Quantification of myosin heavy chain mRNA in somatic
and branchial arch muscles using competitive PCR
HAK HYUN JUNG,1,2 RICHARD L. LIEBER,3 AND ALLEN F. RYAN1,4
of Surgery/Otolaryngology, 3Departments of Orthopedics and Bioengineering, and
4Department of Neuroscience, University of California at San Diego School of Medicine and
Veterans Affairs Medical Center, La Jolla, California 92093; and 2Department of Ear, Nose,
and Throat/Head and Neck Surgery, Korea University, Seoul, Korea
1Department
Jung, Hak Hyun, Richard L. Lieber, and Allen F.
Ryan. Quantification of myosin heavy chain mRNA in somatic and branchial arch muscles using competitive PCR.
Am. J. Physiol. 275 (Cell Physiol. 44): C68–C74, 1998.—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 (b-cardiac, 2A, 2X, 2B, extraocular, embryonic, and
neonatal) as well as b-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 b-cardiac MHC (87.8 6
2.6%). Extraocular MHC was found only in the extraocular
and thyroarytenoid muscles and in relatively small proportions (7.4 6 1.5% and 4.0 6 0.7%, respectively). Neonatal
MHC was identified in extraocular (7.9 6 0.3%), thyroarytenoid (4.4 6 0.4%), and masseter (1.0 6 0.2%) muscles, and
embryonic MHC was identified both in extraocular (1.2 6
0.5%) and, unexpectedly, in soleus (0.6 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 b-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
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 a- and b-cardiac isoforms (14,
16). These isoforms differ in their functional properties
and are expressed in a tissue-specific and developmen-
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C68
tally 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 (10218 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.
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MYOSIN HEAVY CHAIN QUANTIFICATION BY COMPETITIVE PCR
Fig. 1. Schematic construct of competitor used in current study. Nucleotide sequence of consensus sense primer
(CSP) is AGAAGGCCAAGAAAGCCAT instead of a degenerate primer. b-Car, b-cardiac; EO, extraocular; Neo,
neonatal; Emb, embryonic. MHC, myosin heavy chain.
MATERIALS AND METHODS
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 38 end with a short sequence that is
highly conserved between all sarcomeric MHC genes (13). A
b-actin sense primer was ligated to the 58 end to enable
measurement of a constitutively expressed product for internal calibration. Then, eight antisense primers [specific to
b-actin and to the 38 untranslated region of the EO (also
known as type 2L; unpublished observations and Ref. 17), 2B,
2X, b-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).
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 38
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).
RT-PCR and competitive PCR of rat muscle. Adult SpragueDawley 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 (2159°C) and
stored for subsequent analysis at 280°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 (10215 mol), 333 amol (10218
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. b-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 regres-
Table 1. Oligonucleotide sequences and expected PCR product lengths
Expected PCR Product Length, bp
Oligonucleotide Sequences
Sense primers
Consensus
b-Actin
Antisense primers
b-Actin
2A
2B
2X
EO
Neonatal
Embryonic
b-Cardiac
Target isoform
Competitor
Target Mass, g/mmol
300
644
624
645
605
658
659
629
419
519
458
478
437
539
558
498
187.2
398.1
385.7
398.7
373.2
406.6
407.3
388.6
58-AGAAGGCCAARAARGCCAT-38
58-GAAACTACATTCAATTCCATC-38
58-CTAGAAGCATTTGCGGTGCACGATGGAG-38
58-TTACAATAGGATTAAATAGAA-38
58-TTGATATACAGGACAGTGACA-38
58-TTTTTTATCTCCCAAAGTCG-38
58-CCCAGTCTCCCTCTGCTCT-38
58-AGTCAGCAGTGGGAGAAAAG-38
58-ATGTGGAAAGGGGTTACGT-38
58-TTTCTGCCTAAGGTGCTGT-38
R within nucleotide sequence is degeneracy symbol for A or G nucleotides. EO, extraocular muscle type myosin heavy chain (MHC).
C70
MYOSIN HEAVY CHAIN QUANTIFICATION BY COMPETITIVE PCR
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 b-cardiac myosin heavy chain (MHC) are targets,
and lower bands are competitors, whereas bands for b-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).
sion equation was solved for the condition t 5 c [i.e., log(t/c) 5
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 5 (A · 312.2) 1 (G · 328.2) 1
(C · 288.2) 1 (T · 303.2) 2 61.0.
To estimate quantification error due to each step of the
analysis, a 2 3 3 3 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 3 gels/muscle 3 3 peak
sets/gel 3 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 6 SE.
RESULTS
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
b-cardiac transcripts; the soleus muscle expressed 2A,
2X, embryonic, and b-cardiac transcripts; and the
masseter expressed 2A, 2B, 2X, and neonatal MHC
transcripts.
The b-actin PCR product was identified in all muscles
(data not shown), and was expressed at an essentially
constant concentration of 13.4 6 0.3 amol. Thus it was
deemed unnecessary to correct the MHC transcript
levels to the b-actin expression levels.
Quantification of MHC transcript levels. The MHC
mRNA expression in extraocular muscle was character-
MYOSIN HEAVY CHAIN QUANTIFICATION BY COMPETITIVE PCR
C71
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.
ized by approximately equal proportions of 2B (29.8 6
1.4%), 2A (24.7 6 4.3%), and 2X (21.0% 6 3.5) MHC
isoforms, with much lower proportions of b-cardiac
(8.0 6 0.3%), EO (7.4 6 1.5%), neonatal (7.1 6 0.3%),
and embryonic (1.2 6 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 6 5.0%) and type 2X
(39.8 6 3.0%) MHC transcripts, with a lower percentage of 2A (8.0 6 0.3%), neonatal (4.4 6 0.4%), and EO
(4.0 6 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 6 3.5%), 2A (30.9 6 4.1%), and 2X (24.4 6 2.9%),
but also contained very low levels of b-cardiac MHC
(2.3 6 0.3%). The tibialis anterior contained approximately equal proportions of the adult fast isoforms,
types 2A (39.4 6 4.5%), 2X (36.0 6 1.7%), and 2B
(23.6 6 4.9%), again with very low levels of b-cardiac
MHC (1.0 6 0.3%). The soleus muscle, used in physiological experiments as the ‘‘classic slow-contracting
muscle’’ contained, as expected, almost exclusively
b-cardiac (87.8 6 2.6%), a small proportion of 2A
(11.1 6 1.3%) and 2X (0.5 6 0.1%), and, unexpectedly,
embryonic (0.6 6 0.1%) MHC transcript. The diaphragm contained 2X (46.3 6 5.6%), 2A (28.7 6 4.2%),
b-cardiac (20.7 6 3.8%), and 2B (4.3 6 0.5%) MHC
isoforms, whereas the masseter contained mainly type
2B MHC (73.7 6 4.1%) and a small fraction of type 2X
(16.3 6 1.5%), type 2A (9.0 6 2.0%), and neonatal (1.0 6
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 6 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 6 3.7 pg/0.5 µg RNA) and
thyroarytenoid muscle (85.6 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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MYOSIN HEAVY CHAIN QUANTIFICATION BY COMPETITIVE PCR
Table 2. MHC mRNA composition of seven adult rat skeletal muscles
MHC Transcript Isoform, amol/0.5 µg RNA
Muscle
2A
2B
2X
EO
Neonatal
Embryonic
b-Cardiac
Total MHC
Mass, pg/0.5 µg RNA
EOM
THAR
EDL
TA
Sol
Dia
Mass
53.5 6 .63
17.0 6 2.0
37.8 6 2.9
23.8 6 1.6
17.4 6 1.0
36.5 6 2.5
22.0 6 2.9
68.5 6 1.3
97.5 6 6.5
59.0 6 2.8
16.2 6 0.17
ND
6.2 6 0.36
206 6 8.0
45.5 6 4.3
86.1 6 3.9
32.2 6 0.81
23.6 6 0.64
0.61 6 0.08
63.9 6 4.5
43.5 6 2.2
17.6 6 2.1
9.4 6 0.86
ND
ND
ND
ND
ND
15.2 6 0.37
8.5 6 0.39
ND
ND
ND
ND
2.5 6 0.29
2.3 6 0.52
ND
ND
ND
0.39 6 0.04
ND
ND
16.3 6 0.35
ND
3.0 6 0.19
0.65 6 0.11
82.5 6 1.8
27.5 6 2.9
ND
85.9 6 3.7
85.6 6 5.3
51.8 6 2.6
25.4 6 1.0
25.2 6 0.9
53.1 6 4.1
106 6 5.2
Values are means 6 SE. EOM, extraocular muscle; THAR, lateral aspect of the thyroarytenoid; EDL, extensor digitorum longus; TA, tibialis
anterior, Sol, soleus; Dia, diaphragm; Mass, masseter. Total MHC mass per 0.5 µg total RNA was calculated with assumption of 50% reverse
transcription efficiency. ND, not detected.
DISCUSSION
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
b-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
Fig. 4. Relationship between expression of b-cardiac MHC and type
2B MHC in 7 muscles studied. This relationship is described well by
the equation log[type 2B] 5 0.82 · log[b-cardiac] 1 1.857, where
square brackets indicate concentration (r2 5 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).
MYOSIN HEAVY CHAIN QUANTIFICATION BY COMPETITIVE PCR
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
C73
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 b-cardiac and type 2B MHC transcripts
across the seven muscles that expressed both isoforms.
Muscles that expressed high levels of b-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 b-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 b-cardiac MHC mRNA. The other muscles
tested showed intermediate proportions of mRNAs of
both isoforms. The relationship between b-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
b-cardiac MHC and type 2A or type 2X MHC.
We thank Chunyan Zhang and Bill Blevins for technical assistance
and Dr. Gordon Lutz and Qing Dallas-Yang for helpful comments.
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.
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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