Faculty of Biology, University of Konstanz, D-78457 Constance, Germany
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ABSTRACT |
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To elucidate the
sequence of myosin heavy chain (MHC) transitions in fast-to-slow
transforming rabbit muscle, direct reverse transcriptase-polymerase
chain reaction was applied for detecting mRNAs specific to five MHC
isoforms in single fibers from control and low-frequency-stimulated
tibialis anterior muscles. The detection of MHCIIb, MHCIId(x), MHCI,
and MHCI
mRNAs was based on previously published methods. The RT-PCR
assay for MHCIIa mRNA was based on the identification of a cDNA
sequence in the 3'-region from which specific primers were
derived. Comparisons between rat, rabbit, and human MHCIIa sequences
revealed high degrees of sequence identities. MHC mRNA isoform patterns
in single fibers from stimulated muscles showed hybrid fibers
expressing the following combinations: MHCIId(x) + MHCIIa,
MHCIId(x) + MHCIIa + MHCI
, MHCIId(x) + MHCIIa + MHCI
+ MHCI
,
MHCIIa + MHCI
, MHCIIa + MHCI
+ MHCI
, and MHCI
+ MHCI
. The combination MHCIIa + MHCI
without MHCI
was never seen. These coexpression patterns suggest that the fast-to-slow fiber
transition results from sequential isoform expressions in the order
MHCIId(x)
MHCIIa
MHCI
MHCI
. The
allocation of MHCI
between MHCIIa and MHCI
seems to be in line
with graded differences in sequence identity of the 3'-regions of
these mRNA isoforms.
fiber type transformation; MHCIIa sequence; rabbit skeletal muscle; reverse transcriptase-polymerase chain reaction; single fiber analysis
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INTRODUCTION |
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SKELETAL MUSCLES are composed of a variety of fiber
types differing, among other properties, in their contractile speeds. As shown by single fiber analysis, differences in contraction velocity
correlate with differences in the myosin heavy chain (MHC) complement.
According to measurements on pure fibers from limb muscles of small
mammals, contractile velocity decreases in the order MHCIIb > MHCIId(x) > MHCIIa > MHCI (2, 11, 25), the latter thought to
correspond to the -cardiac MHC isoform (17). Moreover, muscle fibers
exhibit a marked capability of changing their phenotype in response to
altered functional demands, changes in neuromuscular activity, or
hormonal signals (for review see Ref. 19). This phenotypic plasticity
has been convincingly documented by the effects of chronic
low-frequency stimulation (CLFS; see Ref. 20). Stimulation-induced
fast-to-slow isoform transitions of myosin light and heavy chains and
other myofibrillar proteins lead to sequential fast-to-slow fiber-type
transitions (15, 16). In rabbit tibialis anterior (TA) muscle
displaying MHCIId(x) as the predominant fast isoform, the
transitions in MHC expression follow the order MHCIId(x)
MHCIIa
MHCI (1). In rat TA muscle with MHCIIb as the
predominant fast isoform, the fast-to-slow conversion is initiated by
an MHCIIb
MHCIId(x) transition (1).
We have recently shown at both the mRNA (22) and protein levels (13)
that the ultimate step of the fast-to-slow transition in rabbit muscle
encompasses the expression of an -cardiac-like MHC isoform (13, 22).
An
-cardiac-like MHC had previously been detected only in rabbit
extraocular and masticatory muscles (5, 7, 8, 14, 24, 26). Using a
reverse transcriptase (RT)-polymerase chain reaction (PCR) protocol, we
were able to demonstrate low amounts of an
-cardiac-like MHC mRNA
also in several limb muscles of rabbit. A 140-fold upregulation of this isoform, designated as MHCI
, was observed in RNA preparations from
TA muscles exposed to CLFS for 60 days. The CLFS-induced rise of this
isoform was first observed by 20 days and occurred in parallel with a
steep increase of the mRNA specific to MHCI (in the following
designated as MHCI
; see Ref. 22).
The upregulation of MHCI and MHCI
in 20-day stimulated muscles
raised the question as to the precise positions of both mRNAs within
the sequence of MHC isoform transitions. Does the upregulation of
MHCI
occur in parallel with MHCI
or does it occur independently, does it occur in same or different fibers, and finally, does it precede
or follow that of MHCI
? The answer to these questions is complicated
by the fact that skeletal muscles exhibit a mixed-fiber-type composition. Also, when interanimal differences in fiber-type composition and fiber-type-specific responses to the imposed
contractile activity during fast-to-slow transformation are taken into
account, mRNA analyses on whole muscle preparations cannot be expected to yield unambigous answers. We therefore set out to investigate in the
present study in single fiber fragments from control and low-frequency-stimulated muscles mRNA and protein expression patterns of MHCI
and MHCI
, as well as of the other sarcomeric MHC
isoforms. This approach made it possible to study the distribution of
MHCI
and MHCI
in individual fibers, to elucidate their
coexistence, and thus to derive their position within the sequence of
fast-to-slow MHC transitions from their combinations with other MHC
isoforms.
The analysis of the various MHC mRNA isoforms was based on a highly
sensitive and reproducible direct RT-PCR assay (23). Because sequence
information for the fast MHC mRNA isoforms in rabbit muscle is as yet
limited to MHCIIb and MHCIId(x) (23), it was necessary to identify as
an important prerequisite for our study a sequence specific to the
third fast isoform, MHCIIa. Using a modified oligo(dT) primer and a
rat-specific sense primer for RT-PCR, we were able to identify a
sequence specific to the 3'-region of the rabbit MHCIIa mRNA and,
thus, included a total of five MHC mRNA isoforms, namely MHCIIb,
MHCIId(x), MHCIIa, MHCI, and MHCI
, in our study. A
sequence specific to
-skeletal actin mRNA served as an endogenous
reference unit.
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MATERIALS AND METHODS |
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Animals and muscles. Contralateral (control) and low-frequency-stimulated TA muscles were obtained from adult male New Zealand White rabbits. To collect fibers at different stages of the transformation process, TA muscles were obtained from animals exposed to CLFS for 10, 20, 30, and 50 days, using the same stimulation protocol (10-Hz, 0.2-ms impulse width, 12 h daily) previously described (15). Two animals were studied for each time point. The animals were anesthetized by intravenous injection of Nembutal and transferred to a surgical table, and TA muscles from contralateral and stimulated legs were dissected for subsequent freeze-clamping, using aluminum blocks cooled in liquid nitrogen. The frozen muscles were cut longitudinally into two halves, one for single fiber dissection and the other for studies on homogenates. The contralateral unstimulated TA muscles served as controls.
Dissection of single fibers and classification
according to MHC complement. The frozen muscles were
split longitudinally in a cryostat (25°C) to yield thin
fiber bundles. These were transferred to precooled aluminum holders and
freeze-dried at
38°C. Single fibers (5-15 mm long) were
isolated at random by freehand dissection under a stereomicroscope.
Small pieces were cut freehand from individual fibers, weighed on a
quartz fiber balance, and analyzed for mRNA and protein complements.
The pieces for direct RT-PCR ranged from 50 to 100 ng, and those for
protein analysis ranged from 150 to 200 ng dry weight. The fibers were
typed according to their MHC isoform complement. Two fragments of each
fiber were extracted (30), and MHC isoforms were electrophoretically
separated in a homogeneous sodium dodecyl sulfate-polyacrylamide gel
system as previously described (12). This system was used for
electrophoresis of single fiber fragments and muscle homogenates.
Because of similar or identical mobilities of MHCI
and MHCI
,
these two isoforms could not be distinguished by protein
electrophoresis. Therefore, their common electrophoretic band was
designated as MHCI.
Direct RT-PCR on muscle fiber
fragments. At least two pieces from each fiber were
investigated. An oil well technique was used for mRNA analysis by
direct RT-PCR (23). Briefly, the fiber fragment was transferred into
0.28 µl of a high-salt extraction medium [50 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl (pH 9.0), 250 mM KCl, 10 mM MgCl2, 10 mM dithiothreitol, and 1 U/µl human placenta ribonuclease inhibitor
(Boehringer Mannheim), complemented with 5 mM ribonucleoside vanadyl
complexes (Sigma)] under mineral oil and incubated for 60 min at
4°C to allow extraction of total RNA. Subsequently, the assay
mixture was diluted to yield optimum conditions for reverse
transcription at 42°C by adding 0.86 µl of the following
solution: 50 mM Tris · HCl (pH 9.0), 10 mM
MgCl2, 10 mM dithiothreitol, 1.3 mM dNTP, 2.5 µM oligo(dT)15
primers, and 1 U/ml RT from avian myeloblastosis virus (Boehringer
Mannheim). The assay was transferred into 30 µl PCR buffer (1×
buffer, Expand High-Fidelity PCR System; Boehringer Mannheim). Three
microliters were used as template for separate PCR assays (25-µl
volume) of the six sequences. The PCR medium consisted of (final
concentrations) 1× Expand buffer, 0.25 mM of each dNTP, 0.12 µM
of each primer, and 0.8 units of Expand polymerase (Boehringer
Mannheim). MgCl2 concentrations
were 2 mM for MHCIIb, MHCIIa, MHCI, and actin and 3.5 mM for
MHCIId(x) and MHCI
. To ascertain that amplification from
contaminating DNA did not occur, control assays were run in the absence
of RT. The PCR products were detected after 33 cycles for
-skeletal
actin and 36 cycles for the MHC isoforms. To estimate possible
differences in efficiency of the amplifications, purified PCR products
(103 molecules) were run in
parallel assays for each sequence. After the amplification, 2.0 µl of
each of the six assays performed on a single fiber fragment were
combined, subjected to electrophoresis, and visualized by silver
staining or ethidium bromide (21).
Oligonucleotide primers for MHCIIb, MHCIId(x), MHCI, and
-skeletal actin were the same as previously described (23)
[MHCIIb, GenBank accession no. X05958; MHCIId(x), GenBank
accession no. U32574; and MHCI
, GenBank accession no. J00672].
The primers for the detection of the MHCI
(GenBank accession no. K01867) were the same previously reported (22). With the exception of
MHCI
, all primers for the MHC mRNA isoforms were derived from the
3'-region: MHCIIb sense primer, AGA GGC TGA GGA ACA ATC CA
and antisense primer, ACT TGA TGC ACA AGG TAG TG;
MHCIId(x) sense primer, ACT GCA AGC CAA GGT GAA AT and
antisense primer, TTA TCT CCC AGA ATC ATA AG; MHCIIa sense primer, CAC
AAA TCT ATC TAA ATT CC and antisense primer, TCC TTT GCA GTA GGG TAG;
MHCI
sense primer, GGA TCC CTG GAG CAG GAG AA and antisense primer, CTT GCA TTG AGG GCA TTC AG; MHCI
sense primer, TCA AGG CCT ACA AGC
GCC AG and antisense primer, TTG CGG GTT AAC AAG AGC GG;
-skeletal actin (GenBank accession no. J00692) sense primer, CGC GAC ATC AAA GAG
AAG CT and antisense primer, GGG CGA TGA TCT TGA TCT TC. These primers
yielded PCR products of 249, 289, 240, 173, 227, and 367 nucleotides
(nt), respectively.
Identification of a cDNA sequence specific to rabbit MHCIIa mRNA 3'-region. Total RNA was prepared from whole muscle powder of 20-day-stimulated rabbit TA muscle (6), and RNA concentration was determined spectrophotometrically. The poly(A)+ fraction of 1 µg of total RNA was reverse transcribed as previously described (22), using avian myoblastosis virus RT and the 37-mer primer CAT TAT GCT GAG TGA TAT CCC GTT TTT TTT TTT TTT T. This primer served for introducing a specific 3'-sequence into the synthesized first-strand cDNA, following the protocol for 3'-end amplification of cDNAs (10; Fig. 1). One microliter of the RT assay was amplified using a standard PCR reaction mixture (21) containing 2 mM MgCl2 and the following primers: sense, TAT CCT CAG GCT TCA AGA TTT G, specific to the rat MHCIIa sequence (GenBank accession no. L13606) in the 3'-translated region and antisense, CAT TAT GCT GAG TGA TAT CCC G, the specific sequence of the primer for cDNA synthesis. Optimum product yield and specificity were obtained with an annealing temperature of 56°C. The resulting major product was purified by gel extraction and reamplified with the same primers to yield a specific PCR product. Sequence analysis was performed on both strands of the PCR product from two independent RT-PCRs. The primers for the amplification of a 240-nt product from the rabbit MHCIIa mRNA were chosen from the sequence data. Amplification was performed at 2 mM MgCl2, and annealing temperature was 58°C.
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RESULTS |
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Identification of a cDNA sequence specific to the
rabbit MHCIIa mRNA 3'-region. Sequence
information was available for rabbit MHCIIb, MHCIId(x), MHCI, and
MHCI
(22, 23). To investigate the complete set of sarcomeric MHC
isoforms in rabbit muscle, it was necessary to establish an RT-PCR
assay for the MHCIIa mRNA. Because several attempts failed to
amplify a specific rabbit sequence with primer pairs derived from
published sequences specific to rat MHCIIa mRNA, the following strategy
was applied (Fig. 1): the
poly(A)+ fraction of rabbit RNA
was reverse transcribed with a modified oligo(dT) primer, introducing
an MHC-unrelated sequence to the 3'-region of the synthesized
first-strand cDNA. This sequence served a specific annealing site for
the antisense primer in the subsequent PCR, allowing the amplification
of a rabbit MHCIIa sequence in the 3'-region in combination with
a rat-specific sense primer. This procedure yielded a major product of
~450 nt. To test the specificity of this product, we used the
gel-extracted fragment as a template for amplifications with primers
specific to the other rabbit MHC isoforms. No specific products were
formed with primers for MHCIIb, MHCIId(x), MHCI
, and MHCI
,
indicating that the selected rat-specific primer did not anneal to
isoforms other than MHCIIa mRNA.
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After sequence analysis of the amplified fragment, a homology search
with regard to sequences for human, rabbit, and rat (EMBL and GenBank)
yielded the following results. The identities between our fragment and
the corresponding regions of human and rat MHCIIa amounted to
89 and 84%, respectively (Fig. 2).
Compared with rabbit MHCIId(x) and MHCIIb 3'-regions, the
identities were 86 and 81%, respectively. Smaller identities existed
for MHCI and MHCI
, amounting to only 75 and 64%, respectively.
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These results strongly indicated that the amplified fragment was specific to a segment of rabbit MHCIIa mRNA consisting of the 3'-untranslated region and extending into the translated region. On the basis of this sequence data, a new primer pair specific to the rabbit MHCIIa mRNA was selected (Fig. 2), which yielded a product of 240 nt. The specificity of the product was verified by restriction analysis (Fig. 3), yielding fragments of expected lengths after digestion with Sau I (146 and 94 nt), Hae III (47 and 193 nt), and Alu I (21, 11, and 208 nt). The restriction site for Sau I proved to be unique for rabbit MHCIIa. In addition, sequence analysis proved the specificity of the product.
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Confirmation of the MHCIIa specificity by direct
RT-PCR on electrophoretically identified fiber
fragments. To further confirm the isoform specificity
of the obtained sequence, we investigated the MHC mRNA complement of
pure fiber types from control muscles, identified according to their
electrophoretically assessed MHC protein isoforms. Pure fiber types
containing MHCIId(x) (n = 6), MHCIIa
(n = 22), or MHCI
(n = 4) displayed single
signals of 289, 240, and 173 nt, respectively (Fig.
4). No pure fibers of the MHCIIb protein
phenotype were detected, which is in agreement with previous
observations on rabbit TA muscle (1). Also, no hybrid fibers displaying
the MHCIIa + MHCI protein phenotype were found in control TA muscles.
All investigated hybrid fibers characterized at the protein level by
coexistence of MHCIId(x) and MHCIIa (n = 15) displayed both the 289 and 240 nt signals (Fig. 4). Many fibers
(15 out of 22) containing only the MHCIIa protein displayed, in
addition to the MHCIIa mRNA, the signal for MHCIId(x) mRNA. However,
fibers from control TA muscle (n = 52)
never yielded the 227-nt signal specific to MHCI
mRNA. A distinct
signal for
-skeletal actin mRNA (367 nt) was detected in all pure
and hybrid fibers. Taken together, mRNA and protein analyses on
fragments of the same fibers confirmed the specificity of the selected
primer pair for the newly established MHCIIa sequence.
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MHC isoform expression in single muscle fibers during fast-to-slow conversion. Electrophoretic analyses of homogenates from muscles exposed to CLFS for different time periods showed fast-to-slow transitions in the MHC isoform pattern (data not shown) similar to those previously described (15). To study sequential changes in myosin expression during the fast-to-slow conversion, fibers displaying specific MHC protein isoform patterns were selected for mRNA analysis. The studies at the protein level revealed a pronounced expansion of the hybrid fiber population. Two major protein patterns of coexisting MHC isoforms were detected [MHCIId(x) + MHCIIa and MHCIIa + MHCI], the latter corresponding to the so-called C fibers (27). Although the first combination was also observed in fibers from control TA muscles, the latter obviously represented a major transitory state during the induced fast-to-slow conversion.
Similar to the findings on control muscle, hybrid fibers in stimulated muscles, as defined by MHC mRNA analysis, were more numerous than defined by MHC protein studies. In addition, mRNA analysis revealed a larger spectrum of coexpressed MHC isoforms than detected in the same fibers by protein electrophoresis. Hybrid fibers from stimulated TA muscles contained up to four MHC mRNA isoforms, whereas only two isoforms were seen in hybrid fibers from control TA muscles (Figs. 4 and 5). The expansion of the hybrid fiber population became evident already in 10-day stimulated muscles and tended to increase with prolonged stimulation.
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A large fraction of hybrid fibers displayed the 227-nt signal specific
to the MHCI mRNA isoform. The following combinations were
distinguished in single fibers from stimulated muscles (Figs. 5 and
6): 1)
MHCIId(x) + MHCIIa; 2) MHCIId(x) + MHCIIa + MHCI
; 3) MHCIId(x) + MHCIIa + MHCI
+ MHCI
; 4)
MHCIIa + MHCI
; 5) MHCIIa + MHCI
+ MHCI
; and 6) MHCI
+ MHCI
.
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In contrast to control muscle, fibers from transforming muscles exhibited less correspondence between mRNA and protein patterns and represented a markedly heterogeneous population. Generally, the fast-to-slow transition appeared to be more advanced at the mRNA level than at the protein level. A detailed analysis of the mRNA patterns in fibers of defined protein phenotypes is given in Tables 1-3. Fibers characterized by their unique MHCIIa protein complement were found to display, in addition to the pure MHCIIa mRNA complement, up to four different coexpression patterns (Table 1). A similar heterogeneity was found for hybrid fibers of the MHCIId(x) + MHCIIa protein phenotype (Table 2) and the so-called C fiber population (Table 3).
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Of special interest was the finding that MHCI mRNA was present in
all C fibers and in 70% of the investigated fibers uniquely displaying
MHCIIa protein. MHCI
mRNA was also detected in fibers with the
MHCIId(x) + MHCIIa protein complement. In all fibers examined, MHCI
was never detected as the unique mRNA isoform but was found in
combination with MHCIIa, with MHCIIa + MHCIId(x), with MHCI
, with
MHCIIa + MHCI
, or in combination with all of these mRNA isoforms.
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DISCUSSION |
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The analysis of MHC mRNA and protein isoforms in single fibers from
low-frequency-stimulated muscles is a suitable approach for defining
the position of individual MHC isoforms in their sequential expression
during induced fiber-type transitions. Previous studies on protein
extracts from low-frequency-stimulated rabbit TA muscles suggested a
sequential expression of MHCIId(x) MHCIIa
MHCI (15).
The detection of the MHCI
isoform during this process (13, 22)
raises the question of its allocation in this sequence. Our previous
mRNA data obtained from whole muscle analyses suggested that this
isoform was induced shortly before or in synchrony with MHCI
in
muscles stimulated for >20 days (22). In this conjunction, it is of
interest whether the transition from MHCIIa to MHCI
occurs directly
or includes the expression of MHCI
. Analyses at the single fiber
level and the elaboration of the RT-PCR for MHCIIa mRNA were major
prerequisites for answering these questions.
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The results of the present study suggest the following sequence of MHC
isoform expression during fastto-slow transition in rabbit muscle:
MHCIId(x) MHCIIa
MHCI
MHCI
.
This sequence follows from the various combinations of MHC mRNA
isoforms in individual fibers of transforming muscle (Table
4). The allocation of MHCI
between MHCIIa and MHCI
matches the failure to detect a direct transition from MHCIIa to
MHCI
, which would postulate the MHCIIa + MHCI
combination in the absence of MHCIa. However, this combinatorial
pattern was never seen in the fibers under study. On the other hand,
the combinations of MHCIIa + MHCI
and of MHCI
+ MHCI
mRNAs
assign MHCI
as intermediate between MHCIIa and MHCI
. Its
intermediate position is further supported by the coexpression of three
or even four isoforms extending from MHCIId(x) to MHCI
or MHCI
in
some fibers and from MHCIIa to MHCI
in others. The existence of the
combination MHCIIa + MHCI
without MHCI
seems to be relevant also
with regard to the time point of the induction of MHCI
. Its
induction appears to precede the expression of MHCI
, suggesting that
both isoforms are regulated independently.
The finding that the expression patterns of various MHC isoforms differ in transforming fibers may be explained by the fact that TA muscle is composed of various fiber types. Thus, depending on the fiber type, the stimulation-induced fast-to-slow conversion starts at different points of the MHC isoform spectrum. The observation that fibers defined by identical protein patterns display up to five different expression patterns of MHC mRNA isoforms indicates that fibers of the same type may not respond in a uniform manner to the imposed contractile activity.
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The coexpression of more than two MHC mRNA isoforms in transforming
fibers is not unexpected in view of our previous observation that a
large fraction of fibers in control rabbit muscles contains two MHC
mRNA isoforms (23). Coexpression always comprises isoforms identified
as next neighbors as deduced from measurements of contractile properties or adenosinetriphosphatase (ATPase) activities in single fiber studies (3, 4, 11). Thus combinations such as MHCIId(x) + MHCI or
MHCIIa + MHCI were not observed. The coexistence of several MHC
isoforms in fibers transforming under the influence of forced
contractile activity may relate to various factors, e.g., at the mRNA
level to differences in transcriptional rates or stability and at the
protein level to differences in turnover rates. This may lead to an
overlap of different isoforms during their sequential expression. In
addition, heterogeneous expression of MHC isoforms along muscle fibers
may occur. Nonuniform myosin expression at the mRNA level has been
shown in control rabbit muscle fibers (23). As revealed by quantitative
myofibrillar actomyosin ATPase histochemistry, this heterogeneity is
greatly enhanced during CLFS-induced fiber-type transformation (29). Such nonuniform distribution might explain some of the differences between MHC isoform patterns at the mRNA and protein levels.
Differences in mRNA and protein turnover rates might also contribute to
the observed discrepancies.
Our findings seem to be relevant also with regard to the C fiber
population. It is commonly accepted that these fibers represent the
MHCIIa + MHCI protein phenotype (9, 18, 27, 28). We now show at the
mRNA level that, in transforming rabbit muscle, MHCI
represents an
additional isoform present in these fibers. Although no data exist on
the contractile properties of MHCI
-containing fibers in limb
muscles, the suggested intermediate position of MHCI
between MHCIIa
and MHCI
seems to be supported by the functional properties of
MHCI
-containing fibers in rabbit masseter muscle. Such fibers have
been shown to be slower contracting than MHCIIa-containing fibers but
are faster than fibers displaying the MHCI
complement. MHCI
would
thus bridge a functionally large gap between the fast MHCIIa and the
slow MHCI
(14, 26). According to immunohistochemical studies, hybrid
fibers of the masseter muscle contain only combinations of MHCIIa + MHCI
or MHCI
+ MHCI
(14). In this conjunction, it is of
interest that, according to sequence alignment of the 3'-regions,
MHCIIa is more similar to MHCI
than to MHCI
.
The upregulation of MHCI in the transforming muscle is remarkable
because this MHC mRNA isoform is present in normal fast-twitch and
slow-twitch limb muscles of rabbit only at very low levels (22). Also,
MHCI
mRNA was not detected in single fibers from control TA muscle
(present study). Its pronounced elevation in transforming muscle
correlates with the expansion of the C fiber population (27). All C
fibers studied contain MHCI
mRNA. Its upregulation thus occurs when
the major fraction of fast-twitch fibers converts into slow-twitch
fibers, passing through the stage of C fibers where MHCI
may bridge
the gap between MHCIIa and MHCI
.
In summary, we report on the identification of the 3'-region of
the rabbit MHCIIa mRNA isoform and the elaboration of a direct RT-PCR
for this isoform in single fibers. On this basis, the complete sequence
of MHC mRNA isoform transitions during induced fast-to-slow conversion
could be elucidated. Our results reveal sequential exchanges in the
order MHCIId(x) MHCIIa
MHCI
MHCI
.
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ACKNOWLEDGEMENTS |
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We thank Nina Hämäläinen and Michael Schuler for helpful suggestions.
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FOOTNOTES |
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This study was supported by Deutsche Forschungsgemeinschaft, SFB 156.
Address reprint requests to H. Peuker.
Received 7 August 1997; accepted in final form 14 November 1997.
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