Regulation of dihydropyridine receptor gene expression in mouse skeletal muscles by stretch and disuse

Tatiana L. Radzyukevich and Judith A. Heiny

Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267-0576

Submitted 19 November 2003 ; accepted in final form 30 July 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study examined dihydropyridine receptor (DHPR) gene expression in mouse skeletal muscles during physiological adaptations to disuse. Disuse was produced by three in vivo models—denervation, tenotomy, and immobilization—and DHPR {alpha}1s mRNA was measured by quantitative Northern blot. After 14-day simultaneous denervation of the soleus (Sol), tibialis anterior (TA), extensor digitorum longus (EDL), and gastrocnemius (Gastr) muscles by sciatic nerve section, DHPR mRNA increased preferentially in the Sol and TA (+1.6-fold), whereas it increased in the EDL (+1.6-fold) and TA (+1.8-fold) after selective denervation of these muscles by peroneal nerve section. It declined in all muscles (–1.3- to –2.6-fold) after 14-day tenotomy, which preserves nerve input but removes mechanical tension. Atrophy was comparable in denervated and tenotomized muscles. These results suggest that factor(s) in addition to inactivity per se, muscle phenotype, or associated atrophy can regulate DHPR gene expression. To test the contribution of passive tension to this regulation, we subjected the same muscles to disuse by limb immobilization in a maximally dorsiflexed position. DHPR {alpha}1s mRNA increased in the stretched muscles (Sol, +2.3-fold; Gastr, +1.5-fold) and decreased in the shortened muscles (TA, –1.4-fold; EDL, –1.3-fold). The effect of stretch was confirmed in vitro. DHPR protein did not change significantly after 4-day immobilization, suggesting that additional levels of regulation may exist. These results demonstrate that DHPR {alpha}1s gene expression is regulated as an integral part of the adaptive response of skeletal muscles to disuse in both slow- and fast-twitch muscles and identify passive tension as an important signal for its regulation in vivo.

dihydropyridine receptor mRNA; decreased use; passive tension; denervation; tenotomy; hindlimb immobilization


ADULT SKELETAL MUSCLE, although terminally differentiated, remains a remarkably plastic tissue that can dynamically match its output to changing demands. This remodeling involves the altered expression of a number of molecules that determine contractile performance, including the myosin isoforms, metabolic enzymes, and proteins involved in Ca2+ handling. The result is a shift in contractile performance along a number of indexes including metabolic type (glycolytic or oxidative), contraction speed (fast or slow), and muscle size (hypertrophy or atrophy). These adaptations are accomplished both at the level of protein translation and by altered gene expression (2–4, 20, 32).

In contrast to the established role of regulated gene expression for the contractile and metabolic proteins, much less is known about the regulation by use of genes that specify membrane proteins involved in muscle activation, although they also contribute to contractile performance. Muscle activation is controlled by voltage-driven movements of the dihydropyridine receptor (DHPR), an L-type calcium channel that associates with the ryanodine receptor/calcium release channel at specialized internal junctions with the sarcoplasmic reticulum (10). The DHPR is absolutely required for excitation-contraction coupling (37), and its content in phenotypically different skeletal muscles is a marker of contractile speed, being more abundant in fast- compared with slow-twitch muscles (18).

Previous studies of the regulation of DHPR gene expression by muscle use produced mixed results. Those studies focused primarily on the slow-twitch soleus (Sol) muscle subjected to disuse produced by removing mechanical load (18) or nerve input (28). DHPR {alpha}1s mRNA in the Sol muscle increases to levels normally seen in the extensor digitorum longus (EDL) muscle after 28 days of unloading by hindlimb suspension (18) or 50-day denervation (28). A few studies using phenotypically fast-twitch muscles report weak or no regulation of DHPR mRNA during denervation-induced disuse and led to the proposal that DHPR gene expression is not highly regulated in fast-twitch muscles. DHPR mRNA increases moderately in the mouse Sol muscle and less in the flexor digitorum longus after 15-day denervation (36) and does not change in the rat EDL muscle during long-term (50 day) denervation (28) or mechanical unloading (18).

A few studies have examined the effect of increased use on DHPR gene expression. DHPR {alpha}1s mRNA and protein both decrease significantly when a fast-twitch muscle is stimulated chronically in a pattern typical for slow-twitch muscle (27), suggesting that regulated DHPR gene expression can occur in association with known phenotype transitions. Additionally, DHPR protein content does not change in the rat plantaris muscle subjected to increased mechanical load (19) and increases in fast- and slow-twitch rat muscles after 12 wk of moderate exercise training (33), but the contribution of regulated gene expression to these adaptations is not known.

Collectively, these studies establish that DHPR gene expression is regulated during altered muscle activity, especially by decreased activity or load. However, they do not reveal a clear relationship between the change in use and the change in DHPR mRNA, suggesting that important physiological signals that control its regulation remain to be identified.

To address this question, we examined further the regulation of DHPR gene expression in phenotypically different skeletal muscles during physiological adaptations to disuse in vivo. We focused on disuse because it produces a measurable end point, atrophy, that has broad clinical relevance. We subjected the Sol, tibialis anterior (TA), gastrocnemius (Gastr), and EDL muscles of the mouse to denervation, tenotomy, or limb immobilization and measured changes in DHPR {alpha}1s mRNA with quantitative Northern blot analysis. DHPR {alpha}1s protein in immobilized Gastr and TA muscles was measured by Western blot. Each of these models produces disuse by a different mechanism, thereby allowing us to dissociate the contributions of nerve input and passive or active tension to DHPR gene regulation. Denervation eliminates nerve input, both electrical and trophic, thereby rendering the muscle inactive while preserving tendon attachments and resting tension. Tenotomy preserves nerve input at near-normal levels (39) but unloads and shortens the muscle, preventing it from generating either passive (resting) or active tension. Immobilization preserves both nerve input and tendon attachments, while allowing resting tension to be manipulated by fixing the muscle in a lengthened or shortened position (16). Resting tension decreases in shortened but immobile muscles and increases significantly in lengthened but immobile muscles (16). Tenotomy and limb immobilization have not been used previously to study DHPR gene expression. A preliminary report of this work has appeared (29, 30).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Surgery and tissue preparation. Experiments were carried out on 62 female ND4 Swiss-Webster mice (6–8 wk old, 20–25 g). All procedures were performed following the guidelines for the care and use of animals at the University of Cincinnati. Surgery, casting, and tissue removal were performed under anesthesia (Avertin, 2.5% tribromoethanol; Aldrich). Skeletal muscles of the hindlimb were denervated unilaterally by cutting and removing 1–2 mm of the sciatic nerve in the proximal area of the thigh or the peroneal nerve at the knee. The sciatic nerve innervates upper and lower hindlimb muscles of the knee and ankle; the peroneal nerve branches from the sciatic above the knee to innervate the ventral muscles of the ankle. There was no reinnervation during the 2-wk protocol, as confirmed by microscopic examination when the tissue was harvested. Tenotomy was performed by a bilateral cut of the Achilles tendon to the Gastr and Sol muscles and the distal tendons of the TA and EDL muscles. The tenotomized muscles shortened to two-thirds of the length of intact controls. Unilateral immobilization of the muscles of the lower leg was achieved by fixing the leg for 4 days in a full dorsiflexed position with a plaster cast. The Sol, TA, Gastr, and EDL muscles are composed of fiber types that increase progressively from predominantly slow oxidative to predominantly fast oxidative-glycolytic and fast glycolytic (6). Muscles from untreated age-matched littermates and the contralateral muscles of treated animals were used for controls. There was no significant difference in body weight between treated and untreated animals up to 2 wk after surgery. The muscles were harvested, weighed, homogenized immediately in TRI reagent (Molecular Research Center, Cincinnati, OH), and stored at –80°C for later RNA extraction.

Selection of control muscles for denervation measurements. It is widely accepted that the contralateral muscles of animals subjected to unilateral denervation can hypertrophy because of extra weight bearing by the untreated limb, with associated changes in metabolism or gene expression. However, hypertrophy of the contralateral muscles does not always occur (40). Consequently, we examined which muscles would best serve as control under the conditions of this study. We found that DHPR {alpha}1s mRNA content of the TA muscle of untreated mice is comparable to that in the contralateral TA muscle of mice subjected to 14-day unilateral denervation of the opposite leg (Fig. 1 and Table 1). Overall, the DHPR mRNA of contralateral muscles tended to stay the same (TA, Gastr) or to decrease by 11–15% (Sol, EDL) compared with control, untreated muscles. The wet weight of contralateral muscles tended to decrease, but the changes did not reach statistical significance. Thus, under our conditions, the contralateral muscles of mice denervated for 14 days do not undergo any significant hypertrophy. Additionally, the small decrease in DHPR {alpha}1s content in the contralateral Sol and EDL muscles would tend to overestimate any potential increase in DHPR mRNA caused by denervation. For these reasons, the muscles of untreated mice were used as controls for analysis of changes in the DHPR mRNA content of 14-day treated mice. Additionally, these measurements establish that our assay can detect statistically significant changes in DHPR {alpha}1s mRNA content as small as 11–15% in mouse skeletal muscle samples. DHPR mRNA is more abundant in the mouse EDL than Sol muscle, with a ratio of {alpha}1s in Sol to {alpha}1s in EDL of 0.66, similar to that in rat (ratio = 0.65; Ref. 27).



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Fig. 1. A: representative Northern blot of dihydropyridine receptor (DHPR) {alpha}1s mRNA and 18S rRNA in the tibialis anterior (TA) muscle of untreated control mice (Cont) and in the contralateral (C-L) muscle of mice subjected to 14-day unilateral section of the sciatic nerve to the opposite leg. Total RNA was pooled from 4 muscles and loaded (5 µg/lane) in duplicate (indicated by brackets). Transcript sizes: DHPR {alpha}1s 6.5 kb; 18S 1.9 kb. B: same membrane stained with methylene blue before hybridization showing that the RNA was undegraded and that similar amounts of total RNA were loaded per lane. Methylene blue improved hybridization efficiency compared with ethidium bromide.

 

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Table 1. Percent change in DHPR {alpha}ls content and wet weight of contralateral muscles of denervated compared with untreated mice and wet weight of untreated muscles

 
Northern blot. Total RNA was isolated by a single-step method (7) using the TRI reagent protocol as described previously (8). Yields of total RNA were 0.67–0.85 µg/mg muscle tissue in control muscles, 0.87–1.31 µg/mg muscle tissue in denervated muscles, 0.54–0.98 µg/mg muscle tissue in tenotomized muscles, and 0.45–1.34 µg/mg muscle tissue in the immobilized muscles, without obvious differences between samples. Muscles from two to four mice were pooled for each measurement. Briefly, 5 µg of total RNA was size-fractionated in duplicate in a 1% agarose-0.4 M formaldehyde gel, transferred to a positively charged nylon membrane (Nytran Super Charge; Schleicher & Schuell, Keene, NH) by downward capillary blotting, fixed to the membrane by baking 1 h at 80°C, and visualized by methylene blue staining. The membranes were hybridized to a DHPR {alpha}1s cDNA probe (8) labeled with 32P-labeled dCTP by random priming to a specific activity of ~5 x 109 dpm/µg. Hybridization was carried out in a 50% formamide solution (High-Efficiency Hybridization Solution; Molecular Research Center) for 48–66 h at 42°C. Membranes were washed, exposed on a phosphor screen, quantified by densitometry (ImageQuant; Molecular Dynamics, Sunnyvale, CA), and then stripped and reprobed with an 18S ribosomal RNA probe (Ambion, Austin, TX). In some experiments, membranes were probed simultaneously with the DHPR {alpha}1s and 18S probes. DHPR {alpha}1s signal intensity was normalized to the intensity of the 18S band and expressed relative to control levels.

Organ culture. Freshly dissected Sol and EDL muscles were fixed with pins to the bottom of a 4-ml culture dish, four muscles/dish, and incubated for 19–24 h in a medium optimized for adult skeletal muscle culture [M199 (GIBCO-BRL), as modified in Ref. 41, at 37°C in an atmosphere of 95% air-5% CO2]. The muscles were fixed either at resting length or stretched to 1.1–1.25 times resting length. Resting lengths of the EDL and Sol muscles were 12.7 ± 0.1 (n = 10) and 9.3 ± 0.1 (n = 9) mm, respectively.

The average sarcomere length of stretched and unstretched muscles was measured at the end of the culture period by measuring the average of 10 sarcomere spans under a light microscope at x400 magnification from different regions of the muscle. This was done to verify that the muscles retained the imposed length change throughout the culture period and as a control for the possibility that length changes in nonmuscle elements such as a tendon could have occurred (e.g., if a tendon lengthened by tearing on the pins used to fix the muscle while the muscle shortened). Sarcomere lengths at 24 h were 13% greater in stretched EDL than EDL muscles cultured without stretch and 17% greater in stretched Sol than Sol muscles cultured without stretch (Table 2), confirming that the stretched muscles retained the lengthened position throughout the culture period. Before harvesting, each muscle was stimulated electrically to confirm that it retained the ability to generate evoked contractions.


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Table 2. Sarcomere length in mouse Sol and EDL muscles cultured at resting or stretched length

 
Western blot. Microsomal membrane samples were prepared and resolved by SDS-PAGE as described previously (8). Briefly, proteins were electrophoresed for 1.5 h on 7.5% precast polyacrylamide minigels (GeneMate Express Gels; ISC BioExpress, Kaysville, UT) with Bio-Rad prestained SDS-PAGE standards and blotted onto a polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Millipore, Billerica, MA). The blots were incubated for 1 h at 37°C with a skeletal muscle-specific monoclonal antibody to DHPR (MA3-920, 1:500 dilution) and incubated for 1 h at 37°C with a peroxidase-conjugated goat anti-mouse secondary antibody (1:10,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA). Calsequestrin expression was measured as a technical control for linearity and protein loading with primary polyclonal antibody (PA1-913 at 1:1,000; Affinity Bioreagents) and anti-rabbit IgG, heavy and light chain goat secondary antibody (1:10,000; Cortex Biochem, San Leandro, CA) incubated for 1 h at room temperature. Blots were developed with an enhanced chemiluminescence system (Amersham-Pharmacia, Piscataway, NJ), visualized on X-ray film (Kodak BioMax MR), and quantified by densitometry (ImageQuant; Molecular Dynamics). Linearity of protein detection was verified by running control immunoblots for six protein amounts (5–30 µg).

Statistical analysis. Values are given as means ± SE. Differences in mean values were evaluated with an unpaired Student’s t-test, with significance accepted at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
DHPR {alpha}1s mRNA and atrophy after sciatic nerve cut or tenotomy. Figure 2, A and B, compares DHPR {alpha}1s mRNA levels in the Sol, TA, EDL, and Gastr muscles after 14 days of disuse produced by sciatic nerve section or tenotomy. In the denervated muscles, DHPR mRNA content increased in the Sol and TA muscles (to 162 ± 6% and 155 ± 27% of control, respectively) but was unchanged on average in the EDL and Gastr muscles (104 ± 32% and 102 ± 4% of control). DHPR mRNA content was more variable in the EDL muscle and could increase or decrease in different samples. In contrast, DHPR mRNA content decreased significantly in all muscles after tenotomy, to 38 ± 6%, 76 ± 7%, 48 ± 8%, and 53 ± 2% of control in the Sol, TA, EDL, and Gastr muscles, respectively.



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Fig. 2. Effect of denervation (Den) or tenotomy (Ten) on DHPR {alpha}1s mRNA and wet weight of the mouse soleus (Sol), TA, extensor digitorum longus (EDL), and gastrocnemius (Gastr) muscles compared with control. A: representative Northern blots. B: mean % change between denervated or tenotomized samples compared with control; 12–16 muscles and 3–5 membranes were used for each muscle type. C: mean % change in muscle mass of the same samples. *Statistical significance at P < 0.05.

 
Both models of disuse produced significant atrophy (Fig. 2C), which was comparable in the denervated and tenotomized groups and independent of the fiber type composition of the muscles. Mean wet weight decreased to 69 ± 9%, 55 ± 4%, 68 ± 3%, and 62 ± 8% of control in denervated Sol, TA, EDL, and Gastr muscles, respectively (n = 5 experiments; each experiment used a pooled sample of 4–5 muscles). It decreased to 73 ± 9%, 72 ± 5%, 78 ± 5%, and 70 ± 4% of control values in tenotomized Sol, TA, EDL, and Gastr muscles, respectively. Thus there is no consistent relationship between the degree of atrophy and the change in DHPR mRNA content, which can increase or decrease in different muscles and models of disuse. These results demonstrate that DHPR {alpha}1s mRNA content is significantly regulated during physiological adaptations to disuse but suggest that its regulation may occur by mechanisms that are unrelated to the change in muscle mass.

DHPR {alpha}1s mRNA in TA and EDL muscles after peroneal nerve cut. The finding that denervation can preferentially upregulate DHPR mRNA in the Sol but not EDL muscle is consistent with previous reports that show a greater effect of denervation on slow-twitch compared with fast-twitch muscles (28). However, the finding that DHPR mRNA is strongly upregulated in the fast-twitch TA muscle contrasts with previous interpretations that the regulation by denervation occurs mainly in slow-twitch muscles. To investigate the basis for this difference, we removed nerve input to a more limited subset of muscles by performing a 14-day section of the peroneal nerve. In contrast to sciatic nerve section, which denervates all four muscles, peroneal section selectively denervates the TA and EDL muscles. Figure 3 shows that DHPR {alpha}1s gene expression is remarkably upregulated in both TA (1.8-fold) and EDL (1.6-fold) muscles after denervation compared with the corresponding innervated controls. DHPR mRNA tended to increase in the TA muscle after sciatic compared with peroneal nerve cut (1.8-fold compared with 1.6-fold). The TA muscle underwent comparable paralysis and atrophy in both treatments. Muscle mass decreased by 57% (n = 18 muscles) after peroneal section and by 55% after sciatic section (n = 16 muscles); the differences were not significant. The DHPR mRNA content of the EDL muscle increased significantly after peroneal nerve section (1.6-fold) compared with no change after sciatic section (1.05-fold; significantly different at P < 0.05). These results suggest that an important factor other than nerve input or denervation-related disuse per se contributes to DHPR gene regulation under these conditions; for example, DHPR mRNA is upregulated in the TA muscle after sciatic nerve section and in the TA and EDL muscles after peroneal nerve section but unchanged in the Gastr muscle, whether denervated (sciatic cut) or innervated (peroneal cut) (data not shown). We hypothesized that the effect of denervation on DHPR gene expression may depend on the passive tension on the denervated muscle, which can be influenced by the surrounding muscles. The mice continue to ambulate after both sciatic and peroneal nerve section but do so in different ways. All lower leg muscles are inactive after sciatic cut, and the animal uses them in variable and unpredictable ways for support and balance, whereas after peroneal cut the dorsal muscles including Gastr and Sol muscles remain innervated and contract during ambulation. In this case, active shortening of the Gastr and Sol muscles is expected to lengthen the TA and EDL muscles in a consistent manner.



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Fig. 3. Effect of peroneal nerve section on DHPR {alpha}1s mRNA in the mouse TA and EDL muscles. A: representative Northern blots in control and denervated muscles. B: mean % change in DHPR {alpha}1s mRNA in the TA and EDL muscles compared with control after either sciatic or peroneal nerve cut. *Statistical significance at P < 0.05.

 
DHPR mRNA after limb immobilization. To test the hypothesis that passive tension is a key determinant of DHPR mRNA expression, we turned to limb immobilization, which allowed us to impose a change in passive tension that is not secondary to other changes. Immobilization renders defined muscles largely inactive but maintained at a greater or less than normal passive tension while simultaneously preserving normal innervation, tendon attachment, and muscle mass. This model was imposed for 4 rather than 14 days to favor changes in DHPR gene expression over changes related to cell size, which peak later and are mediated largely by translational processes (11). Well-characterized adaptations in gene expression occur within 5 days of immobilization (24), before changes in size. In contrast to denervation or tenotomy, maintained immobilization leads to opposite effects on muscle size—atrophy in shortened and hypertrophy in lengthened muscles.

As expected, unilateral immobilization of one limb for 4 days did not produce atrophy or hypertrophy in either the immobilized or contralateral muscles, and the contralateral muscle was used as reference. Figure 4 compares changes in DHPR {alpha}1s mRNA content in the Sol, TA, EDL, and Gastr muscles after unilateral limb immobilization in a maximally dorsiflexed position, which maintains the Sol and Gastr muscles in a lengthened position (increased passive tension) and the TA and EDL muscles at a shorter than normal resting length (decreased passive tension). DHPR {alpha}1s mRNA changed dramatically in all muscles, being upregulated in the Sol and Gastr muscles and downregulated in the TA and EDL muscles. Sorting these results by the change in passive tension (Fig. 4B) reveals that the change in DHPR mRNA correlates directly with the passive tension on the muscle. DHPR {alpha}1s mRNA increases in the lengthened Sol and Gastr muscles (to 229 ± 23% and 151 ± 21% of control, respectively) and decreases in the shortened TA and EDL muscles (to 73 ± 6% and 75 ± 4% of control, respectively). This result demonstrates that resting tension per se is an important physiological regulator of DHPR gene expression.



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Fig. 4. DHPR {alpha}1s gene expression in hindlimb muscles immobilized for 4 days in a dorsiflexed position. A: representative Northern blots for the contralateral and immobilized (I) muscles. B: % change in DHPR {alpha}1s mRNA in each muscle, grouped by the imposed change in resting length (Sol and Gastr lengthened; TA and EDL shortened).

 
DHPR {alpha}1s mRNA in mouse muscles cultured at different lengths. To further define the influence of resting tension on DHPR {alpha}1s mRNA expression, we subjected the Sol and EDL muscles to short-term stretch in vitro by culturing the muscles for 19–24 h at defined resting lengths. DHPR mRNA declined in both muscles when cultured at resting length, to 30% and 50% of paired, freshly isolated controls in the Sol and EDL muscles, respectively, after 24 h. However, much of this loss could be prevented by applying even a small amount of passive tension to the muscle during culture. DHPR mRNA was significantly greater in the stretched Sol (140 ± 6% of paired control) and EDL (129 ± 10% of paired control) muscles cultured at sarcomere lengths of 13% and 17%, respectively, above resting length compared with EDL and Sol muscles cultured without stretch (Fig. 5). This result demonstrates that even a small increase in resting length maintained for a relatively short time can significantly upregulate DHPR {alpha}1s gene expression. This confirms the in vivo findings in immobilized muscles (Fig. 4) and supports the idea that the change in DHPR gene expression depends more strongly on the change in muscle length than on the method of disuse or initial fiber type. Together the in vivo and in vitro findings suggest that resting tension is a sensitive, physiological regulator of DHPR gene expression.



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Fig. 5. Effect of stretch on DHPR {alpha}1s mRNA content in muscles in vitro. A: representative Northern blots of DHPR {alpha}1s mRNA in the Sol and EDL muscles cultured for 24 h either at resting length or in a stretched position. Sarcomere lengths of stretched muscles were 13% and 17% of control values in the Sol and EDL muscles, respectively, after 24 h in culture. Four muscles were pooled for each measurement. B: mean % change (n = 5) in DHPR {alpha}1s expression in the Sol or EDL muscle stretched for 19–24 h. Values are normalized to the DHPR {alpha}1s mRNA content of muscles incubated at resting length. *Statistical significance at P < 0.05.

 
DHPR {alpha}1s protein expression in mouse muscles immobilized in vivo. Figure 6 examines whether stretch can also modify DHPR protein expression in immobilized muscles. Immobilization was used because it provides a test of both positive and negative stretch-induced changes in DHP mRNA in the same animal. When the Gastr muscle is immobilized for 4 days in a lengthened position, DHPR protein tends to stay the same or decrease slightly, 0.81 ± 0.12 compared with contralateral (n = 4), but the differences are not significant (P > 0.2). In the shortened TA muscle, DHPR protein is variable, being 1.4% and 0.7% of contralateral in two samples. The variability may reflect the fact that immobilization without stretch stimulates pathways leading to muscle atrophy and is consistent with the finding that protein recovery is more variable in the TA muscle (usable in 2 of 4 samples). Thus at 4 days there is dramatic induction of mRNA without a corresponding change in DHP protein, indicating that the regulation of DHP mRNA and protein are not closely synchronized and that DHPR expression may be regulated at multiple levels. Immobilization for 4 days was chosen in this study to favor detection of early changes in mRNA over changes in muscle size, which occur later and go in opposite directions in the different muscles.



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Fig. 6. Representative Western blot of DHPR {alpha}1s protein expression in muscles immobilized (Imm) for 4 days in a lengthened (Gastr) or shortened (TA) position. Microsomal membrane samples (20 µg) were loaded in duplicate and probed with an antibody against the DHPR {alpha}1s subunit. Calsequestrin expression was probed as a loading control and used to normalize the expression of all samples before further analysis. Size markers (first lane), contralateral (CL) Gastr, immobilized Gastr, contralateral TA, and immobilized TA.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Regulation of DHPR gene expression in muscle disuse. This study demonstrates that DHPR gene expression is regulated as part of the adaptive response of skeletal muscle to disuse in three independent models. The regulation occurs in both slow- and fast-twitch muscles and is surprisingly robust. The changes are comparable in magnitude to those of the myosin heavy chain (MHC) isoform mRNAs whose regulation by activity is well established. For example, DHP mRNA changed in the Sol muscle by –2.8-fold, +1.6-fold, and +2.2-fold during tenotomy, denervation, and immobilization, respectively, and in the TA muscle by –1.3-fold, +2.5-fold, and +1.5-fold, respectively. In comparison, the mRNAs of MHC isoforms increase or decrease by 1.5- to 2.5-fold in various slow- and fast-twitch muscles of the rat after 1- to 2-wk denervation or immobilization (17). The changes in DHPR mRNA are also comparable to other models of disuse. DHPR mRNA changes –1.5-fold to +3-fold in the rat Sol muscle unloaded by hindlimb suspension (18) or TTX paralysis (31); and it changes up to 1.5-fold in rat hindlimb muscles after denervation (28, 31). The present study adds the first demonstration that DHPR {alpha}1s gene expression is regulated in tenotomized or immobilized muscles during disuse and that it can be significantly regulated in a denervated fast-twitch skeletal muscle. Importantly, it demonstrates that passive tension can regulate DHPR gene expression in muscle independently of inactivity, nerve input, fiber type composition, or associated changes in cell size.

DHPR gene expression and control of muscle size during disuse. Sustained disuse of skeletal muscles is associated with a loss in mass, or atrophy, which can vary in severity in phenotypically different muscles and models of disuse (9, 17, 20, 32). Muscle size is controlled by both translational and transcriptional mechanisms, with translational processes playing a greater role (13). Our results show a dissociation between DHPR gene expression and control of cell size in three models of disuse. For example, 2 wk of denervation or tenotomy induces significant and comparable atrophy in both slow- and fast-twitch mouse muscles, whereas DHPR gene expression can be up- or downregulated or unchanged. In the TA muscle after peroneal nerve cut, DHPR mRNA and cell size change in opposite directions. In muscles immobilized for 4 days, DHPR mRNA changes in opposite directions in different muscles without change in muscle size. Other models of disuse also suggest a dissociation between DHPR gene expression and cell size. DHPR {alpha}1s mRNA is upregulated +1.8-fold in the unloaded rat Sol muscle after 24 h, before atrophy develops (18), and it is upregulated to dramatically different degrees in rat Sol muscle subjected to 14-day TTX treatment or 14-day denervation, two models that produce comparable atrophy (31). This consistent dissociation in different models of disuse suggests that DHPR gene expression is regulated independently of the pathways that control muscle size, although the two processes can coincide.

DHPR gene expression and muscle phenotype during disuse. Phenotypic changes occur in association with several models of disuse, mediated in large part by remodeling of MHC isoform gene expression (13). The relationship between DHPR gene expression and muscle phenotype during adaptations to disuse is not known. Our finding that DHPR {alpha}1s mRNA in the denervated Sol muscle increases to levels normally found in the fast EDL muscle is similar to results in the denervated rat Sol muscle (28) and in the unloaded rat Sol muscle (18), in which MHC isoform mRNAs shift toward a phenotypically faster profile (17). This could occur if common signals control DHPR gene expression and fiber phenotype. Our finding that DHPR mRNA can increase dramatically in phenotypically fast-twitch muscles after denervation was unexpected, because denervation of fast-twitch muscles shifts MHC mRNAs toward a slower profile (17), a change that predicts less DHPR mRNA. Other studies of DHPR mRNA in slow- and fast-twitch rat muscles report little or no change after denervation (31). The present study reexamined this question with both global and selective denervation and included three muscles that are composed predominantly of fast fiber types (6). Our results demonstrate clearly that DHPR gene expression can be regulated in fast-twitch muscles during denervation disuse but show no consistent relationship between regulation of DHPR gene expression and muscle phenotype or nerve input per se. It increases in immobilized, lengthened muscles, decreases in immobilized, shortened muscles, and declines in all tenotomized muscles, in every case independent of phenotype. Thus DHPR {alpha}1s gene expression is regulated in both fast- and slow-twitch muscles during disuse, but the regulation is not linked to the change in muscle phenotype that can accompany these models.

Passive tension regulates DHPR gene expression. Results obtained with cast immobilization reveal that DHPR {alpha}1s mRNA is highly sensitive to the passive tension on the muscle, a parameter that is not controlled in the other models. Short-term cast immobilization allowed us to subject phenotypically different muscles to sustained disuse in either a shorter or a lengthened position while preserving innervation and tendon attachments, in each case before detectable changes in muscle size. In vitro measurements confirm the effect of stretch and further suggest that some degree of resting tension is required to maintain DHPR mRNA expression. Cultured muscles, which lack all tendon attachments and resting tension, downregulate DHPR mRNA dramatically within 24 h; however, much of this loss can be prevented by applying even a small amount of stretch. Collectively, these results demonstrate that passive tension is an important physiological signal that controls DHPR gene expression in all muscle types. Increased stretch to a muscle upregulates DHPR gene expression, whereas decreased tension downregulates it.

DHPR gene expression in the denervated and tenotomized muscles can be understood in this context. After sciatic nerve section, the sampled muscles do not generate active contraction or undergo the length changes that accompany normal ambulation. In this case, the change in DHPR mRNA is variable and does not correlate with muscle phenotype or size. However, when the TA and EDL muscles are selectively denervated by peroneal nerve cut, the Gastr and Sol muscles remain innervated and generate active contractions during body movements, which in turn can passively stretch the inactive TA and EDL muscles. The finding that DHPR mRNA is dramatically upregulated in inactive TA and EDL muscles suggests that passive stretch, more than nerve input or inactivity per se, is the major stimulus for DHPR gene expression under these conditions. In the case of tenotomy, all affected muscles shorten and cannot undergo passive stretching during body movement (1) and DHPR mRNA decreases dramatically in all muscles. Thus shortening a muscle or decreasing resting tension by any of these manipulations downregulates DHPR gene expression.

This conclusion differs from a previous study in which it was proposed that a decrease in chronic tension stimulates increased DHPR gene expression (18). DHPR mRNA increases significantly in the rat Sol muscle unloaded by hindlimb suspension, before atrophy develops. Because passive tension is not fixed, it is possible that the chronic tension on the Sol muscle may be different than assumed or that another signal may act synergistically with passive tension in this model.

Regulation of DHPR gene expression by stretch compared with other muscle genes. Previous studies have highlighted the importance of passive stretch as a signal for muscle growth, phenotypic differentiation, and size (11, 14, 15). Maintained stretch is required for the growth and survival of muscles in culture (26, 38). Stretch alone, independent of nerve input or other mechanical signals, can serve as a stimulus for both phenotypic remodeling and muscle size. Increased stretch leads to hypertrophy even if a muscle is inactive, and, conversely, a muscle that is stimulated chronically can atrophy if stretch is not imposed (12, 16). Stretch can oppose the atrophy that occurs in immobilized and shortened muscles (32), and this model has been used to identify early and downstream genes involved in stretch-induced hypertrophy (21, 22, 34), which may use independent but interacting pathways (34). Identified stretch-responsive skeletal muscle genes include the MHC isoform genes Ankrd2 (21), Smpx (22), and Serhl (34), IGF-1, mechanogrowth factor (42), and muscle regulatory factors (5, 23, 24). Of these, the MHC genes are the best characterized. Stretch makes a greater contribution than activity alone to MHC gene expression (13) and can influence MHC gene expression in denervated muscles in a muscle-specific manner (25). Stretch is required to maintain expression of the slow and neonatal MHC mRNAs that predominate in slower or less differentiated fiber types and to repress expression of the fast MHC mRNAs (13, 14, 24). In this respect, our finding that increased passive stretch promotes increased DHPR mRNA, a direction toward a faster muscle phenotype, is a direction opposite to the regulation of MHC mRNAs by stretch.

Relationship between DHP mRNA and protein in response to stretch. The finding that DHPR mRNA and protein do not follow closely at 4 days suggests that DHPR expression is controlled at multiple levels—including both transcription and protein translation/turnover. This result does not mean that changes in DHPR protein do not occur but rather that, if they do occur, their turnover rate is not tightly synchronized with DHPR mRNA. In general, multiple levels of regulation are expected for a protein as essential to muscle function as DHPR. Studies of DHPR mRNA and protein in other models of disuse are not consistent on this point. DHPR mRNA is increased in 25- to 50-day denervated rat muscles (28), but DHPR mRNA and protein are not changed detectably at 10 days after denervation (31). In denervated chick skeletal muscles, DHPR protein increases within 3 days of denervation, peaks at 15 days, then declines as atrophy becomes evident (35). More measurements of the stability and turnover of both DHPR mRNA and protein, in isolation from changes in cell size or phenotype, are required to evaluate this question.

In conclusion, this study adds DHPR to the growing list of muscle genes whose expression is regulated by stretch. A direction for future research is to identify the molecular stretch sensors and signaling pathways that control DHPR gene expression and their relationship to the pathways that control muscle phenotype and size.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. A. Heiny, Dept. of Molecular and Cellular Physiology, Univ. of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267-0576 (E-mail: heinyja{at}uc.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.


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