INTRODUCTION

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TROPONIN I (TnI) is the inhibitory subunit of the troponin complex that regulates calcium-mediated contraction of striated muscle (18). TnI exists in three isoforms, each encoded by a separate gene. Cardiac TnI is restricted to the adult heart while slow TnI (TnIs) and fast TnI (TnIf) are expressed in slow and fast skeletal muscle fibers, respectively (9). TnIs is activated transcriptionally during the differentiation program in newly formed myofibers (13). However, on innervation, the properties of the motoneuron determine whether the expression of TnIs will be augmented (slow nerve) or repressed (fast nerve) (12).

Adult skeletal muscle is highly plastic, allowing the phenotype of muscle fibers to adapt to various conditions and functional demands. A primary mechanism for this adaptive response involves the differential expression of various isoforms of many muscle-specific genes, including TnI. Contractile activity and the loading history of skeletal muscle are important determinants of its contractile protein isoform profile. Numerous investigators have reported that decreased loading of a skeletal muscle results in a downregulation of the slow isoforms and a concomitant increase in the fast isoforms (3, 6, 7, 11a). This alteration in adult skeletal muscle phenotype has a profound impact on the functional characteristics of the muscle. Although these changes are clearly adaptive to the decreased usage pattern, muscle function is compromised on return to the normal loading condition (3). Esser and Hardeman (6) have shown that the fast and slow contractile protein isoform genes are regulated independently of one another in response to skeletal muscle unloading during spaceflight. This is also true during myogenesis (15); however, it is not clear whether the same cis-acting DNA elements are responsible for the regulation of the TnI isoforms during both development and adaptation of adult muscle to unloading.

Experiments with transgenic mice have indicated that the 5'-promoter region [4,200 to +12 base pairs (bp)] of the TnIs gene is sufficient to direct slow fiber type-specific expression of a reporter gene in adult muscle and to respond to slow nerve innervation as does the endogenous TnIs gene (10). Furthermore, this transgene was found to be expressed properly in the developing skeletal muscle of embryonic, neonatal, and postnatal mice. However, some aberrant expression was noted in the fetal heart and primordial axial skeleton (17). Corin et al. (5) have described a 157-bp enhancer region in the TnIs promoter (from 1,031 to 875) that is sufficient to direct slow fiber-type expression of a reporter gene. Clearly the 4,200 to +12 bp sequence of the TnIs gene is sufficient to direct normal expression during development and responds normally to neural innervation, resulting in fiber type-specific expression in adult muscle. Using the above-mentioned 4,200 TnIs-chloramphenicol acetyltransferase (CAT) transgenic mouse model, we sought to examine whether the TnIs promoter region was sufficient to respond to the fiber type-transition stimulus accompanying skeletal muscle unloading.

	METHODS

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Animals. We used 18 female transgenic mice (6 mo old) carrying 4,200 to +12 bp of the human TnIs gene linked to the bacterial CAT-coding region (10) to assess the responsiveness of the TnIs promoter region to mechanical unloading of the mouse soleus muscle. For this experiment, we used a single transgenic line (no. 712/12) that has previously been tested for responsiveness to innervation during regeneration (10). The transgenic mice were randomly divided into two groups: weight-bearing (WB) controls (n = 9) and hindlimb-unloaded (HU) mice (n = 9) that were tail suspended for 7 days. For analysis of endogenous TnI expression in the mouse soleus, 64 nontransgenic mice were also randomly divided into WB controls (n = 32) and HU groups (n = 32).

HU. Mechanical unloading of the hindlimbs was performed by using a modification of the protocol used by Babij and Booth (1) for tail suspension of rats. Each mouse was weighed and anesthetized with an intraperitoneal injection of a cocktail containing ketamine (73.9 mg/ml), xylazine (3.7 mg/ml), and acepromazine (0.7 mg/ml) at a dose of 1.8 ml/kg body mass. Two strips of Elastoplast elastic adhesive bandage (Beiersdorf, Norwalk, CT), ~15 × 0.5 cm each, were cut. The bandages were wrapped around the tail in a helical pattern starting at the base of the tail and extending 2 cm past the tip of the tail. After the mouse had recovered from the anesthetic, a swivel hook was placed through the bandage just distal to the tip of the tail. The hook was then raised so that the hindlimbs were elevated just off the cage floor, producing an ~30° head-down tilt. Forelimbs remained in contact with the cage floor, allowing the mouse to move freely through a 360° circle around the tail-suspension apparatus. Mice had ad libitum access to chow and water throughout the HU protocol.

Tissue sampling. At 7 days of HU, mice were lowered and then immediately anesthetized as described previously. Post-HU body mass was measured, and the soleus muscles were removed, weighed on an analytic balance (Mettler), and quick frozen in liquid nitrogen. After this procedure was completed, the anesthetized mice were killed by cervical dislocation.

RNA isolation. Right and left soleus muscles from eight nontransgenic mice were combined for RNA isolation to produce four observations per treatment group. Total RNA was extracted from ~100 mg of muscle by using the guanidine thiocyanate method of Chomczynski and Sacchi (4) with Trisolve (Biotecx Laboratories, Houston, TX). The extracted RNA was dissolved in diethylpyrocarbonate-treated water, and the RNA concentration was determined spectrophotometrically at 260-nm wavelength. Total RNA was also isolated from the combined left soleus muscles from control and HU transgenic mice for analysis of CAT transcript abundance.

Northern blot analyses. Northern blot analysis was used to assess the relative abundance of TnIs, TnIf, CAT mRNAs, and ribosomal 18S RNA in the soleus muscles of WB and HU mice. Extracted total RNA for each sample (20 µg for TnIs and TnIf; 10 µg for CAT) was loaded on a denaturing 1% agarose-formaldehyde gel [1 × 3-(N-morpholino)propanesulfonic acid and 6.7% formaldehyde] and electrophoresed at 4 V/cm for 3 h. The RNA was then transferred to a nylon membrane (Hybond-N+; Amersham, Arlington Heights, IL) by capillary action and was ultraviolet cross-linked to the membrane.

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DNA probes. TnI probes have been described in Sutherland et al. (12). For TnIf, a 200-bp Bgl I/Rsa I restriction fragment was used that contains amino acids 11-78 of the protein-coding region of the human cDNA. For TnIs, a 250-bp Pst I restriction fragment of the human cDNA was used that consists of the 5'-untranslated region (UTR) and a short region of proximal protein-coding sequence. The CAT probe is a 1,499-bp Hind III-Hpa I fragment containing the CAT-coding region (2).

CAT enzymatic assay. Individual right soleus muscles from the transgenic mice were homogenized in 1 ml of homogenate buffer (in mM: 25 tris(hydroxymethyl)aminomethane (Tris), 10% glycerol, 2 dithiothreitol, 1 phenylmethylsulfonyl fluoride, 1 EDTA, 1 benzamide, 0.01 mg/ml leupeptin, and 0.01 mg/ml pepstatin) by using a motor-driven glass-on-glass tissue grinder. The homogenate was then centrifuged (10,000 g for 10 min at 4°C), and the protein concentration of the supernatant was determined (Bio-Rad DC protein assay). CAT activity was assayed in 2 µg protein by using a reaction mixture containing 192 mM Tris (pH 7.8), 1.8 mM acetyl CoA, and 0.2 µCi [¹⁴C]chloramphenicol. Samples were incubated at 37°C in a total reaction volume of 140 µl. One-half of the reaction mixture (70 µl) was removed at 20 min and one half at 40 min; each half was placed on ice, and 250 µl of cold ethyl acetate were added to stop the reaction. The acetylated chloramphenicol was separated by using thin-layer chromatography (30 min, 20°C) in a tank containing 95% chloroform-5% methanol, and it was then visualized by autoradiography. The portions of the plates containing the acetylated and unacetylated chloramphenicol for each sample were excised and quantified by scintillation counting. The percent conversion per minute was determined and was used to calculate the activity in nanomoles chloramphenicol catalyzed per minute per milligram protein.

Statistics. Analysis of variance with repeated measures (pre- vs. posttreatment) was used to analyze changes in body mass for WB and HU groups. Student's t-tests were used to test for differences between group means for muscle mass, mRNA abundance, and CAT activity. Statistical significance was established at P < 0.05. All values are reported as means ± SE.

	RESULTS

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Body mass and muscle mass. Pre-HU body mass did not differ between treatment groups. Mean body mass declined 6.11% during the unloading period for the HU group, whereas there was no significant change in body mass of the WB control group (Fig. 1). Absolute soleus muscle mass was significantly reduced by 25% in the HU group compared with the controls (5.54 ± 0.17 vs. 7.40 ± 0.13 mg, respectively). When normalized to body mass, the soleus mass remained significantly lower (16%) in the HU group than in the control group (Fig. 2).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Change in body mass for control and hindlimb-unloaded (HU) mice over the 7-day treatment period. Values are means ± SE; n = 32 mice per group. Pre, pretreatment; Post, posttreatment. * Significantly different from pretreatment mean; P < 0.05.

View larger version (12K): [in this window] [in a new window]   Fig. 2.   Mass of soleus muscle normalized to posttreatment body mass for control and HU mice. Values are means ± SE; n = 32 mice per group. * Significantly different from control group; P < 0.05.

Endogenous TnI mRNA abundance. HU resulted in a decrease in the abundance of TnIs mRNA and an increase in the abundance of TnIf mRNA. TnIs mRNA concentration, expressed as arbitrary units per microgram of total RNA and normalized to ribosomal 18S abundance, was 54% lower in the HU group compared with the controls (74,841 ± 2,782 vs. 163,314 ± 13,418, respectively). TnIf mRNA concentration per microgram total RNA was 64% higher in the HU group compared with the controls (121,572 ± 4,642 vs. 72,619 ± 2,917, respectively).

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Concentration (per mg muscle mass) of slow troponin I (TnIs) and fast troponin I (TnIf) mRNA in soleus muscles of control and HU mice. Arbitrary units obtained from phosphorimager scans and corrected for 18S rRNA amount were multiplied by total RNA concentration (µg/mg muscle) for each sample. Values are means ± SE; n = 4 mice per group. * Significantly different from TnIs concentration in control group; P < 0.05.

TnIs transgene activity. CAT activity driven by the 4,200 TnIs promoter in the soleus muscles of the transgenic mice tended to be lower in the HU group (15%), but this difference did not reach statistical significance (Fig. 4).

View larger version (10K): [in this window] [in a new window]   Fig. 4.   Chloramphenicol acetyltransferase (CAT) activity produced by the TnIs-CAT transgene in soleus muscles of control and HU transgenic mice. Values are means ± SE; n = 9 mice per group. Group means were not statistically significant; P > 0.05.

CAT transcript abundance. The values for transgenic CAT mRNA abundance in the soleus, corrected for 18S abundance and obtained from four separate blots, are as follows: control = 3,167 arbitrary units; HU = 4,525 arbitrary units. Error values could not be determined because the samples from each group were pooled before total RNA isolation.

	DISCUSSION

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Differential regulation of genes encoding various isoforms of muscle-specific proteins represents an important mechanism of skeletal muscle adaptability. It is well established that the unloading of postural skeletal muscles, such as occurs during spaceflight or prolonged bed rest, causes a downregulation of slow isoforms and an upregulation of fast isoforms (6). The concomitant changes in skeletal muscle function leave muscles weaker and more apt to fatigue on return to normal WB (3). TnI is a component of the troponin complex that regulates calcium-mediated muscle contractions. Therefore, the regulation of the isoforms of TnI in adult skeletal muscle is important to our understanding of muscle plasticity. The present study is the first to indicate that the cis-acting DNA elements sufficient to direct sensitivity of the TnIs gene to mechanical unloading differ from elements required to direct the fiber type-specific expression and slow nerve responsiveness of this gene.

Our data gathered from mice after 7 days of tail suspension agree with the report of Esser and Hardeman (6), who found that TnIs mRNA is downregulated and TnIf mRNA is increased in the rat soleus after 9 days of unloading during spaceflight. Data from the transgenic mouse line used in this study (10) and from plasmid-injection experiments (5) indicate that the 5'- flanking region of the human TnIs gene contains the elements necessary to direct slow skeletal muscle fiber-specific expression of reporter genes in adult rat and mouse muscle. However, we did not find convincing evidence that the 4,200 TnIs promoter sequence was sufficient to mediate the unloading-induced downregulation of this gene.

Our finding that the 4,200 TnIs promoter is unable to respond to unloading of the soleus in a manner similar to the endogenous mRNA was surprising, given that this promoter region directs fiber type-specific expression. Yutzey et al. (16) found that an internal enhancer region located in the first intron of the quail TnI gene was required for normal expression of this gene during myogenesis. Furthermore, an element in the second intron of the human TnIs gene was found to be homologous to this region (8, 11). Corin et al. (5) examined this intronic enhancer in adult rat muscle in vivo and found that it was not sufficient to direct reporter gene expression in a manner similar to the endogenous TnIs mRNA. However, no isoform transition stimulus has been studied. It is conceivable that this intronic element, although not necessary for normal nerve responsiveness of the TnIs gene, may be required for isoform transition during unloading. This possibility warrants further research.

Our data support the notion that, although unloading of skeletal muscle causes a fast-to-slow shift in muscle protein isoform expression, the changes in fast and slow isoforms are regulated independently and are not quantitatively similar (6). We report that 7 days of HU causes a significant decrease (54%) in TnIs mRNA in the soleus and a significant increase (67%) in TnIf mRNA when these values are expressed per microgram of total RNA. However, if mRNA concentration is calculated per milligram of muscle, the increase in TnIf mRNA is reduced to a nonsignificant 34%. Furthermore, if the atrophy of soleus mass with HU is taken into account by calculating total mRNA per sample (an indication of the total number of transcripts present per muscle), the increase in TnIf mRNA is eliminated, and the decrease in TnIs mRNA reaches 73%. These data suggest that the mechanism of fast-to-slow isoform transition in response to skeletal muscle unloading involves a large downregulation of the TnIs gene, while expression of the fast gene is simply preserved at control values. This type of regulation may also occur during the response of the myosin isoforms to hindlimb suspension. Thomason et al. (14) reported an 84% reduction in total slow myosin protein content per rat soleus muscle after 56 days of suspension. This percent reduction represented a loss of 11.3 mg of slow myosin protein per muscle pair. Meanwhile, the fast myosin protein content was increased by 33%, representing a gain of only 0.2 mg per muscle pair. A possible explanation for these data would be that the slow isoform genes, such as TnIs, are very sensitive to the loading status of the muscle. In contrast, the fast isoform genes, such as TnIf, may not only be independent of loading but may also able to maintain near-normal expression during muscle atrophy.

In conclusion, we provide evidence that the elements located in the promoter region of the human TnIs gene, which have previously been reported to direct normal skeletal muscle expression of this gene during development (17) and adulthood (5, 10), are not sufficient to respond to an unloading-induced isoform-transition stimulus in the mouse soleus. Feasible explanations for this finding include the possibility that a previously described intronic enhancer element in the human TnIs gene could be involved in the response of this gene to unloading. Another possibility is that mRNA stability, which is often regulated by the 3'-UTR of a gene, may be involved in this response. Neither the intronic enhancer sequence nor the 3'-UTR of the TnIs gene was included in the transgene construct used in this study. Therefore, future studies will be required to examine this question further. It should be noted that the processes involved in regulation of the human TnIs promoter (used in this transgene) may differ from those of the endogenous mouse gene. Nevertheless, our conclusions are based on comparison with previous work (10) that demonstrated nerve responsiveness of this human promoter sequence in the mouse.

Finally, the expression of TnIs mRNA in the mouse soleus is sensitive to loading of the muscle, whereas the quantity of TnIf mRNA in the unloaded soleus remains essentially unchanged. This regulatory strategy results in the observed decrease in TnIs mRNA and increase in TnIf mRNA concentration in the atrophied soleus.