INTRODUCTION

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THE COLLAGEN NETWORK in skeletal muscle gives mechanical strength, distributes the forces of concentric and eccentric muscle contractions, and serves as a supportive structure during normal muscle growth and regeneration after trauma (28). Skeletal muscle tissue contains four main collagen forms, i.e., I, III, IV, and V (28). In addition, there is evidence of gene expression for some other collagens in tiny amounts (32, 37). Type I collagen is the major fibrillar collagen, which has high tensile strength and limited elasticity and is, therefore, suitable for force transmission. Type III collagen is the other main fibrillar collagen. Its structure and arrangement are similar to that of type I collagen, but it forms thinner and more elastic fibers. The fibers of type III collagen can also form copolymers with type I collagen fibers (11, 16).

Collagen biosynthesis is characterized by the presence of a large number of co- and posttranslational modifications in the polypeptide chains that affect the quality and stability of the collagen molecule. Prolyl 4-hydroxylase (PH) is the key modifying enzyme catalyzing the 4-hydroxylation of prolyl residues. The active enzyme is a tetramer composed of two pairs of nonidentical subunits (₂₂). The rates of synthesis of the - and -subunits are regulated differently (21). The -subunit appears to become incorporated into the tetramer directly after its synthesis and contains the major portion of the catalytic site (18, 21). Its concentration limits the rate of active PH formation. In contrast, the -subunit is produced in excess of the -subunit (3, 15, 27) and enters a pool of free subunits, after which a portion of the -subunit is incorporated into the PH tetramer (21). The -subunit has been found to be a multifunctional polypeptide; e.g., it is identical to both the enzyme protein disulfide-isomerase (22, 30) and the cellular thyroid-binding hormone protein (5).

The level of PH activity generally changes along with the rates of collagen biosynthesis, and assay of PH activity has been used to estimate the rate of collagen synthesis in different experimental and physiological conditions (14, 19, 20, 35). Previous studies by our group demonstrate that cast immobilization of rat hindlimb leads to a decrease in PH activity in skeletal muscle after only 3 days, with the effects lasting at least 6 wk (14, 34, 35). The decrease in enzyme activity is in accordance with our observation that the content of soluble collagen, reflecting mainly new synthesized collagen, decreases after immobilization (14). The changes in total insoluble hydroxyproline content of the muscle are usually small during immobilization lasting for a few weeks, probably reflecting the slow turnover of collagen (14, 34, 35), although increased collagen concentrations have also been observed after immobilization (12).

The hypothesis of this study was that, during immobilization of hindlimbs, the early decrease in expression of the active PH ₂₂- tetramer is caused by a downregulation of mRNA levels of the PH subunit. A further purpose was to investigate the relationship between PH gene expression and type I and III (pro)collagen gene expression at mRNA and protein levels. The results show that mRNA level for the PH -subunit was decreased after only 1 day of immobilization. This was followed by subsequent decreases in PH activity and mRNA levels for type I and III procollagen 2 days later, whereas no changes in insoluble total collagen content or the proportion of type I and III collagen were observed during the 1-wk experimental period.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Adult male Sprague-Dawley rats, weighing 298 ± 1 (SE) g, were used. The rats were housed in individual cages (10:14-h light-dark cycle) and maintained on a diet of standard rodent chow (Astra-Ewos) and water ad libitum. Experimental manipulations were performed under neuroleptic anesthesia (150 µl Hypnorm). Treatment of the animals was in accordance with the European Convention for the Protection of the Vertebrate Animals Used for Experimental and Other Scientific Purposes and was controlled by The Committee of Laboratory Animal Experiments, University of Oulu, Finland.

Immobilization experiments. The rats were randomized into three control and three immobilized groups, 5-8 animals in each group. The right hindlimb was immobilized with plaster of Paris so that the ankle joint was in full plantar flexion (150-160°). Immobilization periods of 1, 3, and 7 days were used.

Tissue preparation. The rats were anesthetized and killed by decapitation. The soleus, tibialis anterior, plantaris, and gastrocnemius muscles from the right leg of both control and experimental rats were excised and frozen in liquid nitrogen. The samples were stored at 70°C for further analysis. For RNA isolation, a portion of individual frozen muscle was pulverized to a fine powder in mortars containing liquid nitrogen. For PH activity and protein measurements, the samples were homogenized with an Ultra-Turrax homogenizer in two 7-s bursts in a cold solution containing 0.2 M NaCl, 0.01% (wt/vol) Triton X-100, 0.01% (wt/vol) soybean trypsin inhibitor, 0.1 M glycine, 50 µM 1,4-dithiothreitol, and 0.02 M Tris · HCl buffer, pH adjusted to 7.4 at 4°C. The homogenates (6-10%, wt/vol) were centrifuged at 12,000 g for 20 min at 4°C, and the supernatants were taken for the assays of the enzyme activity and protein concentration. Pellets were used for hydroxyproline and collagen proportion assays.

RNA isolation and Northern and slot-blot analysis. The weighed muscle powders were rapidly vortexed in sterile polystyrene tubes containing 1.2 ml of denaturing solution consisting of 4 M guanidinium thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5% sarcosyl, and 0.1 M 2-mercaptoethanol. The other steps were performed essentially as described by Chomczynski and Sacchi (6). Total RNA was quantified by absorbance at 260 nm, assuming 40 µg/ml for each unit of absorbance. For the Northern blot assay, 20 µg of total RNA were denatured in loading buffer, electrophoresed in a 1% agarose-formaldehyde gel, and transferred to a nitrocellulose filter (Schleicher & Schuell) following the standard procedures (7). For slot-blot assay, 15 µg total RNA were incubated in a buffer containing formaldehyde for 15 min at 68°C and spotted on the nitrocellulose filter at three different amounts by using a vacuum filtration manifold (Minifold II; Schleicher & Schuell). All the filters were air-dried and heated at 78°C for 2 h to bind the RNA to the filter. Prehybridization of the filters was carried out in a solution containing 35% formamide (vol/vol), 6× sodium chloride-sodium citrate (1× sodium chloride-sodium citrate = 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0), 1× Denhardt's solution, 250 µg/ml ssDNA, and 0.1% SDS (wt/vol) for 4-6 h at 37°C. The RNA-cDNA hybridization was performed for 24 h at 41°C by using the buffer containing the same components as the prehybridization buffer and ³²P-labeled cDNA probe. After hybridization, the filters were washed and exposed to Kodak X-Omat or Cronex R 31 film at 70°C. For comparison of relative amount of mRNA, signal intensity of the bands was scanned by densitometry (Millipore), and the value of integrated optical density was used. The signal obtained by hybridization with a 24-mer oligonucleotide (5' ACG-GTA-TCT-GAT-CGT-CTT-CGA-ACC 3') for 18S ribosomal RNA was used to normalize RNA loading and/or transfer amount. The cDNA probes used were ₁₂, a 2.4-kb-long human cDNA for the ₁(I)-chain procollagen mRNA (26), and E6, a 2.4-kb-long human cDNA for the ₁(III)-chain procollagen mRNA (26). The probes for the mRNAs of - and -subunits of PH, which were DNA fragments corresponding to nucleotides 1,111-1,785 of the PA-49 cDNA clone (10) and 459-1,257 of the S-138 cDNA clone (30), respectively, were amplified by polymerase chain reaction.

PH and protein assays. The assay for PH activity was based on the measurement of the labeled hydroxyproline formed from peptide-bound prolyl residues of unhydroxylated labeled procollagen substrate (20). Supernatant protein was measured by a commercial kit (Bio-Rad).

Hydroxyproline content and collagen proportion assays. Hydroxyproline content was measured by the method of Blumenkrantz and Asboe-Hansen (4) after overnight hydrolysis in 6 M HCl at 110°C. The proportions of type I and III collagens were analyzed after cyanogen bromide digestion essentially as described by Kovanen (23). In brief, digested collagen fragments were separated on SDS-PAGE and stained with Coomassie brilliant blue. Commercial type I and III collagens were used as standards for the measurements of specific fragments of type I and III collagens. The proportion of type I collagen is expressed as percentage of type I + III collagen.

Statistical analysis. Statistical evaluation of the results was performed by using two-way analysis of variance followed by modified Student's t-test, where the confidence limits were determined by the Bonferroni method. Results are expressed as means ± SE.

	RESULTS

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Body and muscle mass and total RNA level. The body mass of the immobilized animals was 5 (P < 0.01), 8 (P < 0.001), and 4% (P < 0.01) lower than the corresponding controls after 1, 3, and 7 days, respectively (Table 1). After 1 day of immobilization, there were no significant differences in muscle mass between the experimental and control groups. After 3 days of immobilization, mass of the soleus, tibialis anterior, plantaris, and gastrocnemius muscles was 23 (P < 0.01), 15 (P < 0.01), 21 (P < 0.001), and 22% (P < 0.001) below the control values, respectively. After 7 days of immobilization the corresponding decreases in muscle mass were 43 (P < 0.001), 18 (P < 0.001), 36 (P < 0.001), and 32% (P < 0.001). Total RNA amounts, calculated by multiplying the yield of RNA per milligram of muscle sample by muscle mass (mg), showed a similar reduction in RNA content as in muscle mass in the three posterior compartment muscles (Table 2). After 3 days of immobilization, total RNA levels were reduced by 21 (not significant), 23 (P < 0.001), and 24% (P < 0.01) of the control values in soleus, plantaris, and gastrocnemius muscles, respectively. At 7 days the corresponding decreases were 54 (P < 0.001), 46 (P < 0.001), and 31% (P < 0.01). In the tibialis anterior muscle there were no significant changes in total RNA content during the experiment.

Steady-state mRNA levels for - and -subunits of PH and PH activity. The pattern obtained by Northern blot hybridization showed specific signals from each probe (Fig. 1) (10, 26, 30). The steady-state mRNA levels for the - and -subunits of PH were analyzed in the gastrocnemius and plantaris muscles by Northern hybridization. Figure 2 shows a representative analysis using the total RNA from plantaris muscle. The mRNA for the -subunit was 49 (P < 0.01) and 41% (P < 0.05) of control values in the gastrocnemius and plantaris muscles, respectively, after immobilization for 1 day (Fig. 3A). The corresponding decrease 2 days later was 55 (P < 0.01) and 39% (P < 0.05). The PH -subunit mRNA concentration was significantly (P < 0.05) below the control values after immobilization for 7 days only in the plantaris. The mRNA for the -subunit of PH increased in the gastrocnemius and plantaris after immobilization for 1 and/or 3 days (Fig. 3B). PH activity in the gastrocnemius and plantaris muscles, respectively, decreased by 34 (P < 0.05) and 47% (P < 0.01) after immobilization for 3 days (Fig. 3C). After 7 days, the corresponding decreases were 35 (P < 0.05) and 39% (P < 0.01).

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Autoradiograms of Northern blots hybridized with different probes. Total RNA from rat skeletal muscle was electrophoresed, transferred to nitrocellulose, and hybridized with specific labeled cDNA probes for mRNAs for (left to right) 1(I) and 1(III) procollagens, mRNAs for - and -subunits of prolyl 4-hydroxylase (PH), and 18S rRNA. Positions of 28S and 18S are indicated by arrowheads.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   Northern hybridization analysis of total RNA from plantaris muscle for - and -subunits of PH and 18S rRNA. Twenty micrograms of total RNA in each lane were electrophoresed, transferred to nitrocellulose, and hybridized with the 32P-labeled specific probes. 1C and 1I, 1-day control and immobilization; 3C and 3I, 3-day control and immobilization; 7C and 7I, 7-day control and immobilization, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   mRNA levels for - and -subunits of PH and specific activity of PH in immobilized gastrocnemius and plantaris muscles. Values are means ± SE; n = 5 control and 7-8 experimental animals. Open bars, control; filled bars, immobilized; GM, gastrocnemius; PL, plantaris. * P < 0.05, ** P < 0.01 vs. corresponding control.

Steady-state mRNA levels for type I and III collagen. Figure 4 shows a representative slot blot using the total RNA from plantaris muscle and cDNA probes for type I and III collagen. The mRNA level for type I collagen was unchanged after immobilization for 1 day (Fig. 5). After 3 days of immobilization, the mRNA concentrations for type I collagen were 56 (P < 0.001), 39 (P < 0.05), 32 (P < 0.01), and 23% (not significant) below the control value in the soleus, tibialis anterior, plantaris, and gastrocnemius muscles, respectively. Type I collagen mRNA concentration had returned to the control level by 7 days. The mRNA levels for type III collagen in the soleus, tibialis anterior, plantaris, and gastrocnemius muscles decreased by 26 (P < 0.05), 44 (P < 0.01), 47 (P < 0.001), and 10% (not significant), respectively, after 3 days (Fig. 6). After 7 days the relative mRNA values were restored to the control levels in three muscles but remained decreased in the soleus muscle.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Typical slot-blot hybridization analysis of mRNAs for type I and III collagen in immobilized rat plantaris muscle. Total RNA (8, 4, 2 µg) was slotted on to a nitrocellulose filter in each slot and hybridized with 32P-labeled cDNA probes for pro 1(I), pro 1(III), and 24-mer oligonucleotide for 18S rRNA.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   mRNA levels for type I collagen in immobilized rat skeletal muscles. Values are means ± SE; n = 5 control and 7-8 experimental animals. Bars are defined as Fig. 3. SO, soleus; TA, tibialis anterior. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. corresponding control.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   mRNA levels for type III collagen in immobilized rat muscles. Values are means ± SE; n = 5 control and 7-8 experimental animals. Bars and symbols are defined as in Fig. 5.

Hydroxyproline concentration and proportions of type I and III collagen. Figure 7 shows hydroxyproline concentration and the percentage of type I collagen [I/(I + III)] in the gastrocnemius and plantaris muscles. No changes were observed in hydoxyproline concentration and the collagen proportions throughout the experiment.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Hydroxyproline concentration and percentage of type I collagen [I/(I + III)] ratio in immobilized gastrocnemius and plantaris muscles. Values are means ± SE; n = 5 control and 7-8 experimental animals. Bars are as defined in Fig. 3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

There is evidence that total RNA decreases in atrophying muscle (17) and increases with hypertrophy (8). In the present cast-immobilization study, the reduction in the total RNA seemed to parallel the decrease in muscle mass in soleus, plantaris and gastrocnemius muscles. Because ~85% of the total RNA is of the ribosomal type, the RNA results suggest that the number of ribosomes, and thus maximum translational capacity, decreases in the three atrophying muscles, roughly paralleling the net degradation of the total protein pool. In contrast, total RNA-to-muscle mass ratio increased in the lengthened tibialis anterior. This is in accordance with earlier findings (8).

The study of PH expression on mRNA and enzyme activity levels is of special interest because PH activity reflects the total collagen synthesis rate. In the present study, a decrease of ~45% in PH -subunit mRNA concentration occurred after only 1 day of immobilization. In earlier studies, decreases in the synthesis rates of some specific proteins, such as cytochrome c, -actin, and -myosin, have been observed before the decreases in their mRNA concentrations in immobilized or non-weight-bearing muscles (1, 29, 36, 38), indicating an early pretranslational regulation (24). We observed that immobilization decreases the concentration of mRNA for the -subunit followed by the decreases in PH activity and the mRNAs for fibrillar collagens. The mRNA level for the -subunit was unaltered or increased during the 1-wk experimental period. The concentration of the PH -subunit is significantly lower than that of the -subunit, and the concentration of the -subunit limits the formation of active ₂₂ tetramer (21). These findings provide novel evidence that, during muscle immobilization, the early decrease in the amount of active PH ₂₂ tetramer is at least partially caused by the downregulation of the mRNA for the PH -subunit. In contrast, the increase in mRNA concentration for the -subunit in the present study may be connected to other functions of the -subunit (see the beginning of this study) rather than to PH formation. It is interesting that, in some in vitro studies where a great upregulation of PH tetramer has been observed, upregulation of the -subunit also appears to be associated with tetramer formation. For example, in the model used by Helaakoski et al. (9), a 50-fold simultaneous increase in mRNA for - and -subunits was observed when F9 cells were treated with retinoic acid in the presence of cAMP. Also, some other in vitro studies show simultaneous upregulation of both subunits of PH by pharmacological means (e.g., Ref. 39).

The data gathered hitherto indicate that the regulation of fibrillar collagen synthesis in cultured cells or in developing organisms occurs primarily by the regulation of the mRNA levels rather than by control of mRNA translation (2). The similar timing and roughly similar degree in the downregulation of PH tetramer (measured as its activity) and in the two most-abundant collagen types, I and III, suggest a tight coordination in the regulation of procollagen chain production and hydroxylation capacity of their proline residues. The reaction products, 4-hydroxyproline residues, allow the formation and stabilization of (pro)collagen triple helixes under physiological conditions (19).

In this study, mRNA concentrations for type I and III collagen showed a similar decrease in tibialis anterior (immobilized in the lengthened position) as in soleus and plantaris muscles (shortened position) after 3 days. Although the mRNA-to-total RNA ratio was similar in control and immobilized groups at day 7, total muscle RNA content was decreased in soleus and plantaris. Also, the total muscle mRNA for type I and III collagen remained decreased in these muscles, whereas in the lengthened tibialis anterior muscle values were at control levels. These results agree with our previous observations that stretch counteracts the decrease or even causes an increase in total collagen synthesis in muscle during immobilization and that the stretch effect is usually not apparent during the first immobilization week (14, 34). The results further suggest that the effect is similar for both type I and III collagens. It is evidence that immobilization results in a greater decrease in collagen synthesis in slow-twitch soleus than in fast-twitch muscles (34, 35). In the present study the decrease in mRNA levels for fibrillar collagens was, however, similar in soleus and plantaris. The response of mRNAs for fibrillar collagens seems to differ from those of myosin heavy chain, where stretch causes activation of the slow myosin genes in tibialis anterior for a few days (8). Further studies are necessary to understand whether the decrease in collagen mRNA levels is caused by changes in transcriptional rate and/or in mRNA stability and to investigate long-lasting effects of stretch on collagen mRNA levels in different types of skeletal muscle of for weeks.

Unchanged or slightly decreased total collagen content of the muscle after immobilization for several weeks has been previously observed (13, 33). In this experiment, no decreases were observed in the hydroxyproline concentration in the muscles after 7 days of immobilization. The results are in accordance with the slow rate of collagen synthesis in rat muscles, which is 1.3%/day (31). The proportions of type I and III collagens observed in controls in this study are in accordance with earlier findings (23). The present study indicates that a relative short period of immobilization does not change the proportions of insoluble type I and III collagen. It should be noted that the composition and the amount of mature collagen, including its phenotype and extent of cross-linking, depend on not only the great number of posttranslational modification steps (e.g., Ref. 19) but also on the fractional degradation of newly synthesized as well as mature collagen (25). Further studies are needed to assess possible changes in proteolytic rate of fibrillar collagens during immobilization in skeletal muscle.

In summary, the present results indicate that immobilization of skeletal muscle causes a decrease in mRNA level of the PH -subunit during the course of the first day, followed by a decrease in PH activity and mRNA levels for force-transmitting type I and III collagen 2 days later. The present data thus suggest that a decreased mRNA level for the -subunit is an important determinant in the decrease in PH activity at the onset of immobilization. Moreover, the downregulation of PH protein seems to be coordinated with the expression of the major fibrillar procollagens.