Quantification of myosin heavy chain mRNA in somatic and branchial arch muscles using competitive PCR

Hak Hyun Jung1,2, Richard L. Lieber3, and Allen F. Ryan1,4

1 Department of Surgery/Otolaryngology, 3 Departments of Orthopedics and Bioengineering, and 4 Department of Neuroscience, University of California at San Diego School of Medicine and Veterans Affairs Medical Center, La Jolla, California 92093; and 2 Department of Ear, Nose, and Throat/Head and Neck Surgery, Korea University, Seoul, Korea

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The purpose of this study was to quantify the type and amount of myosin heavy chain (MHC) mRNA within muscles of different developmental origins to determine whether the regulation of gene expression is comparable. Seven MHC isoforms were analyzed in rat adult limb (extensor digitorum longus, tibialis anterior, and soleus) and nonlimb (extraocular, thyroarytenoid, diaphragm, and masseter) muscles using a competitive PCR assay. An exogenous template that included oligonucleotide sequences specific for seven rat sarcomeric MHC isoforms (beta -cardiac, 2A, 2X, 2B, extraocular, embryonic, and neonatal) as well as beta -actin was constructed and used as the competitor. Only the extraocular muscle contained all seven isoforms. All seven muscles contained type 2A and type 2X MHC transcripts in varying percentages. As expected, the soleus muscle contained primarily beta -cardiac MHC (87.8 ± 2.6%). Extraocular MHC was found only in the extraocular and thyroarytenoid muscles and in relatively small proportions (7.4 ± 1.5% and 4.0 ± 0.7%, respectively). Neonatal MHC was identified in extraocular (7.9 ± 0.3%), thyroarytenoid (4.4 ± 0.4%), and masseter (1.0 ± 0.2%) muscles, and embryonic MHC was identified both in extraocular (1.2 ± 0.5%) and, unexpectedly, in soleus (0.6 ± 0.1%) muscles. Absolute MHC mRNA mass was greatest in the masseter (106 pg/0.5 µg RNA) and least for the tibialis anterior (64 pg/0.5 µg RNA). These values suggest that MHC mRNA represents from 4 to 17% of the total mRNA pool in various skeletal muscles. Differences in MHC profile between somatic and branchial arch muscles suggest that the developmental origin of a muscle may, at least in part, be responsible for the MHC expression program that is implemented in the adult. An inverse relationship between the expression of beta -cardiac and type 2B MHC transcripts across muscles was noted, suggesting that the expression of these two isoforms may be reciprocally regulated.

gene expression; rat; polymerase chain reaction

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MYOSIN HEAVY CHAIN (MHC) is the most abundant contractile protein in skeletal muscles and is encoded by a family of genes consisting of 2A (13, 20), 2B (13, 20), 2X (6), extraocular (EO) or 2L (17, 30), embryonic (27), neonatal (21), and alpha - and beta -cardiac isoforms (14, 16). These isoforms differ in their functional properties and are expressed in a tissue-specific and developmentally regulated manner. Isoform transitions are influenced by various factors including hormone levels, exercise, physical damage, and aging, as recently reviewed (26).

The MHC composition within a muscle can be inferred from functional, histochemical, immunohistochemical, and electrophoretic analysis. Since MHC transcript levels can change within hours, MHC transcript studies are becoming increasingly popular for study of the control of MHC gene expression. A few studies have reported MHC composition of muscles at the mRNA level, but the methods used (primarily Northern blotting, in situ hybridization, and PCR) are semiquantitative (4, 6, 31). Absolute transcript levels are more desirable for the investigation of the mechanisms involved in MHC regulation. Although numerous studies have demonstrated the existence of various MHC transcripts in adult skeletal muscles, these are typically reported only for limb muscles and using a restricted set of adult isoforms. In the study of MHC regulation, it is of interest to define MHC content in muscles of various developmental origins using all known MHC isoforms. This is because tissues of different developmental origin may be either more permissive or more restrictive with regard to the expression of different numbers of MHC isoforms.

Recently, competitive PCR was developed and used to quantify absolute amounts of mRNA transcripts encoding several different gene products (8). This method was shown to be reproducible, rapid, and very sensitive, capable of detecting and quantifying transcript levels down to attomolar (10-18 M) concentrations. Competitive PCR is considered the most rigorous method for quantitation of transcript levels (5, 8, 29). This method eliminates the potentially confounding effects of differences in amplification efficiency between primer sets. Such differences could result in overestimation of transcript levels due to efficient amplification or in underestimation of transcript levels due to inefficient amplification. Competitive PCR also dramatically reduces the potential effects of competing messages. This is especially important for MHC transcript quantification, since multiple, highly homologous MHC isoforms exist (26).

The purpose of this study was to develop a competitive PCR assay for detection of all known MHC mRNA transcripts in rat skeletal muscle. We also applied this method to study the MHC transcript levels present across a wide range of rat muscle types representing different embryonic origins and physiological characteristics.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Construction and use of the competitor. The backbone of the MHC competitor fragment (Fig. 1) consisted of 400 bp of the rat type 2A MHC gene (Genbank accession no. L13606), beginning 673 bp from the 3' end with a short sequence that is highly conserved between all sarcomeric MHC genes (13). A beta -actin sense primer was ligated to the 5' end to enable measurement of a constitutively expressed product for internal calibration. Then, eight antisense primers [specific to beta -actin and to the 3' untranslated region of the EO (also known as type 2L; unpublished observations and Ref. 17), 2B, 2X, beta -cardiac, neonatal, and embryonic MHC types; Table 1] were successively added in sequential PCR reactions using partially overlapping 40- to 80-bp oligonucleotides containing the antisense primers. The competitor was ligated into the pGEM-T vector (Promega, Madison, WI), amplified, and sequenced using an ABI 373A automated DNA-sequencing system (ABI, Foster City, CA).


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Fig. 1.   Schematic construct of competitor used in current study. Nucleotide sequence of consensus sense primer (CSP) is AGAAGGCCAAGAAAGCCAT instead of a degenerate primer. beta -Car, beta -cardiac; EO, extraocular; Neo, neonatal; Emb, embryonic. MHC, myosin heavy chain.

                              
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Table 1.   Oligonucleotide sequences and expected PCR product lengths

Subsequent PCR amplifications used a consensus sense primer [CSP; a degenerate oligonucleotide from a highly conserved region occurring from 620 to 660 bases from the 3' end of all known rat MHC genes (13, 17)] and isoform-specific downstream primers (Table 1). Using native mRNA, these reactions produced PCR products of the expected lengths, ranging from 613 to 683 bp (Fig. 2). When the competitor was used with these primer sets, product lengths were different from the native reaction products by 119-168 bp (Table 1).


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Fig. 2.   Competitive PCR of cDNA from rat extraocular muscle (EOM). Lane 1 is a 100-bp ladder, with bright band in ladder representing 600 bp. Lane 2 is a traditional PCR band of mRNA without competitor; cont, control. Initial concentration of competitor is 1,000 amol (lane 3), and 3-fold serial dilutions are shown in lanes 4-12. Upper bands of PCR products from 2A, 2B, 2X, EO, neonatal, embryonic, and beta -cardiac myosin heavy chain (MHC) are targets, and lower bands are competitors, whereas bands for beta -actin are reversed (see Table 1 for exact lengths and masses). Arrows point to approximate competitor concentrations at which target and competitor are at same concentration. Rat EOM thus has predominantly type 2A and type 2X MHC transcripts (~111 amol), with very low levels of embryonic MHC mRNA (~1.4 amol).

RT-PCR and competitive PCR of rat muscle. Adult Sprague-Dawley rats (200-250 g) were anesthetized deeply with rodent cocktail (in mg/kg: 50 Ketamine, 5 Rompun, and 1 acepromazine), and seven skeletal muscles were removed (extraocular, lateral aspect of the thyroarytenoid, extensor digitorum longus, tibialis anterior, soleus, diaphragm, and masseter). For each type of muscle, two to six muscles from different animals were analyzed. Tissues were immediately frozen in isopentane cooled by liquid nitrogen (-159°C) and stored for subsequent analysis at -80°C. Total RNA was isolated from frozen samples using TRIzol extraction (GIBCO BRL, Gaithersburg, MD), and 0.5 µg RNA/reaction was reverse transcribed using the Superscript preamplification system (GIBCO BRL); 1 µl from the resulting 80 µl of cDNA solution was amplified. For initial determination of which of the seven MHC isoforms was present within each muscle, traditional PCR amplification of the cDNA of each muscle was performed using the upstream CSP and one of each of the seven specific downstream antisense primers. When a reaction product of the expected length was obtained, competitive PCR was performed for that isoform using threefold serial dilutions consisting of 1 fmol (10-15 mol), 333 amol (10-18 mol), 111 amol, 37.0 amol, 12.3 amol, 4.12 amol, 1.37 amol, and 0.457 amol, 0.152 amol, and 0.051 amol. In addition, after the approximate MHC transcript quantity in a muscle had been defined, twofold serial dilutions of competitor across a more restricted range were used. Competitive PCR was performed three or more times for each isoform present in each individual muscle sample. The PCR cycle protocol consisted of 94°C for 30 s, 55°C for 30 s, and 72°C for 90 s, and this protocol was repeated for 33 cycles. beta -Actin and plasmid cDNA clones of each MHC were also amplified as positive controls. PCR reactions containing Taq polymerase, the primer combination but no cDNA or competitor were used as negative controls.

Quantification of PCR reaction products. All PCR reaction products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. Polaroid photographs were optically scanned at 300 dots/in., using an HP Deskscan II (Hewlett-Packard, Palo Alto, CA) and analyzed using the National Institutes of Health Image software (v.1.61) with the available gel macros. For quantitation, the positive reaction portion of each lane was outlined manually and scanned to generate an optical density vs. distance graph. Each peak was defined by manual editing, through identification of the peak baseline as well as the nadir between the two peaks. One peak within the scan represented the target MHC (t), whereas the other represented the competitor reaction product (c). Optical densities were linearly corrected for product length (Table 1). The logarithm of t/c was plotted as a function of the logarithm of known competitor concentration, and the resulting linear relationship was analyzed by linear regression (Statview 4.0, Abacus Concepts, Berkeley, CA). The regression equation was solved for the condition t = c [i.e., log(t/c) = 0] to yield the target DNA concentration. Absolute mass of each isoform (in g/mol) was explicitly calculated for each double-stranded DNA product using the known nucleotide sequence and the equation Mass = (A · 312.2) + (G · 328.2) + (C · 288.2) + (T · 303.2) - 61.0.

To estimate quantification error due to each step of the analysis, a 2 × 3 × 3 × 3 nested ANOVA was performed on a subgroup of data obtained from two tibialis anterior muscles for the type 2X isoform. Three separate reactions were performed on each muscle, yielding six gels. Each gel was scanned 3 times, peaks were defined on each scan 3 times, and each set of peaks was then analyzed 3 times, yielding a total of 36 data points (2 muscles × 3 gels/muscle × 3 peak sets/gel × 3 analyses/peak set). The total repeated measure coefficient of variation within each muscle was 18%, which was partitioned as 6.3% muscles, 3.8% scanning, 6.5% peak selection, and 1.4% analysis. The total 18% coefficient of variation was deemed acceptable, and the final protocol consisted of scanning each gel once, selecting peaks twice, and analyzing each peak set once, since the majority of the error within the analysis of a muscle consisted of peak selection. All data are reported in the text as means ± SE.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

MHC transcripts present in rat muscles. Initial screening of the muscles by PCR demonstrated, as expected, that the MHC transcripts were differentially expressed by the various muscles (Fig. 3). For example, the extraocular muscle was the only muscle to express all seven MHC transcripts. The thyroarytenoid muscle expressed 2A, 2B, 2X, EO, and neonatal isoform transcripts; the extensor digitorum longus, tibialis anterior, and diaphragm muscle expressed only 2A, 2B, 2X, and beta -cardiac transcripts; the soleus muscle expressed 2A, 2X, embryonic, and beta -cardiac transcripts; and the masseter expressed 2A, 2B, 2X, and neonatal MHC transcripts.


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Fig. 3.   MHC transcript isoform distribution in 7 muscles studied. Slowest contracting, most oxidative muscle is soleus, and fastest contracting, most glycolytic muscle is masseter.

The beta -actin PCR product was identified in all muscles (data not shown), and was expressed at an essentially constant concentration of 13.4 ± 0.3 amol. Thus it was deemed unnecessary to correct the MHC transcript levels to the beta -actin expression levels.

Quantification of MHC transcript levels. The MHC mRNA expression in extraocular muscle was characterized by approximately equal proportions of 2B (29.8 ± 1.4%), 2A (24.7 ± 4.3%), and 2X (21.0% ± 3.5) MHC isoforms, with much lower proportions of beta -cardiac (8.0 ± 0.3%), EO (7.4 ± 1.5%), neonatal (7.1 ± 0.3%), and embryonic (1.2 ± 0.5%) MHC transcripts (Figs. 2 and 3). The relatively high proportion of the type 2 isoforms in the extraocular muscle (~80%) supports the physiological evidence of its rapid contraction speed. However, the presence of all other isoforms is puzzling in light of the fairly restricted expression of MHC transcripts in most adult skeletal muscles. The thyroarytenoid muscle was characterized by approximately equal proportions of type 2B (44.3 ± 5.0%) and type 2X (39.8 ± 3.0%) MHC transcripts, with a lower percentage of 2A (8.0 ± 0.3%), neonatal (4.4 ± 0.4%), and EO (4.0 ± 0.7%) transcripts. Again, this muscle would be considered fast contracting, with a large number of isoforms expressed.

The extensor digitorum longus muscle contained >95% adult fast isoform MHC transcripts, types 2B (42.6 ± 3.5%), 2A (30.9 ± 4.1%), and 2X (24.4 ± 2.9%), but also contained very low levels of beta -cardiac MHC (2.3 ± 0.3%). The tibialis anterior contained approximately equal proportions of the adult fast isoforms, types 2A (39.4 ± 4.5%), 2X (36.0 ± 1.7%), and 2B (23.6 ± 4.9%), again with very low levels of beta -cardiac MHC (1.0 ± 0.3%). The soleus muscle, used in physiological experiments as the "classic slow-contracting muscle" contained, as expected, almost exclusively beta -cardiac (87.8 ± 2.6%), a small proportion of 2A (11.1 ± 1.3%) and 2X (0.5 ± 0.1%), and, unexpectedly, embryonic (0.6 ± 0.1%) MHC transcript. The diaphragm contained 2X (46.3 ± 5.6%), 2A (28.7 ± 4.2%), beta -cardiac (20.7 ± 3.8%), and 2B (4.3 ± 0.5%) MHC isoforms, whereas the masseter contained mainly type 2B MHC (73.7 ± 4.1%) and a small fraction of type 2X (16.3 ± 1.5%), type 2A (9.0 ± 2.0%), and neonatal (1.0 ± 0.2%) MHC transcripts.

Absolute mass of total MHC mRNA was calculated per 0.5 µg of total RNA with the assumption that reverse transcription efficiency was 50% (Table 2). Reverse transcription efficiency was not explicitly measured but was not expected to vary systematically between isoforms, since the poly-T primer was used for all reverse transcription reactions. Total MHC mRNA varied significantly between muscles. The highest MHC mRNA level was observed in the masseter muscle (106 ± 5.2 pg/0.5 µg RNA); this level was significantly greater (P < 0.05; Table 2) than those observed in the extraocular muscle (85.9 ± 3.7 pg/0.5 µg RNA) and thyroarytenoid muscle (85.6 ± 5.3 pg/0.5 µg RNA). All of these branchial arch muscles contained significantly more (P < 0.05; Table 2) MHC mRNA than did limb muscles, which contained 25-50 pg MHC mRNA/0.5 µg RNA.

                              
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Table 2.   MHC mRNA composition of seven adult rat skeletal muscles

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

The present study is the first to establish the quantity of each of the seven skeletal muscle MHC isoforms in muscles of both branchial arch and somatic origin using competitive PCR. These data clearly demonstrate that competitive PCR can differentiate among even small differences in MHC transcript amount, even though expression levels may be well below the detection levels achievable using Northern blots, in situ hybridization, and RNase protection assays. When substantial amounts of mRNA were present, the MHC composition calculated from these mRNA levels was similar to the MHC protein composition reported for those muscles in which protein levels have been determined (1, 10, 11, 28, 30). However, for the tibialis anterior muscle, some discrepancy between mRNA and protein data exists, since 68-77% of MHC is typically reported as 2B at the protein level (1, 10) but approximately equal proportions of 2A MHC (37.1%), 2B MHC (25.2%), and 2X MHC (36.7%) were detected by our analysis at the mRNA level. It is possible that this discrepancy is related to the large spatial heterogeneity of MHC expression within the tibialis anterior, because the deeper portion of tibialis anterior clearly has more 2A MHC and the superficial portion has more 2B (13). In addition, we detected small but highly reproducible levels of transcripts for which protein has not been detected, presumably due to the lack of sensitivity of immunoblots.

The results revealed several novel patterns of MHC expression. For example, neonatal MHC is found in rat limb muscles during development but by 3-4 wk of age cannot be detected in normal rats (23). However, neonatal MHC has been detected in adult extraocular muscle (22). In this regard, it is interesting to note that neonatal MHC transcripts were detected in all three of the adult branchial arch muscles studied (extraocular, masseter, and thyroarytenoid; Table 2). This result suggests that the developmental origin of a muscle may, at least in part, be responsible for the MHC expression program that is implemented in the adult.

One of the most interesting muscles studied was the extraocular, in that it contained all seven MHC isoforms (Figs. 2 and 3). Previous reports of adult limb muscles typically reveal expression of, at most, four adult isoforms (types 1, 2A, 2X, and 2B), with many muscles expressing only two to three isoforms. Thus the presence of all seven isoforms within extraocular muscle is intriguing, as it is believed that the presence of the different isoforms is a reflection of the functional requirements of a muscle (26). The extraocular muscle is required for precise and rapid positional control of the eye and thus would be expected to have a large proportion of fast isoforms. Indeed, ~80% of the MHC pool consisted of MHC transcripts of types 2A, 2X, 2B, and EO. The fact that they are present in equal proportions (assuming that this reflects the protein levels) suggests that the extraocular muscle is used across a wide range of tasks, since the type 2B isoform predominates in muscles that are used very briefly and infrequently (e.g., tibialis anterior), whereas the beta -cardiac isoform predominates in muscles that are used frequently (e.g., soleus). Even more intriguing is the presence of both embryonic and neonatal isoforms. These isoforms have been most frequently studied in the context of developmental regulation, and it has been shown that they disappear 15-20 days after birth (23). Thereafter, at least in adult limb muscles, these developmental isoforms are only reexpressed in the case of muscle injury and regeneration (9, 24, 25). Thus the stable presence of almost 10% of the total MHC transcript pool as developmental myosin is puzzling. This is unlikely to represent a constant state of muscle fiber turnover in these muscles due to repeated injury and repair. Alternatively, it may reflect the unique innervation pattern of this muscle. In normal motor units, one or two MHC isoforms may be expressed by fibers within a single motor unit and these MHC types are usually closely related (6). The large number of motor units present within extraocular muscle affords the opportunity for expression of a wide range of MHC isoforms. In addition, it is possible that precise control of extraocular muscle length imposed by the need for precise control of ocular orientation requires the expression of multiple MHC isoforms.

Interestingly, the type EO transcript represented 7.4% of the extraocular muscle MHC transcript pool and 4.0% of the thyroarytenoid muscle and was only detected in these two muscles. This may simply reflect the fact that these two tissues have a unique developmental origin compared with the other muscles studied, being derived from the branchial arch. Alternatively, it is possible that EO might be related in terms of expression to the neonatal isoform, since both EO and neonatal MHC transcripts are expressed in the extraocular and thyroarytenoid muscles, albeit as very small percentages. This argument does not explain why the EO MHC isoform is not expressed in the masseter muscle. This could relate to the small size of the extraocular and thyroarytenoid muscles.

The existence of type 2X MHC transcript at levels >15% in all muscles except soleus makes it the most common isoform present in the rat muscles studied and implies that this myosin is important in the function of fast-contracting muscles. This isoform was only recently determined to be distinct from types 2A and 2B, and current experimental evidence suggests that it can function as a transition form between these two isoforms during fiber type transformation (6). However, because the isoform distribution in normal adult muscle is stable under conditions of consistent use, type 2X MHC must serve a different function. Physiological data suggest that, in terms of contraction speed, the three fast isoforms are arranged in the order 2B > 2X > 2A (2), although the magnitudes of the differences in contraction speed are relatively small and may be modified by the presence of the myosin light chains. It is therefore not clear whether the multiple isoforms present represent a fine tuning of the functional requirements of the muscle or redundant expression of duplicated genes generated by parallel evolution (18, 19).

The absolute levels of MHC mRNA ranged from 25 (tibialis anterior and soleus) to 106 (masseter) pg/0.5 µg RNA. It is interesting that the three muscles of branchial arch origin all expressed relatively large amounts of these transcripts (85-106 pg/0.5 µg RNA) compared with limb muscles (25-50 pg/0.5 µg RNA). Indeed, the major isoform within the masseter muscle (type 2B) is present at a level over twice as great (206 pg/0.5 µg RNA) as the total MHC mRNA in any of the limb muscles studied. One interpretation of these differences is that the branchial muscles have a greater need for myosin synthesis.

The mass of MHC mRNA can be used to estimate the fraction of the total mRNA pool that represents MHC within skeletal muscle. Under the assumption that reverse transcription efficiency is 50% and that mRNA represents 2% of the total RNA pool within skeletal muscle, the percentage of MHC mRNA ranged from 4.1% (tibialis anterior) to 17.0% (masseter) of the total mRNA. These values suggest that MHC mRNA is relatively abundant, consistent with the obvious need for MHC within functioning skeletal muscle.

Finally, we noted an inverse relationship between the expression of beta -cardiac and type 2B MHC transcripts across the seven muscles that expressed both isoforms. Muscles that expressed high levels of beta -cardiac MHC mRNA expressed low levels of 2B MHC mRNA. For example, the soleus muscle, by far the slowest contracting when tested functionally, showed the highest proportion of beta -cardiac MHC isoform (88%) but had no detectable 2B MHC mRNA. Conversely, the masseter and thyroarytenoid muscles showed the highest proportions of 2B MHC mRNA (74 and 43%, respectively) but no detectable beta -cardiac MHC mRNA. The other muscles tested showed intermediate proportions of mRNAs of both isoforms. The relationship between beta -cardiac and 2B mRNA within each muscle tested was well fit by an exponential function (Fig. 4). Regression analysis indicated a highly significant (P < 0.0001) inverse correlation between the expression of these two isoforms. The linear regression model explained >80% of the experimental variability when the data were log transformed, indicating that the form of the inverse relationship is exponential. This inverse correlation may represent the differing functions of the tested muscles. Certainly the soleus is physiologically one of the slowest contracting muscles (7), whereas the thyroarytenoid muscle (15) and masseter (3) are very rapidly contracting muscles. The correlation also suggests that the expression of these two isoforms may be reciprocally regulated. Such a relationship was not observed between beta -cardiac MHC and type 2A or type 2X MHC.


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Fig. 4.   Relationship between expression of beta -cardiac MHC and type 2B MHC in 7 muscles studied. This relationship is described well by the equation log[type 2B] = 0.82 · log[beta -cardiac] + 1.857, where square brackets indicate concentration (r2 = 0.80, P < 0.0001). Muscles studied were diaphragm (Dia), extensor digitorum longus (EDL), EOM, masseter (Mass), soleus (Sol), tibialis anterior (TA) and thyroarytenoid muscle (THAR).

    ACKNOWLEDGEMENTS

We thank Chunyan Zhang and Bill Blevins for technical assistance and Dr. Gordon Lutz and Qing Dallas-Yang for helpful comments.

    FOOTNOTES

This study was supported by Korea University, the Research Service of the Dept. of Veterans Affairs, and National Institutes of Health Grants AR-40050, DC-00139, and DC-00129.

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. §1734 solely to indicate this fact.

Address for reprint requests: R. L. Lieber, Dept. of Orthopaedics (9151), UCSD School of Medicine and VA Medical Center, 3350 La Jolla Village Dr., San Diego, CA 92161.

Received 16 January 1998; accepted in final form 27 March 1998.

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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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