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Eur. J. Biochem. 247, 30-36 (1997)0 EBS 1997

Quantitative analyses of myosin heavy-chain mRNA and protein isoforms insingle fibers reveal a pronounced fiber heterogeneity in normal rabbit musclesHeidemarie PEUKER and Dirk PETTEFakultat fur Biolog ie, Universitat Kanstan z, Germany(Received 11 Febm ary/l7 April 1997) - EJB 97 0220/1

A highly sensitive method of reverse-transcriptase polymerase chain reaction (RT-PCR) was estab-lished to study myosin heavy-chain (MHC) mRNA isoform expression in single fibers of rabbit limbmuscles. In combination with myofibrillar adenosine triphosphatase histochemistry and electrophoreticseparation of MHC protein isoforms in fragments of the same fibers, the direct RT-PCR method identifiedthe pMHC20-40 and pMHC24-79 cDNA sequences as being specific to MHCIIb and MHCIId/x isoforms,respectively. In addition, a direct RT-PCR was established for determining relative amounts of MHCmRNA isoforms by using a sequence specific to a-skeletal actin as an endogenous reference. Analysesof large amounts of single fibers revealed an unexpected heterogeneity of the fast fiber population withregard to numerous fibers coexpressing MHCIIb and MHCIIdx. Based on quantitative RT-PCR, thepercentages of MHCITWMHCIId hybrid fibers amounted to approximately 55% in the deep portion ofgastrocnemius, to 43% in the adductor magnus, and to 12% in psoas muscle. Moreover, the two MHCmRNA isoforms were nonuniformly distributed along the fiber length. Qualitative RT-PCR detected evenhigher amounts of hybrid fibers in the three muscles. The percentages of hybrid fibers identified at theprotein level were smaller in adductor magnus muscle (25%) and psoas muscle ( 5 % ) , but equaled thatof the mRNA analysis in gastrocnemius muscle (6170).he detection of high amounts of IIBD and IIDBfibers suggested that hybrid fibers represent functional elements within the fiber spectrum of nornmlmuscles. Our observations on hybrid fibers reveal a heterogeneity within the fiber population of normalmuscles that has not been realized to date.Keywords; coexpression ; direct reverse-transcriptase PCR ;muscle fiber type; myosin heavy-chain iso-form; single fiber analysis.

Skeletal muscle is an extremely heterogeneous tissue com-posed of different fiber types. Therefore, the validity of bio-chemical data obtained from studies on muscle homogenates islimited. For this reason, single fiber studies have been increas-ingly used, e.&. for metabolic studies or the analysis of myo-fibrillar protein isoform patterns in specific fiber types. Variousmethods are commonly used for the classification of fibertypes, e.g. myofibrillar actomyosin adenosine triphosphatase(mATPase) histochemistry [ I ] , immunohistochemistry [2, 3 ,and single fiber electrophoresis of myosin heavy-chain (MHC)isoforms [4]. A s a result, four major fiber types are distinguishedin limb muscles of the rabbit, three fast (types IIB, IID/X, andHA ) and one slow (type 1) [ 5 ] (for reviews see [6, 71). Thesefunctionally different fiber types express different MHC iso-forms. Thus, II B fibers contain MHCIIb, IID/X fibers MHCIId/x, and II A fibers MHCIIa. Studies performed on rat muscle sug-gested that the MHCIId isoform is identical with the MHCIIxisoform [S]. The slow type 1fibers contain MHCI, thought to beidentical to the []-cardiac MHC isoform [9]. Based on mRNA[lo] and protein [ I l l analyses, recent evidence suggests the

Correspondence to H. Peuker, Fakultat fur Biologie, UniversitiitFax: + 4 9 7531 88 39 40.Abbreviations. mATPase, myofibrillar actomyosin ATPase; MHC,Enzymes. RNA-directed DNA polymerase (EC 2.7.7.49) ; DNA-di-

Konstanz, Postfach 5560-M641, D-78434 K onstanz, Germany

myosin heavy chain ; RT, reverse transcriptase.rected DNA polymerase (EC 2.7.7.7).

existence of an additional slow, a-cardiac-like MHC isoform inrabbit skeletal muscle.

In addition to pure fiber types expressing only one MHCisoform, hybrid fibers have been detected. The coexistence oftwo or more MHC isoforms in individual muscle fibers is com-monly interpreted as a sign of fiber type transitions, reflectingphenotypic modulation, as well as the plasticity of gene expres-sion in a terminally differentiated cell. To analyse the phenome-non of coexisting MHC isoforms in individual fibers in moredetail, we studied the expression of MHC isoforms in singlefibers from rabbit muscles at both the mRNA and protein levels.The analysis of MHC mRNA levels was conducted by directreverse-transcriptase polymerase chain reaction (RT-PCR). Wehave used this method previously for a preliminary assignmentof two highly similar cDNA clones specific to two MHC iso-forms in fiber types IIB and IID 112). The present study wasbased on an improved direct RT-PCR [13] displaying a highsensitivity and reproducibility, which is therefore suitable forquantitative analyses at the single fiber level. The improvedmethod encompassed a highly efficient extraction protocol andthe use of a sequence specific to a-skeletal actin mRNA, servingas an endogenous reference unit for determining relativeamounts of MHC inRNA isoforms. In combination withmATPase histochemistry and single fiber electrophoresis ofMHC protein isoforms, this approach confirmed our previousresults on the identity of the two isoforms. Furthermore, the highsensitivity of the direct RT-PCR revealed an unexpected hetero-


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Peuker and Pette ( E m J . Binchem. 247) 31geneity of the fast fiber population, especially with regard tolarge fractions of hybrid fibers in normal muscles. This observa-tion suggests that hybrid fibers may not only be envisaged astransient states during fiber type transitions, but represent ele-ments within a continuum of finely tuned, functionally differentfibers.

MATERIALS AND METHODSAnimals and muscles. Adductor magnus, gastrocnemius,

and psoas muscles were taken from adult male White NewZealand rabbits. Thin muscle strips were slightly stretched andfrozen in melting isopentane (-159C) and stored at -70C.

Dissection of single fibers and histochemical classifica-tion. Thin fiber bundles were prepared from the frozen musclestrips at -25C in a cryostat, transferred to precooled aluminumholders and freeze-dried at -38C. To study the MHC isoformexpression along the fiber length, 5 - 15-mm-long fibers wereisolated by free-hand dissection under a stereomicroscope. Thesewere typed by electrophoretically identifying their MHC isoformcomplement. Consecutive fiber pieces were cut free-hand,weighed on a quartz fiber balance, and subjected alternately tomRNA and protein analyses. The dry masses of fragments fordirect RT-PCR were approximately 50 ng and the fragments forprotein analysis were in the range 150-200 ng.

Fragments of histochemically identified fibers were dis-sected from freeze-dried, thick muscle cross-sections [141(Fig. 1). Fiber typing was performed by histochemical stainingfor mATPase of serial 10-pm-thick cross-sections after incuba-tion at various pH values. Fast fiber types IIB and II D weredelineated after incubation at pH 4.55 [I]. For MHC protein andmRNA analyses, fragments of the histochemically identified fi-bers were dissected under a stereomicroscope from consecutive50- 80-pm-thick freeze-dried cross-sections.

Direct reverse-transcriptase polymerase chain reaction.Oligonucleotide primers for MHCIlb (pMHC20-40), MHCIId(pMHC24-79), and a-skeletal actin were the same as in [I31 andfor MHCI (MHCp174) the same as in [12]. The pMHC20-40(GenBank Accession no. X05958) and pMHC24-79 (GenBankAccession no. U32574) clones were isolated by Maeda et al.[151, and the MHCp174 (GenBank Accession no. 500672)clone was isolated by Sinha et al. [16]. The pMHC20-40 andpMHC24-79 clones were kindly provided by Drs K. Maeda andA. Wittinghofer (Heidelberg). They displayed 84% similarity inthe 3 region from which the following primers were derived;MHCIIb sense primer, AGA GGC TGA GGA ACA ATC CA;antisense primer, ACT TGA TGC ACA AGG TAG TG;MHCIId sense primer, ACT GCA AGC CAA GGT GAA AT;antisense primer, TTA TCT CCC AGA ATC ATA AG. ForMHCI, th e sense primer was GGA TCC CTG GAG CAG GAGAA and the antisense primer was CTT GCA TTG AGG GCATTC AG. For a-skeletal actin (GenBank Accession no. J00692),the sense primer was CGC GAC ATC AAA GAG AAG CT;the antisense primer was GGG CGA TGA TCT TGA TCT TC.These primers yielded PCR products of 249, 289, 173 and 367nucleotides, respectively. The S-ends of the antisense primerswere labeled with digoxigenin (MWG Biotech) to allow chemi-luminescent detection of the PCR products.

The oil well technique [I71 was used for mRNA analysis bydirect RT-PCR [I 21. RNA extraction and reverse transcriptionwere performed as previously described [I31 (see Fig. 1).Briefly, the fiber fragment was picked up under the stereo-microscope using a short piece of hair mounted to a needleholder according to [I81 and transferred into 0.28 p1 high-saltextraction medium under mineral oil and incubated for 60 min

Fig. 1. Scheme for direct RT-PCR on fragments of histochemicallydefined muscle fibers. Fiber typing was performed by histochemicalstaining for my ofibrilku actomyosin adeno sine triphosphatase of serial10-pm-thick cross-sections after incubation at various pH values. Con-secutive thick sections (50-80 pm) were freeze-dried and used formicrodissecting fragments of identified fibers. Myosin heavy-chainmRNA and protein isoforms of fragments of the same fibers were ana-lysed by direct RT-PCR and electrophoretic separation of the MHC pro-tein complement, respectively. For R NA extraction, the fragments w eretransferred into a high salt medium under mineral oil. Reverse transcrip-tion was performed after addition of a dilution medium yielding opti-mum conditions for cDNA synthesis with specific 3 primers. Succes-sively, PCR for the different sequences was performed in separateassays. For product analysis, aliquots of the PCR assays were combinedand products were electrophoretically separated and visualized by silverstaining or by chemiluminescence detection.

at 4C to allow extraction of total RNA. Subsequently, the assaymixture was diluted to yield optimum conditions for reversetranscription, performed for 30 min at 42C in a volume of1.18 pl. These small volumes were pipetted using a previouslydescribed computer-controlled micropipetting system [191. Theassay was then transferred into PCR medium and divided intofour separate PCR assays for amplification of each sequence. Toascertain that amplification from contaminating DNA did notoccur, control assays were run in the absence of reverse tran-scriptase.

Poly(A)-rich RNA isolation from single fibers. Isolation ofpoly(A)-rich RNA from fiber fragments in the range 100-500 ng dry mass was performed using the Dynabeads Biomag-netic Separation system for tissue extraction with strong denatur-ating agents adapted to the microscale [131. Three protocolswere used to introduce the mRNA into the RT-PCR assay: (a)elution of bound mRNA from Dynabead oligo(dT)25 using anelution buffer (2 mM EDTA, pH 8.0) and reverse transcriptionwith specific primers; (b) direct application of the Dynabead-


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32 Peuker and Pette (Eur: J. Biochem. 247)bound inRNA for reverse transcription; (c) elution of the Dyn a-bead-bound mR NA and reverse transcription with oligo(dT) 5 asprimer.

PCR product detection. Two protocols were used foramplif ication and PCR product detection. The assignment ofspecif ic mR NA isoforms to defined f iber types wa s based on aqualitative method of detection. This approach, displaying veryhigh sensitivity, was used to identify specific MHC mRNAspresent at very low levels in single fiber fragments. The PCRproducts were detected in the plateau phase of amplification, i.e.30 cycles for a-skeletal actin and 36 cyc les for the M HC iso-forms. Following the amplification, 2.5 pl of each of the fourassays (for pMHC20-40, pM HC24-79, pM HC Pl7 4, and cr-skele-tal actin) performed on a single f iber fragment were combined,separated by electrophoresis, and visualized by silver staining

A quantitative assay detected the PC R products in the expo -nential phase of amplification, which was determined in a sepa-rate set of experiments 1211. Usually, 23 cycles were performedfor both a-skeletal actin and for the MHC sequences. 1 pl ofeach PCR assay was combined and the products separatedelectrophoretically. Th e digoxigenin-labeled PCR products werevisualized by a chemiluminescent detection system [21]. Forquantitative evaluation of MHC mRNA expression levels, thesignal intensity of a-skeletal actin mRNA served as an endoge-nous control for efficiencies of R NA extraction and reverse tran-scription. In addition, the signal intensities of MHC-specif icmRNAs were corrected for differences in amplif ication eff i-ciencies determined by amplification of identical amounts (10'-lo" molecules) of purified PCR products as external standards[lo]. Thus, the signal intensities after 23 cycles were 1.5-timeshigher for pMHC24-79 than for pMHC20-40, indicating aslightly lower amplification efficiency of the latter. The signalintensities of the PCR products using silver staining or chemilu-minescence detection, were evaluated by integrating densitome-try using the Scanpack software (Biometra).MHC protein analyses in single fiber fragments. Fiberfragments dissected from freeze-dried cross-sections or cut fromdissected single fibers were analyse d by gradient gel electropho-resis for their MH C isoform complemen t as previously described[22]. The silver-stained gels were evaluated densitometrically(see above).

113, 201.

RESULTSValidity of the direct RT-PCR procedure. The eff iciency ofboth total R NA extraction and cDN A synthesis was checked bycomparing our direct RT-PCR method with an m RN A isolationusing the Dynabead Biomagnetic Separation system 1231 andthe use of oligo(dT) for reverse transcription. We adapted theDynabead protocol to the microscale and compared three dif-ferent protocols, i .e . (a) e lution of m RN A from the Dynabeadsand cD NA synthesis with specific pr imers (Fig. 2, lane 1); (b )omitting the elution step and performing cDNA synthesisstar ting from Dynabead-bound mR NA (Fig. 2, lane 2); (c) e lu-tion of mRNA from the Dynabeads and cDNA synthesis witholigo(dT),, (Fig. 2, lane 3). Compared to our direct RT-PCR(Fig. 2, lane 4) , the procedures including the elution step (Fig. 2,lanes 1 and 3) proved to b e less eff icient, most obviously in thecase of the 289 nucleotide signal. As documented by similarsignal intensities specific to a-skeletal actin (367 nucleotides),and the two MHC isoforms (289 nucleotides, 249 nucleotides) ,the use of Dynabead-bound mRN A without e lu tion for cDN Asynthesis proved to be equivalent to the direct RT-PCR (Fig. 2,lanes 2 and 4). Furthermore, iden tical signal intensities were ob-

0 1 2 3 4 0 1 2 3 4 M

367-289-249-

Fig.2. Comparison of direct RT-PCR with different protocols ofpoly(A)-rich RN A isolation by magnetic heads and subsequentcDNA synthesis. The comparison applies to two hybrid fibers of theadductor magnu s muscle. Eight consecu tive fragments of each fiber wereanalysed. 1-4 refer tci four different protocols as follows: 1-3, usingDynabead-based poly(i9)-rich RNA isolation ;4, direct RT-PCR (for fur-ther details see text). I'CR conditions were the same in all procedures,especially with regard to identical amounts of the template. Productanalysis of three fragments (367 nucleotides, a-skeletal actin; 289 nucle-otides, pMHC24-79; 249 nucleotides, pMHC20-40) was performed after36 cycles applying polyacrylamide gel electrophoresis and silver stain-ing; M, molecular mass marker V (Boehringer Mannheim, Germany).

tained when cDNA synthesis in the direct RT-PCR was per-formed with specific primers or oligo(dT),, (data not shown).Taken together , these f indings show ed the com pleteness of theRNA extraction and also indicated similar efficiencies of thefirst-strand cD NA synthesis for the sequences under study.Sensitivity of the direct RT-PCR. To determine the absolutedetection limit of the direct RT-PCR for a specif ic mRNA insingle f iber fragments, we performed PCR on serial dilutions ofthe products obtained from reverse transcription. For example,the minimum sample in which the MHC mRNA specif ic topMHC20-40 was detected in type IIB f ibers corresponded to10-20 pg dry fiber. ,4 ample of this size was assumed to con-tain 40-80 fg total RNA. Based on the quantification of M HCmR NA m olecule numbers in total R NA preparations of adductormagnus muscle (4.4X lo* molecules MHCIIb mRNA/pg to ta lRNA) [21] , we estimated that the direct RT-PCR was sensitiveenough to detect less than 50 molecules of MHCIIb mRNA insingle f ibers. In the case of type IID f ibers, the minimum samplefor detection of mRN,4 specific to pMHC24-79 was in the range20-50 pg dry mass. For a-skeletal actin mRNA, the detectionlimit was in the range 5-10 pg. No differences were detectedbetween different fiber types, which indicated a uniform expres-sion level of a-skeletal actin in the fibers under study.Fiber type and isoform specificity of M HC mRNA detectionby direct RT-PCR. To confirm that muscle f ibers displayingsigna ls for mRNA s spec i fic to the pMHC24-79 and pMHC20-40 clones coexpressed tw o distinct MH C isoforms, we re- inves-tigated the specificity of the selected primer pairs and amplifiedsequences. Pure fibers identified by their histochemicalmATPase staining and protein spectra as either type ILB, typeIID, or type I displayed single signals of 249, 289, and 173nucleotides, respectively (Fig. 3). In the case of type IIA f ibers,no signal was obtained. However, a clear signal was detectedfor a-skeletal actin mKNA. Taken together, these findings con-firmed the specificity of the three selected primer pairs and ex-cluded the possibility that a given primer pair cross-reacted withadditional MH C mR N.4 isoforms. The detection of signals spe-cif ic to both MHC mRNA isoforms in individual f ibers, there-fore, unambiguously identif ied such f ibers a s hybrids.Pure and hybrid fibers. Th e assignment of the two fast MHC-specif ic mRNA sequences to fiber types defined by theirmATPase histochemistry and MH C protein isoform com plement


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APeuker and Pette (EUK . Biochem. 247)

A33

G A S 1 2 3 4 5 6 7 B A D " ' 2 3 L 5 6 7 8 9 10 1 1 G A SBMHC Ila,MHC li d -MHC IMHC IIb

C367-289 -249-173-

C o 1 2 3 L E 6 7

Fig. 3. Direct RT-PCR for MHC mRNA isoforms in histochemicallydefined fibers demonstrates isoform specificity of the chosen prim-ers. Analyses on cross-sections of gastrocnemius muscle were performedas described in the legend of Fig. 1. (A ) Histochemical classification offiber types by mATPase staining after iucubation at pH 4.55 (bar =40 pm). (B ) Electrophoretic analysis of the MHC protein isoform com-plement of the fibers specified in A. GAS, whole muscle extract fromgastrocnemius muscle containing all four MHC isoforms. (C) mRNAanalysis by quantitative RT-PCR. Products for a-skeletal actin (367 nu-cleotides) and the MHC-specific sequences pMHC24-79 (289 nucleo-tides), pMHC 20-40 (249 nucleotides), and pM HCP174 (1 73 nucleotides)were electrophoretically separated after 30 and 36 cycles, respectively,and visualized by silver staining. Co, control assay with a fiber fragmenti n the absence of reverse transcriptase to monitor amplification fromcontaminating DNA.

was based on the qualitative RT-PCR analyses performed onthree muscles. A typical example is given in Fig. 4 for adductormagnus muscle, showing that type IIB fibers containingMHCIIb at the protein level displayed the signal for mRNA spe-cific to pMHC20-40. Type IID fibers containing the MHCIIdprotein isoform displayed the mRNA specific to pMHC24-79.

A large fiber fraction displayed signals for pMHC24-79 aswell as for pMHC20-40 (Fig. 4, lanes 3-6, 8 and 9). As judgedfrom the mRNA signals, only 22% and 9% of the adductor mag-nus fibers examined (n= 170) were identified as pure type IIBand type IID fibers, respectively. Most fibers (69%), however,were identified as hybrids. In gastrocnemius muscle (data notshown), fibers identified as type IID displayed the signal specificto pMHC24-79, whereas fibers identified as type IIB, yieldedsignals for mRNA specific to pMHC20-40. Approximately 37 %

C o 1 2 3 4 5 6 7 8 9 1 0 1 1- 367C- 289- 249

Fig.4. Direct RT-PCR for identification of mRNA isoforms specificto pMHC20-40 and pMHC24-79 in fragments of histochemically de-fined fibers from adductor magnus muscle. Analyses on cross-sec-tions were performed as described in Fig. 3. (A) mATP ase histochemis-try (bar = 40 pm) (B ) Electrophoretic analysis of the MHC protein iso-form complement of the fibers specified in A. ADM and GAS, wholemuscle extracts from adductor magnus and gastrocnemius muscles asmarkers. (C) mRNA analysis by quantitative RT-PCR. Note the highnumber of hybrid fibers displaying signals for both MHC isoforms, espe-cially at the mRNA level.

of the examined fibers ( n = 366) were classified as pure typeIID and 3% as type IIB. We noted that some fibers unambigu-ously identified as type IID displayed variable signal intensitiesfor pMHC24-79. The fraction of hybrid fibers expressing themRNAs specific to MHCIIb and MHCIId amounted to 60% inthe gastrocnemius muscle. In addition, hybrid fibers displayingMHCIId and MHCIla at the protein level, yielded the signal forpMHC24-79. Due to the lack of MHCIIa-specific primers, thecorresponding signal for this isoform was not detected.

Based on the analysis of 262 fibers, the assignment ofpMHC24-79 to type IID fibers was also valid for psoas muscle.In addition, electrophoretically identified hybrid fibers with co-existing MHCIIb and MHCIId yielded a signal for pMHC20-40.In agreement with previous studies on MHC isoform distributionin rabbit psoas muscle [ 5 ] , no pure type IIB fibers were detectedin the present study, neither at the protein nor at the mRNAlevel. It should be noted that, among the several hundred fastfibers studied, the coexistence of mRNA specific toMHCI(pNHCP174) with the mRNAs specific to MHCIIb orMHCIId was never observed. This was also true for 23 histo-chemically and biochemically identified type I fibers from gas-trocnemius muscle.

Hybrid fibers, in which MHCIIb and MHCIId mRNAs co-existed, displayed highly variable ratios of these two isoforms.Examples are shown in Fig. 5 where the mRNA signals werealigned according to their varying intensities to demonstrate acontinuum of hybrid fibers between pure types II D and IIB.


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34 Peuker and Pette ( E m J . Biochem. 247)A G

MHC116-AC

-MHCllb

Fig.5. A continuum of MHCIId and MHCIIb mRNA isoform ex-pression as shown by analysis of fragments from pure type IID andIIB fibers, and of fragments from ten hybrid fibers. mRNA analysiswa s performed for cr-skeletal actin (AC),MH CIId, and MHCIIb by directRT-PCR (see Fig. 3). To demonstrate the continuum between pure typesIID and IIB, samples were aligned according to their varying signalintensities.

AMHClldMHCllb

B

ACMHClldMHCllb

Fig. 6. Nonuniform MHC isoform expression along a hybrid musclefiber. MHC isoform mRNA and protein analyses were alternately per-formed on consecutive fragments of an approximately 4-m m-long hybridfiber from gastrocnemius muscle. (A) Electrophoretic analysis of theMHClId and MHCIIb protein isoform complement. M, whole muscleextract of adductor magnus as marker; (B ) quantitative direct RT-PCR.Products were detetced after 23 cycles (exponential phase) by chemi-luminescence. M, inolecular mass marker VI.

Relative amounts of specific mRNA isoforms in single fiberfragments. A surprisingly high percentage of hybrid fibers wasdetected in the three muscles und er study. In so me hybrid fibers,the coexistence of MHCIIb and MHCIId isoforms was detectedat both the mRNA and protein levels (Fig. 4, lanes 4-6),whereas other hybrid fibers displayed signals of different inten-sit ies for the two mRNA isoforms, but only a single isoformwas detected at the protein level (Fig . 4, lanes 3, 8 and 9). Thus.the qualita tive mR NA detection yielded a higher fraction of hy-brid fibers than the protein analyses. Very low amounts of M H CmR NA isoforms, which were present in addition to the dominantisoform, were probably overestimated by the high sensitivity (36cycles) of the qualitative assay.

To exclude this shortcoming, we established a quantita tiveassay for chemiluminescence detection of PC R products in theexponential phase of amplification (23 cycles) (Fig. 68). Usingthe signal intensity of the a-skeletal actin mRNA as an endoge-nous control for RN A extraction and reverse transcription, com-bined with a correction for differences in amplification efficienc-ies, the quantitative assay proved to be suitable for measuringrelative expression levels of MHCIIb and MHClId mRNA iso-forms in single fibers.Based on this quantita tive approach, we evaluated in moredetail MH C mR NA and protein expression levels in tw o groupsof hybrid fibers, namely IIBD (MHCITb > MHCIId) and IIDB( M HC lI d > MHCIIb) fibers. We then compared the results ofthe quantita tive mRN A detection with th e protein data . In addi-tion, qualita tive mRNA analyses were included to identify the

Table 1. Expression of MHCIIb and MHCIId isoforms at mRNAand protein levels in type IIB and IID fibers from adductor magnusmuscle of the rabbit..96 type I1 fibers were analysed for MHC proteinisoform complement by electrophoresis and for their MHC mRNA iso-forms by qualitative direct RT-PCR (36 cycles, silver staining), as well asby quantitative direct RT-PCR (23 cycles, chemiluminescent detection).Hybrid fibers were separated into two groups accordig to their signalintensities. Data from quantitative mRNA analyses were corrected fordifferences in amplification efficiencies.~~ ~ ~ ~

MHC isoform Amount of fibers Fiberdetermined by type____RT-PCR detection proteinquali- quanti-tative tative

analysis

~~~ ~~ ~ ~ ~

5_ _ _ _MHCIlb 22.9 42.1 51.0 IIBMHCIIb > MHCIId 42.1 28.1 14.6 IIBDMHCIId > MHCIIb 23.9 15.7 10.4 IIDBMHCIId 10.5 13.5 24.0 IID

Table 2. Size of hybrid fiber fractions defined by qualitative andquantitative RT-PCR, and single fiber electrophoresis in three limbmuscles of the rabbit. The values represent the mean values of datacollected from different regions of the muscles. For further explanation.see Table 1.Muscle Hybrid fiber fractionexpressing MHClIb and MHCIIddetermined by

RT-PCR detection proteinquali- quanti-tative tative

analysis

%Adductor magnus ( n = 112) 7 2 43 28

Pw as, red and white ( 1 1 = 184) 37 12 5Gaqtrocnemius, deep (n = 111) 70 55 61Gastrocnemius, middle ( n = 168) 56 38 40

fraction of f ibers expressing one of the two MHC isoforms atvery low levels. Representative results from adductor magnusmuscle are given in Table 1. Qualitative and quantitative RT-PCR yielded high amounts of hybrid f ibers, i.e. 67% and 44%,respectively. Within the hybrid fiber populations, IIBD fiberswere more num erous than IIDB fibers. A much smaller percen-tage of hybrid fibers (25%) resulted from M HC protein analysesin the sam e f ibers. T ~ u , discrepancy existed between the frac-tions of hybrid f ibers detected by mRNA and protein analysis.This discrepancy wa:; most pronounced in psoas fibers and lessobvious in gastrocnemius f ibers (Table 2). Conspicuous differ-ences existed beiween the three muscles w ith regard to hybridfibers detected at the protein level. The highest percentage ofhybrid fibers (6 % ) was found in the deep portion of gastroc-nemius. Contrary to the other m uscles, the hybrid f iber fractionsdelineated by mRNA and protein analyses were similar in gas-trocnemius muscle.Nonuniform MHC isoform expression along the length of hy-brid fibers. To investigate M HC isoforin distr ibution along the


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Peuker an d Pette (ELK J. Biochrm. 247) 35length (5-15 mm) of hybrid fibers, we performed protein andmRNA analyses on consecutive pieces of the same fibers. Inaddition to fibers with a constant ratio of the two fast MHCisoforms along their length, we found fibers with nonuniformexpression of the MHCIIb and MHCIId mRNA isoforms andtheir corresponding proteins. The variations in isoform expres-sion were seen from quantitative MHC mRNA and proteinanalyses over distances in the millimeter range (Fig. 6). Similarmeasurements were performed on a total of 57 hybrid fibers(types IIBD and IIDB) from adductor magnus and gastroc-nemius muscles. Variations in MHC mRNA isoform expressionalong the fiber length were unambiguously detected in 17 fibers(data not shown).

DISCUSSIONA major point of the present study was the determination of

relative amounts of MHC mRNA and protein isoforms at thesingle fiber level. For this purpose, we combined mATPase his-tochemistry, single fiber electrophoresis, and direct RT-PCR toidentify cDNA clones pMHC20-40 and pMHC24-79 as beingspecific to MHCIIb and MHCIId, respectively. This assignmentwas the basis for assessing relative amounts of the two fast MHCmRNA isoforms in microdissected fibers of defined types.

An unexpected finding of our investigations on MHC iso-form mRNA expression was the high content of hybrid fibers i nlimb muscles of normal rabbit. In general, coexpression of dif-ferent MHC isoforms in single fibers is thought to be mainlyrestricted to muscles transforming their phenotype i n response toaltered functional demands. Thus, coexistence of different MHCisoforms at the protein level has been observed in response toincreased or decreased levels of neuromuscular activity, e.g. in122, 241, but small numbers of hybrid fibers have also been re-ported in muscles under steady-state conditions. As judged fromsingle fiber protein analyses, the percentage of hybrid fibers innormal fast-twitch muscles of rat and mouse is in the range 4-10% [25, 261. Higher values (30-50%) were reported for ratfast-twitch muscles [27] and by DeNardi et al. [28] using i n situhybridization. The rabbit data indicate even larger hybrid fiberfractions.

The low detection limits (approximately 50 molecules ofmRNA) made the direct RT-PCR an extremely sensitive tool forthe study of MHC isoform expression in view of their distribu-tion i n different fibers and their distribution along individualfibers. The combination of single fiber protein electrophoresisand direct RT-PCR yielded information on relationships betweenmRNA and protein expression that cannot be provided by in situhybridization and immunohistochemistry. Moreover, the highresolution of the direct RT-PCR disclosed an unexpected hetero-geneity of the fiber population. Thus, considering only MHCIIband MHCIId as two next-neighbour isoforms of the MHCspectrum, we show that the percentage of fibers coexpressingtwo MHC isoforms may exceed the fraction of pure fibers. Thenumber of hybrid fibers would have probably been even higherif coexpression with the third fast MHC isoform, MHCIIa,would have been included in our study. As shown by proteinanalyses on single fibers, MHCIIa coexists with MHCI in so-called C-fibers [14, 291, and, in the rabbit, also with MHCIId intype IIDA and IIAD fibers [ 5 ] .The coexistence of MHCIId andMHCIIa at the mRNA level can, therefore, be anticipated. Suchcombinations have been studied in human muscle fibers at boththe protein and mRNA level [30-321. However, fiber type tran-sitions may occur more frequently in human muscles under theinfluence of altered neuromuscular activity, than in muscles of

the caged and sessile rabbit. The presence of high amounts ofhybrid fibers in rabbit muscle, therefore, emphasizes their roleas functional elements under steady-state conditions.

More than two MHC isoforms at the protein level have beenobserved only during induced fiber type transformations [22,24). The contention that coexpression of MHC mRNA isoformsnormally applies only to next-neighbour isoforms is also sup-ported by our observation that mRNA specific to the slow MHCIisoform was never detected in combination with the fastMHCIIb and MHCIId mRNAs. The coexistence of MHCI andMHCIIa mRNAs, however, occurs in C fibers as shown by insitu hybridization in rat [ 2 8 ] .

The coexistence of MHC mRNA and protein isoforms understeady-state conditions suggests that hybrid fibers may not onlybe regarded as transitory states during fiber type conversion.Obviously, hybrid fibers represent entities allocated betweenpure fibers in a continuum of functionally different fibers [25,331. This continuum has previously been shown by biomechani-cal measurements on single pure and hybrid fibers. Fibers thatcontain two MHC isoforms were allocated according to theircontractile properties as intermediate between their next-neigh-bour pure fiber types [27, 341.

The heterogeneity of muscle fiber phenotypes is further em-phasized by the nonuniform expression of MHC isoforms alonghybrid fibers, which points to a heterogeneity at the level ofthe myonuclear population. This heterogeneity either reflects thecoexistence of differentially programmed myonuclei derivedfrom different inyoblast lineages or the potential of a homogen-eous population of myonuclei to differentially express myofibril-lar protein isoforms. Nonuniform expression of myosin isoformshas been observed mainly in muscle fibers 1351 and myotubes[36] undergoing experimentally induced phenotype transitions.Further studies using in situ RT-PCR will provide informationon the heterogeneity at the myonuclear level.

The present findings do not exclude the possibility thatadditional and as yet unidentified MHC isoforms exist in rabbitmuscle. This suggestion relates to discrepancies between mRNAand protein levels in the case of type IID fibers. Some fibersunequivocally identified by their MHC protein pattern as puretype IID exhibited very weak signals for the pMHC24-79 se-quence (data not shown). This discrepancy, which was also ob-served in previous studies [12,21], was especially obvious whencompared to the relations between MHCIIb mRNA and proteinlevels in pure type IIB fibers. A possible explanation could bethe existence of as yet unidentified subforms of MHCIId, pre-sumably due to splice variants not detected by our primers.Splice variants and multiple polyadenylation sites have beenshown for the a-cardiac MHC of rat [37] and for smooth muscleMHC isoforms [38].In hybrid fibers displaying two MHC mRNA isoforms butonly one MHC protein isoform, the failure to detect the secondMHC isoform may have resulted from insufficient sensitivity ofelectrophoretic protein detection. Another explanation relates tothe possibility of post-transcriptional regulation. For example,very low amounts of message may be detected by the highlysensitive RT-PCK, but must not necessarily be translated.

In summary, a method has been devised to determine expres-sion levels of MHC mRNA and protein isoforms in single fiberfragments. Its high sensitivity revealed an unexpected hetero-geneity of the fiber population, which by far exceeds that dis-closed by the methods of histochemical and/or immunohisto-chemical fiber classification. Although our study has focusedonly on two fast MHC isoforms, we show that a major fractionof fibers in normal muscles expresses more than one MHCisoform. The highly variable ratios of different MHC mRNA


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36 Peuker and Pette (Eur: J. Biochem. 2 4 3isoforms indicate a continuum of hybrid fibers between next-neighbour pure f iber types.

This study was supported by the Deursche ForschunRsgemritaschafr,SF B 156 . The authors thank M rs Barbel Gohlsch for technical assistancein performing the single fiber electrophoreses.REFERENCES

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