SI, decreasing ~90% relative to NC (Fig. 1), while the -215-bp promoter showed the least, decreasing ~39%.

In contrast to the -408-, -299-, and -215-bp promoters, -171-bp promoter activity normalized to the alpha -actin reference promoter was significantly increased in response to SI (Fig. 1). This apparent increase in -171-bp promoter activity with SI is the result of normalization to actin and should not be interpreted as promoter activation. In fact, uncorrected absolute -171-bp type I FLuc activity was still significantly decreased in response to SI (Fig. 2; by 80% relative to NC). However, this decrease in activity was smaller than all other constructs (95-98% decrease) and smaller than the decrease in actin promoter activity (~90% across all experiments). The increase in normalized 171-bp promoter activity in SI was the result of a smaller fractional decrease in 171-bp promoter activity [(SI - NC)/NC] than in actin promoter activity. This higher ratio can be interpreted as attenuated responsiveness of the test promoter. This reduced responsiveness to SI of the -171- compared with the -215-bp promoter suggests that some regulatory elements essential for type I MHC downregulation with SI are likely located between 215 and 171 bp of the type I promoter. One putative cis-acting DNA regulatory element in this region is the beta e3 element (-214/-190 bp) (38), which could confer type I MHC responsiveness to SI. Consequently, to determine the role of the beta e3 element in type I MHC downregulation with chronic inactivity, an additional set of experiments was performed that involved mutation of this element in two promoter fragments.


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Fig. 2.   Absolute reporter activity in soleus muscle of NC and SI rats. Top: absolute FLuc activity of type I MHC promoter in NC and SI soleus muscle. Activity of all deletion constructs significantly decreased in response to SI. Inset: change in FLuc activity with SI relative to NC for each type I MHC promoter construct. Bottom: absolute Renilla luciferase (RLuc) activity of alpha -actin promoter in NC and SI soleus when coinjected with the various type I MHC promoter constructs. RLuc activity was significantly decreased with SI in all promoter fragments. No significant differences existed among NC or SI group. Reporter activity is based on measurements from 5 µl of muscle supernatant integrated over 10 s and corrected to noninjected tissue basal activity (dual-luciferase assay, Promega). Reporter activities are expressed in relative light units (RLU). Values are means ± SE. *P < 0.05 vs. NC.

Mutational analysis of the type I promoter. Mutation of the beta e3 element in the -215-bp type I promoter decreased activity in the NC soleus, confirming the role of the beta e3 element as a positive regulatory element in normal expression of the type I MHC gene in slow-twitch skeletal muscle (Fig. 3). Mutation of this element in the -215-bp promoter construct also attenuated type I MHC downregulation in response to SI. Similar to the results observed with the -171-bp promoter, activity of the -215-bp beta e3 mutant promoter relative to actin significantly increased in response to SI. Absolute FLuc activity of the 215-bp beta e3 mutant promoter was still decreased 65% in response to SI (6,742 ± 1,990 to 2,274 ± 660 RLU); however, this was smaller than the 96% decrease found with the 215-bp wild-type promoter (29,360 ± 12,082 to 1,206 ± 550 RLU). Thus decreased responsiveness to SI when the beta e3 element is mutated suggests that this element is involved in regulating transcriptional downregulation of type I MHC (Fig. 3).


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Fig. 3.   Mutational analysis of -215-bp (A) and -408-bp (B) type I MHC promoters in soleus muscle of NC and SI rats. A: mutation of beta e3 element or its deletion (-171 bp) significantly decreased activity of the -215-bp promoter in NC soleus muscle [vs. wild type (WT); dagger P < 0.05] and decreased promoter responsiveness to SI. Values are means ± SE. *P < 0.05 vs. NC; dagger P < 0.05 vs. 215 WT. B: mutation of beta e3 element significantly decreased normalized activity of the -408-bp promoter in NC soleus muscle (vs. WT) and attenuated promoter responsiveness to SI. Simultaneous mutations of beta e3 and beta e2 elements resulted in similar promoter activity in the NC soleus muscle compared with the beta e3 mutation alone and also attenuated responsiveness to SI. Values are means ± SE. *P < 0.05 vs. NC; dagger P < 0.05 vs. 408 WT.

The same beta e3 mutation in the -408-bp promoter fragment also attenuated type I downregulation in response to SI (Fig. 3). These results further confirm the importance of the beta e3 element in type I downregulation with SI, since the additional upstream elements in the -408-bp promoter were unable to rescue type I responsiveness to SI. In an additional promoter fragment, the upstream beta e2 (-285/-269 bp) regulatory element was simultaneously mutated with the beta e3 element. This second mutation had no additional effect on promoter activity in normal soleus muscle compared with the single beta e3 mutation (Fig. 3), inasmuch as both activities were similarly lower than the 408-bp wild-type promoter construct. If the beta e2 cis-regulatory element was involved in the SI response, the responsiveness of the 408-bp double mutant would be expected to be less than that of the single beta e3 mutant. Collectively, these results with the beta e2/beta e3 mutation demonstrate that beta e3, but not beta e2, positively regulates the type I MHC promoter in NC soleus muscle and that the 408- and 215-bp promoters were responsive to SI via the beta e3 element, the proximal positive enhancer (17).

Normalization of reporter activity to plasmid uptake. As discussed in METHODS, an additional control for pGL3 plasmid uptake normalization was performed in selected experiments. In contrast to actin promoter activity, which was significantly decreased in response to SI (Fig. 2), plasmid uptake was slightly greater in the soleus muscle of SI than NC rats (Table 3). Although normalization to actin reduced reported responsiveness of the type I promoter to SI, normalization to plasmid uptake resulted in type I downregulation, which was more closely matched to the decrease in FLuc activity than to the ratio of FLuc to RLuc activity. Specifically, although the normalized type I MHC activity relative to actin decreased 92 and 39% for the -3500- and -215-bp promoters, respectively, and increased 109% for the -171-bp promoter, the activities were decreased 99, 98, and 80% relative to plasmid uptake. These results clearly demonstrate that significant downregulation of the type I MHC promoter in response to SI was not due to reduced plasmid uptake but, rather, to SI-induced transcriptional regulation.

                              
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Table 3.   FLuc activity, pGL3 plasmid uptake levels, and FLuc activity relative to pGL3 uptake for -3500-, -215-, and -171-bp type I MHC promoters in NC and SI rat soleus

Activity of the type IIb MHC promoter. Activity of a -1400-bp type IIb MHC promoter construct was significantly increased in the soleus muscle of SI rats compared with NC rats (Fig. 4). This upregulation is in agreement with the shift in MHC expression in response to SI (16) and demonstrates that the previously reported increases in type IIb mRNA with SI are transcriptionally regulated. Furthermore, upregulation of the type IIb promoter demonstrates that SI does not cause a global decrease in transcription.


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Fig. 4.   Activity of the -1400-bp type IIb MHC promoter in soleus muscle of NC and SI rats. Chronic inactivity significantly increased activity of the fast IIb MHC promoter. Values are means ± SE. *P < 0.05 vs. NC. Promoter activity is expressed as the ratio of FLuc to RLuc.

Gel mobility shift assays. The findings that 1) mutation of the beta e3 element in the -215- and -408-bp promoter fragments and 2) deletion of this element (-171-bp fragment) decreased type I responsiveness to SI strongly suggest that this element is involved in the SI response.

Thus gel mobility shift assays were used to examine interactions of the beta e3 element with nuclear proteins extracted from the soleus muscle of NC and SI rats (Fig. 5). Although DNA-protein interactions were resolved into several complexes on the gel, on the basis of competition with different DNA probes, only one complex was clearly specific to the beta e3 probe (SpC in Fig. 5). This specific band (SpC) was competed out by 100-fold molar excess of unlabeled beta e3 probe. In contrast, an unlabeled beta e3 mutant probe and an unrelated sequence (GATA) were not able to effectively compete out binding to the radiolabeled beta e3 probe when incubated at 100-fold molar excess. By densitometric analysis, it was determined that there was a significant increase in binding with the SI nuclear extracts compared with NC: 235,800 ± 38,060 and 392,500 ± 48,630 densitometric units in NC and SI, respectively (n = 6). Although the difference in binding to the beta e3 element was relatively small (85% increase with SI vs. NC), it was not due to differences in nuclear protein concentration between groups, inasmuch as binding to probes containing other cis-acting DNA elements (GATA or NFAT) was not different between the two experimental groups (data not shown).


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Fig. 5.   Representative gel mobility shift of extracts from NC (C1 and C2) and SI rat soleus muscle binding to the beta e3 element. Several DNA-protein complexes were formed with a gel mobility shift assay when control soleus nuclear extract was reacted with a 24-bp double-stranded probe representing the rat beta e3 element (lane 1). The 2 slowest-migrating bands are due to nonspecific (NS) binding, inasmuch as they were competed off with a 100-fold excess of cold beta e3 probe (lane 2), cold mutant beta e3 (lane 3), or an unrelated sequence (GATA, lane 4). Examination of the gel strongly suggests that the 3rd complex (SpC) was due to specific interactions of the beta e3 element and muscle nuclear proteins. SpC was not competed off with excess cold mutant beta e3 (lane 3) or an unrelated probe (lane 4). SpC was absent when nuclear extract was reacted to a mutant beta e3 probe (lane 5). A significant 85% increase in specific binding to the beta e3 element (SpC) was seen with extracts from SI (lane 8) soleus muscle compared with NC (C2, lane 6). Despite increases in SpC with SI nuclear extract, the amount of DNA-protein complexes supershifted (SS) by the transcription enhancer factor-1 antibody (TEF-1 Ab) was ~50% less in SI (lane 9) than in NC (lane 7) soleus muscle.

Reactions with an antibody specific to TEF-1 caused a clear supershift of the SpC protein-DNA complex into a heavier supershifted complex (SS in Fig. 5). Although SI significantly increased nuclear protein binding to the beta e3 element by 85%, 50% less protein was supershifted by the TEF-1 antibody in SI samples: 453,000 ± 122,000 vs. 233,000 ± 146,000 densitometric units for NC and SI, respectively.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Several models of chronic decreased activity, such as denervation, hindlimb unloading, spinal cord transection, and SI have clearly demonstrated the ability of skeletal muscles to adapt their MHC phenotype to the imposed alterations in activity and load (for review see Refs. 27 and 36). However, although variable amounts of neuromuscular activity are known to impact muscle fibers in the majority of these models, SI is unique, in that it provides a baseline for complete inactivity while preserving potential neural, but non-activity-related, factors. With the use of this model, the findings reported here provide the first evidence that the complete absence of neuromuscular activation and loading significantly decreases in vivo transcription of the type I MHC gene in a slow-twitch muscle. In addition, deletion and mutation analyses strongly suggest that the beta e3 DNA regulatory element is involved in the type I MHC downregulation associated with chronic inactivity. As evidenced by the supershift assays, this downregulation with SI is potentially mediated by decreased binding of TEF-1 to the beta e3 element. Transcriptional regulation of the MHC genes with chronic inactivity was not confined to the slow type I MHC gene, inasmuch as we also demonstrated a significant transcriptional upregulation of the fast type IIb MHC gene. Thus the previously observed time-dependent MHC shifts at the steady-state mRNA and protein levels (i.e., decreases in slow type I MHC and increases in the fast isoforms) (16) are initiated at the level of transcription. Clearly, neuromuscular activation and loading are essential factors in maintaining normal levels of type I MHC transcription in a slow-twitch rat hindlimb muscle.

The significant depression in type I MHC promoter activity within 7 days of SI has a profound effect on muscle MHC protein expression, as evidenced by the rapid loss of muscle mass and protein/MHC content over the first 8 days of SI. We recently reported that the soleus muscle had lost nearly half of its type I MHC protein after only 8 days (16). As demonstrated by the present study, this rapid loss was primarily due to transcriptional regulation of the type I MHC gene, rather than posttranscriptional and/or translational regulation. With longer-duration SI, the rate of muscle atrophy and MHC protein loss was reduced, and the atrophied muscles then underwent additional remodeling of the myosin phenotype.

This is the first study to demonstrate that decreased type I MHC expression with complete skeletal muscle inactivity is transcriptionally regulated and likely involves the beta e3 regulatory element. The enhancer core of the beta e3 element has been characterized in cardiac myocytes and is also referred to as the myocyte-specific CAT (MCAT) cis-acting element (17, 19). Evidence that beta e3 is an enhancer element in the normal weight-bearing soleus is demonstrated here by the significant decrease in -215- and -408-bp type I promoter activity when the beta e3 element is mutated (Fig. 3). Members of the TEF-1 multigene family bind to this beta e3 (MCAT) element and activate transcription of muscle genes in cardiac myocytes such as type I MHC (17, 18, 34, 40), alpha -MHC (12), and skeletal alpha -actin (19). Recently, a TEF-1-related protein, RTEF-1, was identified and found to be a target of alpha 1-adrenergic signaling in hypertrophied cardiac myocytes (40). Specifically, phosphorylation of a serine residue accounted for a majority of the alpha 1-adrenergic response. Overexpression of TEF-1 activated a -215-bp type I MHC promoter, while overexpression of RTEF-1 activated the -215- and -3300-bp type I promoters (34). An intact MCAT element was necessary for this transactivation of the type I promoter by TEF-1 and RTEF-1.

TEF-1 is also involved in regulating skeletal muscle genes, inasmuch as 3 days of stretch overload in the chicken anterior latissimus dorsi muscle increased TEF-1 binding to the alpha -actin promoter (2). Our supershift assays clearly demonstrate that TEF-1 is also involved in type I MHC regulation in the rat soleus muscle and is responsive to changes in neuromuscular activity (Fig. 5). A TEF-1 antibody supershifted protein complexes binding to the beta e3 oligonucleotide in nuclear extracts from NC and SI soleus muscle. Although total nuclear protein binding to the beta e3 oligonucleotide increased 85% with SI, the protein supershifted by the TEF-1 antibody was only 50% of that observed in NC soleus muscle. Thus chronic inactivity with SI decreased TEF-1 binding, in contrast to muscle overload, which was previously shown to increase TEF-1 binding (2). Consequently, one mechanism whereby SI could downregulate type I MHC is a decrease in TEF-1 binding to the beta e3 element, as evidenced by a significant reduction in downregulation when this element was mutated or deleted. Taken together with previous work, TEF-1 may upregulate muscle-specific genes in response to muscle overload (2) while downregulating these genes in response to chronic inactivity.

Because SI increased nuclear protein binding to the beta e3 oligonucleotide while decreasing the amount of TEF-1 binding, this suggests that the residual binding was composed of other transcription factors that increase in response to SI. One possibility is that SI may increase other TEF-1-related factors (e.g., RTEF-1, DTEF-1, and ETF). However, this is not likely, since this family of transcription factors enhances transcription, while chronic inactivity downregulates type I MHC expression. Another potential transcription factor that may contribute to the increase in nuclear protein binding to the beta e3 oligonucleotide is cAMP-responsive binding protein (CREB). The beta e3 oligonucleotide probe used in the gel mobilty shift assays also contains a putative CREB site (-213/-216 bp; Table 2) adjacent to the TEF binding site (MCAT). Unphosphorylated CREB has been shown to repress activity of the c-jun promoter (20); consequently, deletion of this CREB site could remove another potential regulatory site for decreasing type I MHC expression.

In our previous SI time-course study (16), we reported de novo expression of type IIb MHC mRNA after only 4 days of SI. These findings are supported by the present observation that 1 wk of SI significantly upregulates type IIb MHC promoter activity (Fig. 4). Interestingly, although short-term inactivity and/or unloading is sufficient to activate transcription of the IIb MHC gene, it is unable to induce expression of type IIb protein. For example, in hindlimb-unloaded rats, type IIb MHC was expressed at the mRNA, but not at the protein, level (14). Similarly, 60 days of SI (16), 30 days of denervation (13, 15), or 14 days of tetrodotoxin paralysis (24) did not induce de novo type IIb protein expression, despite increases in its mRNA. Taken together, these observations suggest a marked uncoupling between transcriptional and translational events in the upregulation of type IIb MHC in slow-twitch muscles during the initial stages of unloading. However, more prolonged inactivity (e.g., 90 days of SI) or decreased activity coupled with other factors such as thyroid hormone status have induced type IIb protein expression in the soleus muscle. For example, the combination of hindlimb unloading and triiodothyronine treatment resulted in increases in type IIb mRNA and protein in the rat soleus muscle (4, 14), which was transcriptionally regulated (4).

Collectively, we have used the direct gene transfer approach to characterize the regulation of type I MHC gene expression in response to chronic inactivity. In summary, our results demonstrate that chronic inactivity and unloading of slow-twitch rat hindlimb muscles caused a significant downregulation of type I MHC promoter activity. Deletion and mutation analyses strongly suggest involvement of the beta e3 DNA regulatory element in the type I MHC downregulation associated with SI. Furthermore, decreased TEF-1 binding to the beta e3 element likely contributed to this type I MHC downregulation. Future studies should investigate mechanisms whereby prolonged absence of activation and/or loading alter the amount and binding of TEF-1 to the beta e3 element and how these interactions contribute to type I MHC downregulation with different forms of reduced activity.


    ACKNOWLEDGEMENTS

The authors thank H. Zhong, J. Kim, and M. Herrera for animal care and surgery and A. Qin for plasmid preparation.


    FOOTNOTES

This work was supported by National Institutes of Health Grants NS-16333 (to V. R. Edgerton) and AR-30346 (to K. M. Baldwin).

Address for reprint requests and other correspondence: K. M. Baldwin, Dept. of Physiology and Biophysics, University of California, Irvine, 346-D Med Sci I, Irvine, CA 92697-4560 (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.00355.2001

Received 27 July 2001; accepted in final form 31 October 2001.


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