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J Appl Physiol 81: 1941-1945, 1996;
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Journal of Applied Physiology
Vol. 81, No. 5, pp. 1941-1945, November 1996
EXERCISE AND MUSCLE

Cytochrome c mRNA in skeletal muscles of immobilized limbs

Frank W. Booth, Wei Lou, Marc T. Hamilton, and Zhen Yan

Department of Integrative Biology, University of Texas Medical School, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES

ABSTRACT

Booth, Frank W., Wei Lou, Marc T. Hamilton, and Zhen Yan. Cytochrome c mRNA in skeletal muscles of immobilized limbs. J. Appl. Physiol. 81(5): 1941-1945, 1996.---Even though immobilization of a slow skeletal muscle in a lengthened position prevents muscle atrophy, it is unknown whether this treatment would prevent a decrease in mitochondrial quantity. We found that, regardless of muscle length in immobilized limbs, the mRNA of a marker for mitochondrial quantity, cytochrome c, decreased. Cytochrome c mRNA per milligram of muscle was 62 and 72% less 1 wk after fixation of the soleus muscle in shortened and lengthened positions, respectively, than age-matched controls. Cytochrome c mRNA per milligram wet weight was 36 and 32% less in the tibialis anterior muscle fixed for 1 wk in the shortened and lengthened positions, respectively, compared with age-matched controls. Recently, in the 3'-untranslated region of cytochrome c mRNA a novel RNA-protein interaction that decreases in chronically stimulated rat skeletal muscle was identified.[Z. Yan, S. Salmons, Y. L. Dang, M. T. Hamilton, and F. W. Booth. Am. J. Physiol. 271 (Cell Physiol. 40): C1157- C1166, 1996 [Medline] ]. The RNA-protein interaction in the 3'-untranslated region of cytochrome c mRNA in soleus and tibialis anterior muscles was unaffected by fixation in either shortened or lengthened position. We conclude that, whereas lengthening muscle during limb fixation abates the loss of total muscle protein, the percentage decrease in cytochrome c mRNA is proportionally greater than total protein. This suggests that the design of countermeasures to muscle atrophy should include different exercises to maintain total protein and mitochondria.

atrophy; mitochondria; gene expression

INTRODUCTION

MANY INDIVIDUALS EXPERIENCE the fixation of joints with plaster casts to allow injured bones, ligaments, and tendons to heal. A common secondary effect of this treatment is skeletal muscle atrophy (21), which produces decreased muscle strength. Atrophied muscles also have a reduction in mitochondrial concentration (3), which contributes to decreased endurance capacity for low- and moderate-intensity work (16). Most of our knowledge about the mitochondrial response to limb immobilization comes from muscles fixed in a neutral (i.e., resting) or shortened position (see Refs. 1, 4), as briefly summarized below. After 10 days of limb immobilization, in both fast- and slow-twitch muscles the capacity of whole muscle homogenates to oxidize pyruvate, beta -hydroxybutyrate, palmitate, or glucose is reduced (22). A final common pathway for these substrates is their oxidative phosphorylation in mitochondria. The functional consequence of these events is that atrophied muscles in immobilized limbs had lower ATP levels and an apparent increase in their dependence on anaerobic metabolism, as reflected by the more extensive depletion of glycogen and enhanced lactate production during continuous contractile activity (31).

The length of a skeletal muscle in an immobilized limb determines, in part, whether muscle atrophy occurs. When muscles are fixed at a neutral or shortened position, they atrophy (5, 12, 28, 29). On the other hand, lengthening of skeletal muscle beyond its neutral length during limb fixation either attenuates or prevents muscle atrophy, and in some cases produces hypertrophy (3, 12, 26-29).

Appell (1) has hypothesized that an early decrease in mitochondrial concentration could be of etiological importance in the initiation of atrophy of skeletal muscle in immobilized limbs. Appell's hypothesis predicts that mitochondrial concentration would not decrease in lengthened slow muscles because they do not atrophy. A hypothesis in our present study is that lengthening would attenuate muscle atrophy but would not prevent the loss of cytochrome c mRNA in the soleus muscle of immobilized limbs [contrary to the hypothesis of Appell (1)]. The rationale for our hypothesis is that mitochondrial concentration in skeletal muscle has been shown to be correlated with the daily duration of muscle contraction (8, 10), and because lengthened immobilized muscles do not undergo isotonic contractions, cytochrome c mRNA concentration would decrease in muscles fixed in a lengthened position.

Cytochrome c concentration in skeletal muscle has been shown to be positively correlated with another mitochondrial protein marker, citrate synthase (10). We have used cytochrome c expression as an index for the loss of mitochondria in skeletal muscle that occurs during limb immobilization. Cytochrome c protein and mRNA per whole muscle decrease 40% during the first week of limb immobilization in the shortened position in young rats (19). These observations imply that a change in the pretranslational control of cytochrome c plays a role in its decrease in immobilized limbs. However, little further molecular information on the loss of cytochrome c mRNA is available. In a concurrent study, Yan et. al (32) noted that muscles with less cytochrome c mRNA had more RNA-protein interaction in the 3'-untranslated region (3'-UTR) of cytochrome c mRNA. Thus we hypothesized that a decrease in contractile activity by limb immobilization would increase RNA-protein interaction in the 3'-UTR of cytochrome c mRNA.

MATERIALS AND METHODS

Animals. Pathogen-free Sprague-Dawley female rats were obtained from Harlan (Houston, TX) and assigned randomly at 7-8 wk of age to control group and hindlimb immobilization groups with ankles fixed in either plantar flexion or dorsiflexion. Rats were anesthetized with 1.4 ml/kg of a mixture of ketamine (54 mg/ml), xylazine (2.2 mg/ml), and acepromazine (3.5 mg/ml) during the application of immobilization to both hindlimbs with plaster of paris (5) and at the time of death.

Total protein. Total protein was determined with a detergent-compatible protein assay (Bio-Rad).

RNA isolation. RNA for Northern blot analysis was extracted from soleus and tibialis anterior (TA) muscles of three groups of rats: ankles fixed for 7 days in plantar flexion, ankles fixed for 7 days in dorsiflexion, or controls who were killed at the end of immobilization according to a procedure by Chomczynski and Sacchi (7) with RNAzol B (Biotecx Laboratories) as described earlier (32).

Northern blot analysis. The coding region of the cytochrome c gene was subcloned into pBluescript SK (+) vector (Stratagene). In brief, the BamH I-EcoR I fragment (958-base pair) from pRC4 plasmid (25) was inserted into the polylinker region of pBluescript SK (+) digested with BamH I and EcoR I. The resulting plasmid pRC4 (Bluescript) was used for Northern blot analysis of cytochrome c mRNA as described by Babij and Booth (2). mRNA was quantified by image analysis of the autoradiogram (BioImage, Millipore). Total RNA per milligram wet weight was determined by the alkaline hydrolysis procedure of Munro and Fleck (20).

Cytoplasmic extract. Muscle homogenization was described in Yan et al. (32). The homogenates were centrifuged at 15,000 g for 15 min at 4°C. The supernatant (S15) was snap-frozen in liquid nitrogen and stored at -80°C until analysis. The protein concentrations of S15 were determined by the Lowry method (18).

Radiolabeled-RNA probe. For the cytochrome c 3'-UTR probe, pRC4CAT3' (9) was digested with Bgl II and BamH I followed by ligation with T4 ligase into pBluescript SK (+). A 116-base pair fragment (+1,337 to +1,452) was produced by digestion with Dra I and in vitro transcription with T3 polymerase (Promega) and [alpha -32P]uridine 5'-triphosphate (3,000 Ci/mmol; ICN). After transcription at 37°C for 1 h, RNA transcripts were digested with 1 unit of ribonuclease-free deoxyribonuclease (Promega) for 15 min at 37°C, extracted with phenol-chloroform, and precipitated in ethanol with 20 µg Escherichia coli tRNA as carrier. The radiolabeled RNAs were quantified, and their specific activities (counts · min-1 · µg-1) were determined by liquid scintillation counting after trichloroacetic acid precipitation (24).

Ultraviolet (UV) cross-linking. UV cross-linking assays were performed according to Rondon et al. (23) with slight modification as described earlier (32). In brief, cytoplasmic extracts (15 µg protein) were incubated at 30°C for 10 min with excess 1.5 ng 32P-labeled RNA probe (see above) in a reaction mixture containing 10% glycerol and (in mM) 12 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.9), 15 KCl, 0.25 EDTA, 0.25 dithiothreitol, and 5 MgCl2, as well as E. coli tRNA (200 ng/µl) in a total volume of 15 µl. The reaction mixtures were placed on ice after ribonuclease T1 treatment (0.6 units, 37°C, 20 min) and irradiated in open tubes with UV light (0.12 J) for 5 min in a Stratalinker chamber (Stratagene). After being incubated in Laemmli sample-loading buffer at 95-100°C for 3 min, the reaction mixtures were subjected to electrophoresis on 10% sodium dodecyl sulfate-polyacrylamide gels together with prestained molecular-weight markers (Sigma Chemical). The gels were dried and placed in contact with Kodak X-OMAT film to obtain the autoradiogram.

Statistics. Comparison of the values among different groups was performed with analysis of variance. If a significant difference was found, the Tukey-Kramer multiple-range test was employed. For protein concentration, a two-tailed Student's t-test was employed. P < 0.05 was designated as significant. Values are expressed as means ± SE.

RESULTS

Soleus muscle wet weight and total RNA concentration (µg RNA/mg wet wt) were the same as control values, and the soleus muscle-to-body weight ratio was increased by 39% (P < 0.05) at the end of the 7-day fixation of the muscle in a lengthened position (ankle is immobilized in dorsiflexion) (Table 1). However, soleus muscle wet weight, total RNA concentration, and muscle-to-body weight ratio were 55, 29, and 44% less (P < 0.05), respectively, than control after 7 days of fixation in a shortened position (plantar flexion) (Table 1).

Table 1. Cytochrome c mRNAs in soleus muscles fixed in lengthened or shortened position for 7 days

Position Body Wt, g Wet Wt, mg Wet Wt-to-Body Wt Ratio Total RNA, mg/g Cytochrome c mRNA
IOD/µg RNA IOD/mg wet wt IOD/whole muscle
Control 154 ± 2  62.8 ± 5.3  0.41 ± 0.01  1.7 ± 0.1  0.384 ± 0.023  0.648 ± 0.046  40 ± 4 
Shortened (plantar flexion) 121 ± 1* 28.3 ± 2.1* 0.23 ± 0.01* 1.2 ± 0.1* 0.204 ± 0.051* 0.249 ± 0.058* 7 ± 1*
Lengthened (dorsiflexion) 114 ± 4* 64.6 ± 4.0Dagger 0.57 ± 0.01*Dagger 1.9 ± 0.3Dagger 0.133 ± 0.011dagger 0.179 ± 0.035* 12 ± 2*
Values are means ± SE. Muscle weights for each animal were average of both sides. For cytochrome c mRNA analysis, 2 soleus muscles from a rat were pooled for an observation (control and lengthened groups), and 4 soleus muscles from 2 rats were pooled for a single observation (shortened group). For control and lengthened groups, n = 5; for shortened group, n = 10 rats. Average body weight and soleus muscle weights of 2 rats pooled for mRNA determination were used to calculate body and muscle weights. IOD, integrated optical density units. * P < 0.01 from control; dagger P < 0.05 from shortened group; Dagger P < 0.01 from shortened group.

Although Babij and Booth (2) previously reported that fixation of the soleus muscle in a shortened position for 7 days did not alter its protein concentration, similar data for a 7-day fixation of rat muscle in a lengthened position have not been previously published. An additional set of rats (n = 5) was employed to obtain this information. Fixation in a lengthened position did not alter soleus muscle protein concentration (183.1 ± 6.3 mg/g wet wt for control; 160.3 ± 9.0 mg/g wet wt for 7-day fixation; P = 0.07, two-tailed Student's t-test) and content (15.0 ± 1.1 mg/muscle for control; 12.9 ± 0.8 mg/muscle for fixation; P = 0.17). Previously, Jokl and Konstadt (17) reported that myofibrillar and sarcoplasmic protein concentration were unchanged in cat fast- and slow-twitch skeletal muscle that had been immobilized in the lengthened position for 4 wk.

Cytochrome c mRNA was less in soleus muscles fixed in either a shortened or lengthened position. Cytochrome c mRNA per microgram RNA was 47 and 65% less (P < 0.01) in shortened and lengthened positions, respectively, after 7 days of immobilization compared with the control group of the same age (Table 1, Fig. 1). Cytochrome c mRNA concentration per milligram soleus wet weight was 62 and 72% less (P < 0.01) in shortened and lengthened positions, respectively, compared with the control group (Table 1). Cytochrome c mRNA content per whole soleus muscle was 82 and 70% less (P < 0.01) in shortened and lengthened positions, respectively, than the control group (Table 1).

Fig. 1. Autoradiogram of Northern blot (A) for soleus muscles from rats with no treatment or with hindlimbs fixed in either a shortened (plantar flexion) or a lengthened position (dorsiflexion). Total RNA (10 µg) was added to each lane, electrophoresed on 1% agarose denaturing gels, blot transferred to Hybond N+, and hybridized with a 958-base pair radiolabeled probe from pRC4 rat somatic cytochrome c probe (25). See Table 1 for tabulated data. Ethidium bromide staining (B) of 28S and 18S on gel after electrophoresis and before transfer shows approximate equality of total RNA loading.
[View Larger Version of this Image (70K GIF file)]

To determine whether the observed phenomena are only specific to soleus muscles, we performed the measurements on the TA muscles. The TA muscle wet weight was 37% (P < 0.05) and 13% (P < 0.01) less when it was immobilized in a shortened position (dorsiflexion) and lengthened position (plantar flexion), respectively, compared with control values (Table 2). However, fixation of the TA muscle in a lengthened position maintained the muscle-to-body weight ratio. In a separate set of rats (n = 5), fixation in a lengthened position did not alter TA muscle protein concentration (219.7 ± 1.2 mg/g wet wt for control; 211.1 ± 4.1 mg/g wet wt for 7-day fixation; P = 0.08) but decreased protein content (76.0 ± 2.5 mg/muscle for control; 58.8 ± 1.3 mg/muscle for fixation; P < 0.05). Goldspink (12) found that the protein content of the rat extensor digitorum longus (EDL) muscle was the same as in an age-matched control after 2, 4, and 7 days of immobilization in the lengthened position. Lengthening of the rabbit EDL by immobilization for 3 days did not alter its protein concentration, which indicates that wet weight reflects protein content in this animal (13). Total RNA concentration (µg RNA/mg wet wt) was the same as the control values at the end of the 7-day fixation in both shortened and lengthened TA muscles (Table 2). Cytochrome c mRNA levels were less than the control values in the TA muscles immobilized in both the shortened and lengthened positions as normalized by three methods. Cytochrome c mRNA (integrated optical density unit per microgram of RNA) was 31 and 40% less (P < 0.01) in shortened and lengthened positions, respectively, after 7 days of immobilization compared with controls of the same age (Table 2). Cytochrome c mRNA per milligram wet TA weight was 36 and 32% less (P < 0.01) in shortened and lengthened positions, respectively, than in the controls (Table 2). Cytochrome c mRNA per whole TA muscle was 59 and 40% less (P < 0.01) in shortened and lengthened positions, respectively, compared with control group values (Table 2). These data again demonstrate that muscle atrophy during immobilization can be attenuated by lengthening the muscle, but cytochrome c mRNA levels decrease as long as the muscle is immobilized.

Table 2. Cytochrome c mRNAs in tibialis anterior muscles fixed in lengthened or shortened position for 7 days

Position Body Wt, g Wet Wt, mg Wet Wt-to-Body Wt Ratio Total RNA, mg/g Cytochrome c mRNA
IOD/µg RNA IOD/mg wet wt IOD/whole muscle
Control 140 ± 2  587 ± 48  4.2 ± 0.1  1.4 ± 0.2  0.304 ± 0.043  0.435 ± 0.056  254 ± 29 
Lengthened (plantar flexion) 119 ± 3* 509 ± 33* 4.3 ± 0.1  1.6 ± 0.3  0.182 ± 0.024dagger 0.296 ± 0.077dagger 152 ± 46dagger
Shortened (dorsiflexion) 118 ± 4* 369 ± 40dagger Dagger 3.1 ± 0.2dagger Dagger 1.3 ± 0.1  0.209 ± 0.019dagger 0.278 ± 0.022dagger 103 ± 17dagger
Values are means ± SE (n = 5). Two muscles from a rat were pooled for each observation of muscle weight, RNA, and cytochrome c mRNA. * P < 0.05 from control; dagger P < 0.01 from control; Dagger P < 0.05 from lengthened group.

No consistent change in cytoplasmic protein interaction with a 116-base riboprobe (+1,337 to +1,452) from the 3'-UTR of rat somatic cytochrome c mRNA in cytoplasmic extracts was noted from soleus muscles fixed for 7 days in either lengthened or shortened position (Fig. 2). Similar observations were observed in TA muscle (data not shown).

Fig. 2. RNA-protein interaction in the 3'-untranslated region of cytochrome c mRNA. Equal amounts of protein (15 µg) from whole soleus muscle homogenates were incubated with 1.5 ng of 32P-labeled riboprobe. Autoradiogram from ultraviolet cross-linking assay with radiolabeled riboprobe for 116 bases (+1,337 to +1,452) of the 3'-untranslated region of cytochrome c mRNA. Molecular masses (in kDa; left) were estimated from prestained markers (Sigma Chemical). Groups were control, shortened (plantar flexion) for 7 days, and lengthened (dorsiflexion) for 7 days. FP, free probe.
[View Larger Version of this Image (97K GIF file)]

DISCUSSION

When soleus and TA muscles were immobilized in lengthened position, cytochrome c mRNA levels per whole soleus and TA muscles were 30 and 60%, respectively, of control. In contrast, total protein per whole soleus and TA muscles was 86 and 77%, respectively, of control. Thus fixation of muscle in a lengthened position not only did not prevent loss of cytochrome c mRNA, but the decrease in cytochrome c mRNA predicts a decline in cytochrome c protein that is greater than total protein. Although the attenuation of muscle atrophy when muscles are lengthened in immobilized limbs confirms previous reports (3, 12, 28, 29), the observation that cytochrome c mRNA content is not maintained in muscles immobilized in a lengthened position is new. The disassociation of muscle mass from the loss of cytochrome c mRNA supports the hypothesis of differential gene regulation of mitochondrial and contractile protein pools by different cell signals. Examples are that resistance training enlarges muscle mass whereas mitochondrial concentration remains constant (15), endurance training increases mitochondrial concentration whereas muscle mass is unaltered (6), and muscle mass decreases whereas mitochondrial density increases during chronic stimulation (30). The hypothesis that mitochondrial and contractile proteins are modulated by different signaling pathways from contractile activity deserves further study.

Results of the present study support the idea that neither passive stretch nor isometric contractions can maintain the concentration of mitochondria in skeletal muscle in immobilized limbs. Fournier et al. (11) found that integrated electromyograph (EMG) was unchanged when the soleus muscle was fixed in a lengthened position during immobilization. This EMG activity must reflect isometric contraction because the joints were fixed. Hník et al. (14) found similar results for the soleus muscle and extended these observations to the TA. The fixation of the ankle in neither plantar flexion nor dorsiflexion had any appreciable effect on EMG activity in the TA. Hník et al. (14) concluded that, because immobilization in the lengthened position maintained, but did not increase, EMG activity in either the soleus or TA muscle, passive stretch, rather than EMG activity, appeared to be the factor mainly responsible for lessening atrophy of muscles fixed in the lengthened position. Previous reports indicate a direct correlation exists between duration of low-intensity isotonic contractions and cytochrome c protein concentration in skeletal muscle (8, 10). Hindlimb unloaded muscles undergo random isotonic contractions. Because it has previously been shown that cytochrome c mRNA per whole soleus muscle was decreased 54, 45, and 61% by 7 days of hindlimb unloading, limb immobilization of the soleus muscle in a shortened position, and denervation, respectively (2), the isotonic contractions occurring in the hindlimb-unloaded muscle are insufficient in duration to maintain cytochrome c mRNA.

Because muscles with more contractile activity have higher concentrations of cytochrome c mRNA and less RNA-protein interaction in the 3'-UTR of cytochrome c mRNA as determined by gel mobility shift and UV cross-linking assays (32), we hypothesized that decreasing loaded isotonic contractions of the soleus muscle would increase this RNA-protein interaction. This hypothesis did not hold up. Protein interaction with the 3'-UTR was not changed by fixation of the soleus muscle in either a shortened or lengthened position. Thus the decrease in cytochrome c mRNA was not due to the increase in RNA-protein interaction in the 3'-UTR of cytochrome c mRNA. In summary, passive lengthening of the immobilized soleus muscle does not prevent the loss of cytochrome c mRNA, even though loss in its muscle mass was attenuated; and decreased cytochrome c mRNA levels in the immobilized muscles are not due to increased RNA-protein interaction in the 3'-UTR of cytochrome c mRNA. The greater losses of cytochrome c mRNA (70%) than of total protein (14%) in the whole soleus muscle during immobilization in a lengthened position underscore that countermeasures to atrophy in limb immobilization must include specific exercises to signal increases in both contractile and mitochondrial gene expression.

ACKNOWLEDGEMENTS

We express our deep appreciation for the help we received from the late James Pastore, who assisted our research. We thank Brad Goodwin and his staff for animal care.

FOOTNOTES

   This research was supported by National Aeronautics and Space Administration Grant NAGW 3908.

Address for reprint requests: F. W. Booth, The Univ. of Texas-Houston Health Science Center, Medical School, Dept. of Integrative Biology, 4.100 MSB, 6431 Fannin, Houston, TX 77030 (E-mail: fbooth{at}girch1.med.uth.tmc.edu).

Received 6 November 1995; accepted in final form 22 May 1996.

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