alpha -Cardiac-like myosin heavy chain as an intermediate between MHCIIa and MHCIbeta in transforming rabbit muscle

Heidemarie Peuker, Agnès Conjard, and Dirk Pette

Faculty of Biology, University of Konstanz, D-78457 Constance, Germany

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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), MHCIalpha , and MHCIbeta 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 + MHCIalpha , MHCIId(x) + MHCIIa + MHCIalpha  + MHCIbeta , MHCIIa + MHCIalpha , MHCIIa + MHCIalpha  + MHCIbeta , and MHCIalpha + MHCIbeta . The combination MHCIIa + MHCIbeta without MHCIalpha was never seen. These coexpression patterns suggest that the fast-to-slow fiber transition results from sequential isoform expressions in the order MHCIId(x) right-arrow MHCIIa right-arrow MHCIalpha right-arrow MHCIbeta . The allocation of MHCIalpha between MHCIIa and MHCIbeta 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

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 beta -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) right-arrow MHCIIa right-arrow MHCI (1). In rat TA muscle with MHCIIb as the predominant fast isoform, the fast-to-slow conversion is initiated by an MHCIIb right-arrow 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 alpha -cardiac-like MHC isoform (13, 22). An alpha -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 alpha -cardiac-like MHC mRNA also in several limb muscles of rabbit. A 140-fold upregulation of this isoform, designated as MHCIalpha , 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 MHCIbeta ; see Ref. 22).

The upregulation of MHCIalpha and MHCIbeta 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 MHCIalpha occur in parallel with MHCIbeta or does it occur independently, does it occur in same or different fibers, and finally, does it precede or follow that of MHCIbeta ? 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 MHCIalpha and MHCIbeta , as well as of the other sarcomeric MHC isoforms. This approach made it possible to study the distribution of MHCIalpha and MHCIbeta 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, MHCIalpha , and MHCIbeta , in our study. A sequence specific to alpha -skeletal actin mRNA served as an endogenous reference unit.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 MHCIalpha and MHCIbeta , 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, MHCIalpha , and actin and 3.5 mM for MHCIId(x) and MHCIbeta . 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 alpha -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), MHCIbeta , and alpha -skeletal actin were the same as previously described (23) [MHCIIb, GenBank accession no. X05958; MHCIId(x), GenBank accession no. U32574; and MHCIbeta , GenBank accession no. J00672]. The primers for the detection of the MHCIalpha (GenBank accession no. K01867) were the same previously reported (22). With the exception of MHCIbeta , 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; MHCIbeta sense primer, GGA TCC CTG GAG CAG GAG AA and antisense primer, CTT GCA TTG AGG GCA TTC AG; MHCIalpha sense primer, TCA AGG CCT ACA AGC GCC AG and antisense primer, TTG CGG GTT AAC AAG AGC GG; alpha -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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Identification of a cDNA sequence specific to the rabbit MHCIIa mRNA 3'-region. Sequence information was available for rabbit MHCIIb, MHCIId(x), MHCIalpha , and MHCIbeta (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), MHCIalpha , and MHCIbeta , indicating that the selected rat-specific primer did not anneal to isoforms other than MHCIIa mRNA.


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Fig. 1.   Schematic representation of the procedure used for identification of the 3'-region of rabbit myosin heavy chain (MHC) IIa cDNA. A modified oligo(dT) primer (37 mer) was used for reverse transcription of total mRNA. This primer served for introducing a specific 3'-sequence (XXXX) into the synthesized first-strand cDNA. This cDNA was used as template in a PCR with a rat-specific primer for MHCIIa and the specific sequence of the primer for cDNA synthesis (XXX), which yielded a product of ~450 nt. After sequence analysis of this product, primers specific to the rabbit MHCIIa sequence were chosen to yield a 240-nt product.

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 MHCIalpha and MHCIbeta , amounting to only 75 and 64%, respectively.


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Fig. 2.   Sequence alignment of the identified rabbit MHCIIa sequence with human and rat MHCIIa sequences. Hum, human MHCIIa (EMBL accession no. Z32858; GenBank S73840, positions 176-490); rat, rat MHCIIa (GenBank accession no. L13606, positions 365-672); and rab, rabbit MHCIIa (cDNA positions 1-319 nt).

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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Fig. 3.   Restriction analysis of the 240-nt polymerase chain reaction (PCR) product as analyzed by polyacrylamide gel electrophoresis and ethidium bromide staining. Lanes 1 and 5, undigested sequence; lane 2, Sau I digest with 146-and 94-nt fragments; lane 3, Hae III digest with 47- and 193-nt fragments; lane 4, Alu I digest with 21-, 11 (not visible)-, and 208-nt fragments; M, DNA marker V (Boehringer Mannheim). Molecular masses of the marker are specified at right.

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 MHCIbeta (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 MHCIalpha mRNA. A distinct signal for alpha -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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Fig. 4.   MHC isoform expression patterns in single fibers from control rabbit tibialis anterior muscle. Fragments from 10 fibers were analyzed. One-half of a fiber fragment was used for protein analysis (A; silver-stained electrophoresis) and the other one-half for mRNA analysis [B; electrophoretically separated and silver-stained products from direct reverse transcriptase (RT)-PCR]. The last lane in A shows the MHC pattern of a total extract from normal gastrocnemius muscle. The last lane in B shows purified PCR products used as standards for each sequence. Actin, 367 nt; MHCIId, 289 nt; MHCIIa, 240 nt; and MHCIbeta , 173 nt. According to protein analysis (A), fibers in lanes 1-3 contain MHCIId(x), fibers in lanes 4 and 6 are hybrids containing MHCIId(x) + MHCIIa, fibers in lanes 5, 7, and 8 contain MHCIIa, and fibers in lanes 9 and 10 contain MHCI. mRNA patterns match the protein patterns except for fibers in lanes 3 and 5, which display, in addition to the major isoform, an additional isoform, namely MHCIIa and MHCIId(x), respectively.

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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Fig. 5.   MHC isoform expression patterns in 10 single fibers from low-frequency-stimulated rabbit tibialis anterior muscles. Duration of chronic low-frequency stimulation was 10 days for fiber in lane 2; 20 days for fibers in lanes 1, 4, and 5; 30 days for fibers in lanes 3, 6, 8, and 9; and 50 days for fibers in lanes 7 and 10. A: silver-stained protein electrophoresis; last lane on right is total extract from gastrocnemius muscle. B: electrophoretically separated and silver-stained products from direct RT-PCR; purified PCR products were used for each sequence as standards (last lane on right). Actin, 367 nt; MHCIId, 289 nt; MHCIIa, 240 nt; MHCIalpha , 227 nt; and MHCIbeta , 173 nt. For further explanations, see Fig. 4. According to protein analysis (A), fibers in lanes 1, 5, and 6 contain MHCIIa, fiber in lane 2 is hybrid containing MHCIId(x) + MHCIIa, fibers in lanes 3, 4, and 7-10 are hybrids containing MHCIIa + MHCI. Generally, the mRNA patterns of these transforming fibers do not match the protein patterns but contain various combinations of mRNAs specific to slower isoforms than the predominant protein isoform. Note that all fibers display the signal for MHCIalpha .

A large fraction of hybrid fibers displayed the 227-nt signal specific to the MHCIalpha 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 + MHCIalpha ; 3) MHCIId(x) + MHCIIa + MHCIalpha  + MHCIbeta ; 4) MHCIIa + MHCIalpha ; 5) MHCIIa + MHCIalpha  + MHCIbeta ; and 6) MHCIalpha  + MHCIbeta .


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Fig. 6.   Distribution of different MHC mRNA isoform combinations in fibers (n = 115) from low-frequency-stimulated tibialis anterior muscles of rabbit.

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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Table 1.   MHC mRNA complement of fibers from low-frequency-stimulated rabbit TA muscles characterized by unique content of MHCIIa protein

Of special interest was the finding that MHCIalpha mRNA was present in all C fibers and in 70% of the investigated fibers uniquely displaying MHCIIa protein. MHCIalpha mRNA was also detected in fibers with the MHCIId(x) + MHCIIa protein complement. In all fibers examined, MHCIalpha was never detected as the unique mRNA isoform but was found in combination with MHCIIa, with MHCIIa + MHCIId(x), with MHCIbeta , with MHCIIa + MHCIbeta , or in combination with all of these mRNA isoforms.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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) right-arrow MHCIIa right-arrow MHCI (15). The detection of the MHCIalpha 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 MHCIbeta in muscles stimulated for >20 days (22). In this conjunction, it is of interest whether the transition from MHCIIa to MHCIbeta occurs directly or includes the expression of MHCIalpha . 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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Table 2.   MHC mRNA complement of hybrid fibers from low-frequency-stimulated rabbit TA muscles characterized by coexistence of MHCIId(x) and MHCIIa protein isoforms

                              
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Table 3.   MHC mRNA complement of hybrid fibers from low-frequency-stimulated rabbit TA muscles characterized by coexistence of MHCIIa and MHCI protein isoforms

The results of the present study suggest the following sequence of MHC isoform expression during fastto-slow transition in rabbit muscle: MHCIId(x) right-arrow MHCIIa right-arrow MHCIalpha right-arrow MHCIbeta . This sequence follows from the various combinations of MHC mRNA isoforms in individual fibers of transforming muscle (Table 4). The allocation of MHCIalpha between MHCIIa and MHCIbeta matches the failure to detect a direct transition from MHCIIa to MHCIbeta , which would postulate the MHCIIa + MHCIbeta 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 + MHCIalpha and of MHCIalpha  + MHCIbeta mRNAs assign MHCIalpha as intermediate between MHCIIa and MHCIbeta . Its intermediate position is further supported by the coexpression of three or even four isoforms extending from MHCIId(x) to MHCIalpha or MHCIbeta in some fibers and from MHCIIa to MHCIbeta in others. The existence of the combination MHCIIa + MHCIalpha without MHCIbeta seems to be relevant also with regard to the time point of the induction of MHCIalpha . Its induction appears to precede the expression of MHCIbeta , 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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Table 4.   Combinatorial patterns of MHC mRNA isoforms in single fibers of rabbit TA muscle during fast-to-slow transition by chronic low-frequency stimulation

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 + MHCIbeta 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 + MHCIbeta protein phenotype (9, 18, 27, 28). We now show at the mRNA level that, in transforming rabbit muscle, MHCIalpha represents an additional isoform present in these fibers. Although no data exist on the contractile properties of MHCIalpha -containing fibers in limb muscles, the suggested intermediate position of MHCIalpha between MHCIIa and MHCIbeta seems to be supported by the functional properties of MHCIalpha -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 MHCIbeta complement. MHCIalpha would thus bridge a functionally large gap between the fast MHCIIa and the slow MHCIbeta (14, 26). According to immunohistochemical studies, hybrid fibers of the masseter muscle contain only combinations of MHCIIa + MHCIalpha or MHCIalpha  + MHCIbeta (14). In this conjunction, it is of interest that, according to sequence alignment of the 3'-regions, MHCIIa is more similar to MHCIalpha than to MHCIbeta .

The upregulation of MHCIalpha 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, MHCIalpha 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 MHCIalpha 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 MHCIalpha may bridge the gap between MHCIIa and MHCIbeta .

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) right-arrow MHCIIa right-arrow MHCIalpha right-arrow MHCIbeta .

    ACKNOWLEDGEMENTS

We thank Nina Hämäläinen and Michael Schuler for helpful suggestions.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Cell Physiol 274(3):C595-C602
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