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J Appl Physiol 85: 246-253, 1998;
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Vol. 85, Issue 1, 246-253, July 1998

Developmentally regulated expression of cytochrome-c oxidase isoforms in regenerating rat skeletal muscle

Chalermporn Ongvarrasopone and John M. Kennedy

Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, Illinois 60612

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The developmental expression of tissue-specific isoforms of cytochrome-c oxidase (COX) subunit VIII [heart (COX VIII-H) and liver (COX VIII-L)] and the influence of innervation were examined in regenerating fast [extensor digitorum longus (EDL)] and slow (soleus) muscles. In adult muscles, COX VIII-H was the predominant isoform. The COX VIII-L mRNA was expressed 3 days after induction of regeneration, and it progressively decreased after 7, 10, 14, and 30 days of regeneration in both muscles. In contrast, the expression of COX VIII-H mRNA accumulated as myogenesis proceeded to the myotube stage between 7 and 10 days of regeneration and progressively increased to near control levels by 30 days. The influence of innervation on the expression of COX VIII and alpha -actin isoforms was examined in control, innervated, and denervated regenerating muscles at 3 and 10 days. The relative expression of COX VIII-L mRNA in denervated regenerating EDL muscles was significantly greater, while that of COX VIII-H was significantly less than in innervated regenerating EDL muscles after 10 days of regeneration. Similarly, cardiac alpha -actin mRNA levels were elevated in denervated regenerating EDL muscles after 10 days of regeneration. In conclusion, motor innervation influences the transition from the COX VIII-L to COX VIII-H isoform during myogenesis in regenerating muscles.

muscle regeneration; gene expression; nerve

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

IN MAMMALS, cytochrome-c oxidase (COX) consists of 13 different polypeptide subunits. The three largest subunits (subunits I, II, and III) are encoded by mitochondrial DNA and form the catalytic core of the enzyme. The remaining 10 subunits (subunits IV, Va, Vb, VIa, VIb, VIc, VIIa, VIIb, VIIc, and VIII) are encoded by nuclear DNA. Three of the ten nuclear-encoded subunits (VIa, VIIa, and VIII) have been shown to exist as tissue-specific isoforms in mammals. The nomenclature that has been adopted designates the isoforms for each subunit as the liver (L) or heart (H) isoform. The L-isoform of COX is predominantly expressed in liver and nonmuscle tissues, whereas the H-isoform is expressed almost exclusively in heart and skeletal muscles (5). One exception to this rule is exhibited in brown adipose tissue of rats; this tissue expresses the muscle-specific H-isoform of COX subunit VIII (13). In rodents, such as rats and mice (35), only COX subunits VIa and VIII exist as tissue-specific isoforms. In humans, only COX VIa and VIIa have L- and H-isoforms, while the COX VIII isoform that is expressed is more similar to the L-isoform found in other species (31). In the cow, in contrast to other mammals, all three subunits (COX VIa, VIIa, and VIII) exist as tissue-specific isoforms (14).

These nuclear-encoded subunits of COX in various species are developmentally regulated. A switch of the L- to the H-isoform of COX VIa has been clearly demonstrated during in vivo and in vitro muscle development. COX VIa-L is expressed in heart and skeletal muscle in cow (10) and in humans (3) during fetal development. The transcript of COX VIa-L is downregulated during development, and the transcript of COX VIa-H is the predominant isoform that is expressed in adult heart and skeletal muscles. This transition also occurs during fetal development in the rat heart (17, 23) and during postnatal development in mouse skeletal muscle (16, 27). An incomplete isoform transition of COX VIa also occurs during myogenic differentiation in human skeletal muscle cell cultures (33). Thus the L-isoform of COX may be regarded as the embryonic skeletal muscle isoform. Although a transition of COX VIa isoforms has been clearly demonstrated during myogenesis both in vivo and in vitro, the pattern of COX VIII isoform expression has not been fully examined. Although Lomax et al. (19) showed a decrease in the expression of the L-isoform of COX VIII mRNA during myogenic differentiation in a mouse C2C12 cell line, it is not clear whether an increase in the expression of the COX VIII-H isoform occurs in parallel.

Skeletal muscle regeneration recapitulates the embryonic development of skeletal muscle fibers. The reexpression of embryonic and neonatal isoforms of the contractile protein, myosin heavy chain (MHC), occurs during skeletal muscle regeneration. In addition, the transition from the expression of these developmentally regulated isoforms of MHC to the adult isoform exhibits nerve dependence (38). The accumulation of the slow MHC isoform in the soleus (Sol) muscle is entirely dependent on the nerve (9, 38). These results indicate that motor innervation plays an important role in the determination of the adult fast and slow muscle phenotype.

In the present study, we employed a model of muscle regeneration to examine the expression of COX VIII isoforms during myogenesis in vivo and to elucidate the role of motor innervation as a possible regulatory mechanism underlying the switch of the L- to the H-isoform of COX VIII during development in fast- and slow-twitch skeletal muscles. The results indicate that the expression of the embryonic or L-isoform of COX VIII mRNA is dominant during the early period of skeletal muscle regeneration. A transition from the embryonic to adult isoform of COX VIII occurs in regenerated muscles in both the presence and absence of the nerve. However, this transition is not complete in the absence of the nerve, suggesting that innervation influences the expression of COX subunit VIII.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regeneration surgery. Surgery was performed on male Sprague-Dawley rats (180-200 g) under ketamine anesthesia (90 mg/kg) containing acepromazine maleate. The muscle regeneration protocol was modified from the methods of Carlson et al. (7) and Esser et al. (9). Briefly, the proximal (origin) and the distal (insertion) tendons of extensor digitorum longus (EDL) and Sol muscles as well as the associated blood vessels were cut. If the muscles were to regenerate in the presence of nerves (RI groups), the nerves to the muscles were allowed to remain intact. If the muscles were to regenerate without innervation (RD groups), the nerves were cut close to the muscles, and a nerve segment of ~1 cm was removed to prevent reinnervation. The muscles were sutured into their original location and injected with a myotoxic agent [0.5% (wt/vol) bupivacaine hydrochloride (Marcaine) solution]. This procedure results in complete degeneration of all muscle fibers without pharmacological denervation (11). However, an initial loss of motor neuron activity in the regenerating muscles with the nerve intact is expected because of the loss of blood supply (11). The neuromuscular connection is generally accepted to return after 4-5 days of regeneration (7), and neural activity can be detected at about day 7. By day 10, contractile activity is restored (7). To examine the direct involvement of the nerve on the expression of COX subunits, we surgically denervated the hindlimb muscles of an additional group (DD) by removing a segment of the sciatic nerve (0.5-1 cm) within the upper thigh. The muscles were not injected with Marcaine solution in this DD group. All animal procedures followed a reviewed and approved protocol.

RNA isolation and Northern blot analysis. Tissues were removed under anesthesia. Connective tissues were removed, and muscle samples were rapidly frozen between two blocks of dry ice and stored at -80°C for subsequent mRNA analysis. Total cellular RNA was isolated by guanidium isothiocyanate followed by phenol-chloroform extraction according to a method modified from Chomczynski and Sacchi (8) and Kennedy et al. (15). The final RNA concentration was determined by spectrophotometry at 260 nm. The absorbance ratio of A260/A280 was >1.8, indicating that the RNA samples were relatively pure.

For Northern hybridizations, 15 µg total RNA of each sample were size fractionated by electrophoresis on 1.5% agarose gels containing 3.5% formaldehyde. The RNA in gels was transferred to Zeta probe membranes (Bio-Rad Laboratory, Richmond, CA) by capillary transfer techniques and cross-linked to membranes by exposure to ultraviolet light (UV Stratalinker; Stratagene, La Jolla, CA). The blots were prehybridized for 2-4 h and hybridized with the alpha -[32P]dATP-labeled cDNA probe overnight at 42°C. Prehybridization and hybridization solutions were prepared according to Kennedy et al. (15). All COX and 18S Northern blots were washed according to specific published protocols for individual probes (15, 21, 23). Both alpha -actin blots were washed 4 × 15 min in 2× sodium saline citrate solution (SSC; 1× SSC = 0.15 M NaCl and 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS) at room temperature. Cardiac alpha -actin blots were then washed 2 × 20 min in 0.5× SSC-0.1% SDS at 56°C, whereas the skeletal alpha -actin blots were washed 3 × 20 min in 0.5× SSC-0.1% SDS at 56°C and subsequently washed in 0.1× SSC-0.1% SDS for 15 min at 56°C. Blots were exposed to Kodak XAR-5 film at -80°C in the presence of intensifying screens. The hybridization signal was quantified on autoradiograms by scanning densitometry (Personal Densitometer model no. PDSI-PC; Molecular Dynamics, Sunnyvale, CA). The autoradiograph was corrected for background signal on the film, and the relative steady-state level of COX mRNA transcription was normalized by the 18S rRNA signal to correct for loading differences. After the film was exposed to the blots, the blots were stripped 2 × 20 min in 0.1× SSC-0.5% SDS at 100°C and sequentially hybridized with additional cDNA probes.

Probes. The expression of various mRNAs was measured by using cDNA probes for the following: COX III, COX VIc, COX VIII-H, COX VIII-L, skeletal alpha -actin, cardiac alpha -actin, and 18S rRNA. COX cDNA clones were provided by B. Kadenbach of Philipps-Universitat (COX VIII-H and COX VIII-L) and R. Zak of the University of Chicago (COX III and COX VIc). Actin clones were provided by K. Esser of the University of Illinois. cDNA clones were isolated from plasmid vectors by restriction endonuclease digestion and were purified by agarose gel electrophoresis by using a Geneclean II Kit (Bio101, Vista, CA). For hybridizations, probes were labeled to high specific activity by using a random primer labeling kit (Stratagene) and alpha -[32P]dATP (Amersham, Arlington Heights, IL). Unincorporated alpha -[32P]dATP was removed by spin-column chromatography.

Statistical analysis. The data were expressed as means ± SE. The statistical analysis of the mean ± SE between RI and RD groups was performed by using the Student t-test in the Sigma Stat program version 1.0. A measurement of P < 0.05 was accepted as statistically significant.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Tissue-specific expression of COX VIII isoforms. The expression of tissue-specific isoforms of COX subunit VIII was examined in a variety of nonmuscle (brain, liver, and kidney), striated muscle (tibialis anterior, diaphragm, and ventricle), and smooth muscle (duodenum and aorta) obtained from the adult rat. Autoradigrams from Northern blots hybridized with cDNAs of the liver (COX VIII-L) and heart (COX VIII-H) isoforms of COX subunit VIII are shown in Fig. 1. All of the tissues examined expressed the COX VIII-L isoform at variable levels. COX VIII-L was expressed at relatively high levels in nonmuscle and smooth muscle tissues but only at very low levels in striated muscle samples. The highest hybridization signal for COX VIII-L mRNA expression was observed in the kidney. COX VIII-H was not expressed in nonmuscle tissues or in intestinal smooth muscle of the duodenum. In contrast, COX VIII-H mRNA was expressed at high levels in adult skeletal (tibialis anterior) and cardiac muscles. Significant levels of both COX VIII-H and COX VIII-L mRNA isoforms were expressed in the smooth muscle of the ascending limb of the aorta.


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  		Fig. 1.   Tissue-specific expression of cytochrome-c oxidase (COX) VIII isoforms. Northern blot analysis of expression of tissue-specific isoforms of COX VIII from nonmuscle tissues [brain (B), liver (L), and kidney (K)], striated muscles [tibialis anterior (TA), diaphragm (D), and ventricle (V)], and smooth muscles [duodenum (Duo) and aorta (A)]. Data were obtained from a single Northern blot which was sequentially hybridized with COX VIII-heart (H) and COX VIII-liver (L) probes.

COX VIII-L expression during regeneration. Northern blots (Fig. 2) demonstrated that COX VIII-L mRNA was expressed at very low levels and that COX VIII-H was the predominant COX VIII isoform expressed in normal adult EDL and Sol muscles. The developmental expression of COX VIII-L mRNA was investigated during skeletal muscle regeneration in fast-twitch (EDL) and slow-twitch (Sol) muscles at 1, 3, 7, 10, 14, and 30 days of regeneration. The autoradiograms demonstrated that a relatively high hybridization signal for COX VIII-L isoform was observed after 1 and 3 days of regeneration and progressively decreased after 7, 10, 14, and 30 days of regeneration in RI-EDL (Fig. 2A) and RI-Sol (Fig. 2B) muscles. However, even after 30 days of regeneration, the level of COX VIII-L mRNA expression appeared to remain slightly higher than in the control EDL and Sol muscles.


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  		Fig. 2.   Transition in COX VIII isoform expression during muscle regeneration. Representative autoradiograms of Northern blot hybridizations of nerve-intact regenerating muscles (RI) at 1, 3, 7, 10, 14, and 30 days of regeneration are shown for RI-extensor digitorum longum (EDL; A) and RI-soleus (Sol; B) muscles. C, control. Time course experiments were repeated 3 times. Blots with total RNA (15 µg) were sequentially stripped and rehybridized with cDNA probes for COX VIII-L, COX VIII-H, cardiac alpha -actin, skeletal alpha -actin, and 18S rRNA. RI-EDL and RI-Sol blots were exposed on same XAR film.

COX VIII-H expression during regeneration. The hybridization signal for COX VIII-H mRNA was extremely low at 1 and 3 days of regeneration in RI-EDL (Fig. 2A) muscles. The COX VIII-H signal was virtually absent at 3 and 7 days in RI-Sol (Fig. 2B) muscles. The mRNA expression of the COX VIII-H isoform in RI-EDL and RI-Sol muscles became the predominant isoform expressed as the regenerating muscle progressed to the myotube stage by 10 days. The COX VIII-H isoform progressively increased to levels approximating those found in control muscles by 30 days as the regenerating muscle matured under the influence of innervation. The hybridization signal of COX VIII-H mRNA expression at day 1 is higher than the level of the expression at day 3, presumably as a result of a delay in the degeneration of some of the mature myofibers after surgical procedures.

Expression of alpha -actin isoforms during regeneration. Transcripts for the cardiac alpha -actin mRNA were observed at high levels at 3 days and to a lesser extent at 7 and 10 days of regeneration in RI-EDL muscles (Fig. 2A). In RI-Sol muscles, a high hybridization signal of cardiac alpha -actin mRNA expression was observed at 7 days. The transcripts for cardiac alpha -actin in RI-Sol muscles were also detected at low levels at 10, 14, and 30 days of regeneration (Fig. 2B). The downregulation of cardiac alpha -actin mRNA expression after 10, 14, and 30 days of regeneration was concomitant with the upregulation of skeletal alpha -actin mRNA expression in RI-EDL (Fig. 2A) and RI-Sol (Fig 2B) muscles. In addition, the appearance of large amounts of the COX VIII-H isoform appeared to correlate with the appearance of the skeletal isoform of alpha -actin. This suggests that the COX VIII-H isoform accumulation was correlated with the transition from the embryonic to neonatal phenotype. The alpha -actin isoforms of actin are expressed only in striated muscle cells. Consequently, since cardiac alpha -actin and/or skeletal alpha -actin are expressed at a time when the COX VIII-L isoform is the only isoform of COX VIII present (Figs. 2-4), then the only COX VIII isoform which could be present in the muscle cells is the COX VIII-L isoform.


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  		Fig. 3.   Effect of innervation on COX VIII isoform expression in regenerating EDL muscles. Northern blots containing total RNA from RI, RD, and surgically denervated (DD) muscles at 3 and 10 days were examined. From muscle samples at 3 and 10 days, 15 µg total RNA were run on the same gel and transferred to the same Zeta probe membrane. Blot was sequentially stripped and rehybridized with cDNA probes of COX VIII-L, COX VIII-H, cardiac alpha -actin, skeletal alpha -actin, and 18S rRNA probe. Blot was exposed on XAR film with or without intensifying screens.


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  		Fig. 4.   Effect of innervation on COX VIII isoform expression in regenerating Sol muscles. Experimental protocol for Sol muscle was identical to that described in Fig. 3 for EDL muscle.

Effect of innervation on isoform expression. The role of motor innervation on the mRNA expression of tissue-specific isoforms of COX subunit VIII during EDL (Fig. 3) and Sol (Fig. 4) muscle differentiation was examined in the Marcaine-treated regenerating muscle with the nerve intact (RI) and without the nerve (RD). In these studies, we compared the tissue-specific expression of COX subunit VIII at 3 and 10 days of muscle regeneration. The day 3 time point was chosen to represent a stage of muscle regeneration associated with satellite cell activation and proliferation and the formation of myoblasts. The day 10 time point was chosen to represent a stage at which functional neuromuscular connections would be made with differentiated myotubes (6). In addition, the direct involvement of the nerve on COX gene expression was also investigated by cutting the sciatic nerve at the upper thigh level, inducing a denervation without surgically manipulating EDL and Sol muscles.

Autoradiograms showing the influence of motor innervation on the expression of the COX subunit VIII isoforms are shown for EDL (Fig. 3) and Sol (Fig. 4) muscles. During muscle regeneration, the COX VIII-L isoform accounted for almost all of the COX VIII isoform expressed in both RI and RD groups at 3 days, and the transcript levels of COX VIII-L were substantially less by 10 days in both groups of muscles. On the other hand, the COX VIII-H mRNA accounted for almost all the COX VIII mRNA detected in the adult EDL (Fig. 3) and Sol (Fig. 4) control muscles. No COX VIII-H mRNA expression was detected in RD-EDL, RI-Sol, or RD-Sol muscles at 3 days of regeneration. Only a very low hybridization signal of COX VIII-H isoform expression was observed in RI-EDL muscles at 3 days. By 10 days of regeneration, the regenerating EDL and Sol muscles expressed much higher levels of COX VIII-H mRNA in the nerve-intact muscle grafts and to a lesser extent in the nerve-absent grafts.

The effect of motor innervation on the expression of alpha -actin mRNAs was also demonstrated in regenerating EDL (Fig. 3) and Sol (Fig. 4) muscles. During muscle regeneration, cardiac alpha -actin mRNA was reexpressed in both RI and RD groups. A relatively high level of cardiac alpha -actin mRNA was observed at 3 days. The expression of cardiac alpha -actin mRNA in RD-EDL muscles remained high at 10 days of regeneration, whereas the level of cardiac alpha -actin mRNA was reduced in RI-EDL muscles. A similar pattern for the expression of cardiac alpha -actin mRNA was demonstrated in the Sol muscle. In the regenerating EDL muscles, the hybridization signal for the skeletal alpha -actin mRNA was much lower than control in both RI- and RD-EDL muscle groups at 3 days (Fig. 3). By 10 days, the transcript levels of skeletal alpha -actin was increased in both RI- and RD-EDL muscles. In the regenerating Sol muscles, the hybridization signal of skeletal alpha -actin mRNA was undetectable at 3 days in RI- and RD-Sol muscles but was relatively abundant by 10 days of regeneration (Fig. 4).

In contrast to the regenerating muscles, the denervated EDL (Fig. 3) and Sol (Fig. 4) muscles in DD groups expressed both the L- and the H-isoform of COX VIII at 3 and 10 days after denervation. The transcript level of COX VIII-L isoform was slightly higher at 10 days than at 3 days after denervation. This was correlated with an increase in the expression of cardiac alpha -actin mRNA after denervation in both EDL and Sol muscles. On the other hand, the transcript levels for COX VIII-H and skeletal alpha -actin were slightly lower than for control muscles both at 3 and 10 days in DD groups. No further accumulation of COX VIII-L mRNA or decrease in COX VIII-H mRNA was apparent in denervated muscles for up to 30 days (data not shown). This suggests that the observed changes were caused by the limited degeneration and regeneration which has been documented to occur in denervated muscle (25).

Quantitative analysis of the effect of motor innervation on isoform expression. The influence of innervation on the expression of COX VIII and alpha -actin isoforms was specifically addressed by a quantitative analysis of differences between RI and RD groups at 10 days of regeneration (Figs. 5 and 6). Significant differences in the relative transcript levels of COX VIII-L, COX VIII-H, and cardiac alpha -actin mRNAs were demonstrated between RI and RD groups in the EDL muscle after 10 days of regeneration (P < 0.05) (Figs. 5A and 6A). COX VIII-L and cardiac alpha -actin mRNAs were greater by 103 and 603%, respectively, while COX VIII-H was 21% less (Fig. 5A). Although the skeletal alpha -actin mRNA level appeared to be 62% less in RD-EDL muscles, this did not prove to be significant (Fig. 6A). No differences in the relative COX VIII-L, COX VIII-H, or skeletal alpha -actin mRNA levels were observed between RI-Sol and RD-Sol groups (Figs. 5B and 6B). However, the relative level of cardiac alpha -actin mRNA was significantly greater (284%) in RD-Sol muscles than in RI-Sol muscles (Fig. 6B).


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  		Fig. 5.   Absence of motor nerve retards the COX VIII isoform transitions in regenerating muscles. Relative steady-state levels of transcripts for isoforms of COX (COX VIII-L and COX VIII-H) in EDL (A) and Sol (B) muscles are shown after 10 days of regeneration. Total RNA (15 µg) of control, RI, and RD muscles (n = 4 in each group) were run on the same gel and transferred to the same Zeta probe membrane. Blots from EDL and Sol muscles were sequentially hybridized, stripped, rehybridized with cDNA probes, and exposed to the same XAR film. Quantitation of the relative steady-state transcript of the genes was achieved by densitometric scanning of autoradiograms. mRNA hybridization signal for RI and RD groups was normalized to 18S rRNA levels and expressed as %mRNA levels expressed in adult control muscles. Statistical analysis was performed on the relative COX VIII mRNA expression by Student's t-test. * Significant difference between RI and RD groups; P < 0.05.


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  		Fig. 6.   Absence of motor nerve retards alpha -actin isoform transition in regenerating muscles. Relative steady-state transcripts for alpha -actin (cardiac alpha - and skeletal alpha -actin) mRNA in EDL (A) and Sol (B) muscles are shown after 10 days of regeneration. Experiment was performed as described in Fig. 5 legend. Relative expression of cardiac alpha - and skeletal alpha -actin mRNA was normalized to 18S rRNA and expressed as %mRNA levels expressed in adult control muscles. * Significant difference between RI and RD groups; P < 0.05.

Effect of innervation on COX III and COX VIc expression. The nerve dependence of expression for the mitochondrial-encoded subunit (COX III) and nuclear-encoded subunit (COX VIc) of COX was also examined during muscle regeneration (Fig. 7). The relative levels of both COX III and COX VIc mRNA expression in RI and RD groups were substantially lower than control levels at both 3 and 10 days of regeneration. The relative level of COX III and COX VIc mRNA expression increased significantly as regeneration progressed from 3 days to 10 days in RI groups (P < 0.05) but did not exhibit a significant increase in RD groups for either EDL or Sol muscles. Although COX III levels were 19 and 21% less and COX VIc mRNA levels were 20 and 9% less in RD-EDL and RD-Sol muscles, respectively, at 10 days of regeneration, only the difference for COX VIc in EDL muscles proved to be significant (P < 0.05).


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  		Fig. 7.   Effect of innervation on expression of COX III (A and C) and COX VIc (B and D) mRNAs in regenerating EDL and Sol muscles. Quantitative analysis of relative transcript level of COX III and COX VIc in control, RI, and RD muscles of EDL (A and B) and Sol (C and D) muscles. Relative COX mRNA expression was normalized to 18S rRNA level in a given sample and expressed as %COX mRNA level in adult control muscle. Statistical analysis was performed on relative COX mRNA expression by Student t-test. * Significant difference between RI and RD group; P < 0.05. # Significant difference between 3 and 10 days in each group; P < 0.05.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study was undertaken to examine the tissue-specific expression of COX VIII and the effect of motor innervation on the regulation of COX VIII isoform expression during skeletal muscle regeneration. In the rat, the L-isoform of COX VIII mRNA is differentially expressed in nonmuscle and muscle tissues, whereas the H-isoform is abundantly expressed in striated muscle and aortic smooth muscle. However, we show here that the smooth muscle of the rat duodenum expresses only COX VIII-L (Fig. 1). Nonmuscle tissues, such as brown adipose tissue of the rat (13) and thymus and lung tissues of the mouse (35), have been shown to contain transcripts for COX VIII-H. However, rat lung tissue does not express COX VIII-H (18). This may indicate that the expression of COX VIII-H is regulated by mechanisms with unique species- and tissue-specific properties.

The physiological significance of different tissue-specific isoforms of COX remains unclear. Merle and Kadenbach (24) have proposed that the different isoforms of the subunits act as tissue-specific modulators of enzyme activity and demonstrated a difference in the steady-state enzyme kinetic properties of the COX enzyme which had been isolated from bovine liver or heart tissue. Other evidence suggests that tissue-specific isoforms of COX are the binding sites of effectors of enzyme activity, such as nucleotides, hormones, and fatty acid (4). The tissue-specific isoform of COX VIa-H has been proposed to contain a nucleotide binding site and mediate the allosteric effect of ADP on the heart enzyme (32). In addition, COX isoform switching has been observed to be regulated by oxygen tension in yeast and Dictyostelium discoideum (2). This may imply that COX isoform switching plays an important role in the fine tuning of COX activity to serve the different energy requirements of specific cell types.

In this study, the results indicate that a transition of the L- to the H-isoform of COX VIII occurs during in vivo skeletal myogenesis in both fast-twitch EDL and slow-twitch Sol muscles. The COX VIII-L isoform is expressed at relatively high levels during the early stages of regeneration and is eventually replaced by the COX VIII-H isoform. The downregulation of the COX VIII-L isoform during myogenesis may be due to an instability of this mRNA isoform. Priess et al. (28, 30) proposed that a downregulation of COX L-form transcript binding protein (COLBP) may underlie the switch of COX isoforms during myogenesis. COLBP, which is present in myoblasts but not in myotubes (28), binds to the 3' untranslated region of the transcript encoding COX L-form polypeptides and stabilizes the message (29). Therefore, the lack of COLBP in myotubes is proposed to cause an instability of COX VIII-L mRNA and result in a decrease in the steady-state level of COX VIII-L mRNA.

Isoform switching from the embryonic to the adult muscle isoform of contractile proteins (1, 9) is commonly seen during skeletal muscle regeneration and in vivo muscle development. In the present study, the transition of the cardiac alpha -actin isoform to the adult skeletal alpha -actin isoform occurred in parallel with the switching of the COX VIII-L to COX VIII-H isoform. Although the molecular mechanisms which control these isoform switches are not fully understood, members of the myoD family of helix-loop-helix proteins and the myoD binding motif (E-box) have been shown to be critical in the transcription of many muscle-specific genes (37). Myocyte enhancer factor-2 (MEF-2) is another candidate for muscle-specific transcription as this factor has also been shown to enhance the expression of muscle-specific genes (26). The 5' upstream cis-regulatory elements of the rat COX VIII-H gene include duplicated E-boxes with a core sequence of CAGCTG. Lenka et al. (18) demonstrated that the binding of myogenic factors to these tandemly duplicated consensus E-box elements is necessary for the transcriptional activation of the COX VIII-H promoter in both skeletal and cardiac muscle cell lines. Like the rat COX VIII-H gene, three potential E-boxes and a MEF-2 element located in the 5' flanking region of the murine COX VIa-H gene have been shown to direct the myotube-specific expression of COX VIa-H (36). Such regulatory motifs may play a significant role in the upregulation of COX VIII-H expression during skeletal muscle development.

We also investigated whether motor innervation influences isoform switching during development in regenerating skeletal muscle. Innervation has been shown to be crucial for the maturation and maintenance of regenerating muscle fibers (7) and to influence the acquisition of fast and slow contractile protein mRNA profiles (9). In earlier studies, Toyofuku et al. (34) demonstrated an alpha -actin isoform switch during skeletal muscle regeneration in which innervation facilitated the recovery of the skeletal alpha -actin mRNA but was not required for the downregulation of cardiac alpha -actin mRNA expression. In contrast, the results presented here (Fig. 6) show that the expression of cardiac alpha -actin mRNA expression is increased by the absence of the nerve in both EDL and Sol muscle grafts. The differences between the two studies are most likely a result of the time points which were examined. The present study focused on the early stages of regeneration (3-10 days), whereas the study of Toyofuku et al. (34) focused on the latter stages (10-40 days). Thus, it seems likely that innervation alters the rate at which cardiac alpha -actin expression is repressed, but innervation may not be necessary for this transition to occur.

In this study, a switch of the L- to the H-isoform of COX VIII occurred in regenerating EDL and Sol muscles with the nerve intact or without the nerve (Figs. 3 and 4). This suggests that the expression of COX VIII isoforms was not solely dependent on the nerve. However, innervation was required for the complete downregulation of COX VIII-L isoform and facilitated the upregulation of COX VIII-H isoform. It is possible that the transition of the embryonic (L) isoform to the adult (muscle-specific) isoform of COX VIII during skeletal muscle regeneration follows a mandatory developmental program as myoblasts fused to form myotubes and mature to adult myofibers. Therefore, it is probable that motor innervation, which influences the rate of these developmental steps and the terminal differentiation of regenerating muscle, may indirectly facilitate COX isoform transitions.

A coordinated increase in the expression of the mitochondria-encoded subunit (COX III) and nuclear-encoded subunit (COX VIc) of COX was demonstrated as regeneration progressed and myoblasts fused to form myotubes (Fig. 7). The accumulation of these transcripts indicates a rapid rate of mitochondrial biogenesis during skeletal muscle regeneration and suggests that the transition from the embryonic (COX VIII-L) to the mature (COX VIII-H) phenotype of COX may be associated with this event. In this study, innervation was shown to influence the process of mitochondrial biogenesis, since the increase in the transcripts of COX III and COX VIc as muscle regenerated from 3 to 10 days was eliminated in EDL and Sol muscles regenerating in the absence of innervation. In other studies, a coordinated increase in the expression of COX III and COX VIc mRNAs also occurred in response to chronic stimulation of rat skeletal muscle (12, 22, 40), whereas denervation caused a coordinated decrease in the expression of these two genes (39). However, expression of these two genes was not coordinated during growth (20) or alteration of thyroid hormone levels (21). Taken together, results from these studies suggest that signals provided by the nerve may have a unique capacity to regulate a coordinated expression of mitochondrial- and nuclear-encoded subunits of COX.

In conclusion, we have demonstrated a developmental transition of tissue-specific isoforms of COX subunit VIII during skeletal muscle regeneration which is influenced by innervation. In addition, the isoform switching of alpha -actin mRNAs during the early stages of muscle regeneration is also nerve dependent, as is the accumulation of COX III and COX VIc mRNAs. Taken together these results suggest that innervation provides cues which either directly or indirectly affect the rate of acquisition of the mature muscle phenotype.

    ACKNOWLEDGEMENTS

We thank Dr. B. Kadenbach for kindly providing COX VIII cDNAs, Dr. R. Zak for COX III and COX VIc cDNA clones, Dr. K. Esser for alpha -actin cDNA clones and for assistance with the regeneration model, Rosemary Clepper for animal care, and Linda Alaniz-Avila for photographic skills.

    FOOTNOTES

Address for reprint requests: J. M. Kennedy, 835 S. Wolcott Ave., Dept. of Physiology and Biophysics, M/C 901, Univ. of Illinois at Chicago, Chicago, IL 60612 (E-mail: JKennedy{at}uic.edu).

Received 3 December 1997; accepted in final form 26 February 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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