The purpose of this study was to investigate whether the previously demonstrated heterogeneity of myosin heavy chain (MHC) in type 28 fibers of the superficial portion of the lateral gastrocnemius (SLG) muscle of the rat may be due to presence of type 2D/X fibers. lmmunohistochemical identification of MHC heterogeneity, histochemical identification of appendicular 2D/X muscle fibers, and immunoblotting of myosins were used. It was found that some, but not all, of the heterogeneity of rat SLG is correlated with the presence of type 2D/X fibers. lmmunoblots with MHC from several muscles revealed the presence of at least two antigenically distinct isoforms of MHC with the same electrophoretic mobility as the “28”band. These results show that a previously undetected type of MHC is expressed in rat skeletal muscle and raise the possibility that there may be as yet undetected MHC isoforms in human muscle which may be clinically important. 0 1992 John Wiley & Sons, Inc.
Key words: myosin heavy chain isoforms skeletal muscle monoclonal antibody MUSCLE & NERVE 15~1349-1353 1992
EVIDENCE FOR NEW ISOFORM OF FAST MYOSIN HEAVY CHAIN IN RAT SKELETAL MUSCLE JUDITH A. SAWCHAK, MD, BETTY LEUNG, BA, and SAYID A. SHAFIQ, PhD
T h r e e fast myosin heavy chain (MHC) isoforms, 2A, 2B, and 2D/X, have been distinguished in rat skeletal and assigned to fiber types (of the same names) by analyzing electrophoretically microdissected fragments of histochemically classified fibers.16 Physiologic studies have shown that 2D/X MHC is correlated with a velocity of shortening intermediate between slow (type 1) and 2B M H C S . ’ Subsequently, ~ it has been demonstrated5 that 2D/X fibers can be identified histochemically in rat limb muscles; they, like 2B fibers, are intermediate in reactivity for myosin ATPase with preincubation at p H 4.6 but, unlikd 2B fibers, display moderate to high reactivity for succinate dehydrogenase. We had demonstrated heterogeneity of MHC content in type 2B fibers of the superficial portion of the lateral gastrocnemius muscle (hereafter abbreviated SLG) of the rat,’ and it was suggested that MHC heterogeneity in SLG may be From the Department of Neurology, State University of New York. Health Science Center at Brooklyn, Brooklyn, New York Acknowledgments Thls work was partially funded by NINCDS ClDA Grant #NSO1125 Address reprint requests to Judith A Sawchak, MD, Department of Neurology, Box 1213, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203 Accepted for publication May 1, 1992 CCC 0148-639x1921121349-05 0 1992 John Wiley & Sons, Inc
New lsomyosin in Fast Muscle
d u e to presence of type 2DlX fibers.14 Identifying 2D/X fibers by the procedure of Gorza5 allowed us to study whether the heterogeneity of MHC expression in the SLG was d u e to the presence of 2D/X fibers. During this investigation, w e found that some, but not all, of the heterogeneity of rat SLG is correlated with the presence of type 2DlX fibers in this muscle. Moreover, we uncovered immunocytochemical evidence to suggest _that a novel type of fast MHC is expressed in rat skeletal muscle. This MHC isoform is absent in the levator ani and superficial vastus lateralis muscles of rats, but is present in SLG and EDL muscles in a subset of type 2B fibers which is distinct from that o f 2 D l X fibers. Immunoblots of gradient gels of MHC prepared from these muscles corroborate that the electrophoretically localized 2B band derived from rat skeletal muscle contains antigenically distinct isoforms of MHC. AND
Six normal adult Sprague-Dawley rats were maintained according to guidelines of the Animal Care and Use Committee of the State University of N~~ York ~ ~ science ~ center l ~prior hto ;heir use in this study. They were killed by decapitation and the lateral gastrocnemius muscle, and portions of the diaphragm, soleus, vastus lateralis (VL), extensor digitorum longus (EDL), and leva-
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tor ani (LA) muscles were dissected from each animal and frozen in liquid nitrogen-cooled isopentane and stored at -70". Ch-yosections of the muscles were reacted for myosin adenosine triphosphatase (ATPase) with preincubation at pH 9.4, 4.6, and 4.3"z'0and also reacted for succinic dehydrogenase (SDH)." Sections serial to those used for histochemical staining were reacted with anti-MHC monoclonal antibodies (MAbs) and visualized by indirected immunofluorescence as described before." T h e MHC MAbs used in this study included HM 1.2, which is reactive with MHC present in a subset of rat type 2 fibers and with all human type 2 fibers,x ALD 47 (specific for adult slow MHC),I8 and AI,D 19 (specific for all fast MHC).' Controls included (a) omitting MAbs from the prot.oco1 for immuriocytochemistry, and (b) processing sections of the different muscles (mounted on the same slide) together so that all muscle sections receive identical treatment. Myosin was extracted from the aforementioned rat muscles as described previously'5 and electrophoresed on 5% to 8% gradient SDS polyacrylamide gels with a stacking gel of 4% per the method of Bar and Pette.* Part of each gel was stained with either Coomassie blue or silver stain (Rio-rad Kit), and the other part was electrophoretically transferred to nitrocellulose (NC) sheets." The NC sheets were then treated with MAb HM 1.2 to detect reactive MHC bands by the avidin-biotin complex (ABC) peroxidase proce-
dure (Vector Laboratories). Controls included omitting treatment with either HM 1.2 or the second antibody complex in the ABC procedure.
RESULTS
In order to investigate the basis of the MHC heterogeneity in the SLG we first sought to determine if MAb HM 1.2 reacts with the 2D/X fibers in this muscle. Figure 1A shows the uniform 2B fiber type composition in the SLG demonstrated by the moderately strong staining of all myofibers when reacted with myosin ATPase with preincubation at pH 4.6. All o f these fibers in the SLG, in accordance with their 2B characteristics,x reacted strongly with preincubation at pH 9.4 and negatively with preincubation at pH 4.3 (data not shown), Figure lB, however, reveals the heterogeneity of MHC content in these fibers when reacted with MAb HM 1.2. At least three grades of reactivity with HM 1.2 are seen; that is, strongly reactive, moderately to minimally reactive, and nonreactive. When an adjacent section is reacted for SDH one can see that some of the fibers that are very strongly reactive with HM 1.2 are also strongly reactive with SDH (Fig. lC), and hence, are 2D/X fibers.5 Thus, some of the heterogeneity demonstrated with MAb HM 1.2 in the SLG can be attributed to the presence of type 2D/X fibers (approximately 13% of the fibers in the SLAG).
FIGURE 1. Serial frozen sections of rat superficial lateral gastrocnemius (SLG) muscle treated with adenosine triphosphatase (ATPase) with acid (pH 4.6) at preincubation (A), indirect imrnunofluorescence with MAb HM 1.2 (B),and reacted for succinic dehydrogenase (SDH). (C). Note the uniform ATPase reaction of the SLG fibers in (A) as contrasted to the varied reactivity with HM 1.2 (B) and SDH (C).The fibers, which are dark with SDH, are type 2DIX fibers labeled 20. Bar = 50 prn.
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However, the remainder of the fibers in the field which have to be considered as “true 2B” (non2D/X) fibers still demonstrate at least two grades of reactivity with HM 1.2 (Fig. 1). Depending on area sampled, 17% to 36% of these “true 2B” fibers react with HM 1.2. In sections of rat EDL muscle, 2A, 2B, and 2D/X fibers could be identified histochemically. When treated with the same protocol as in Figure 1, all 2A and 2D/X fibers reacted positively with HM 1.2, and the pattern of reactivity of “true 2B” fibers with HM 1.2 in the EDL was similar to that seen in the SLG: HM 1.2 reacted with only a subset of the “true 2B” fibers (not shown). In contrast, the only HM 1.2 positive fibers in the homogenous field of 2B fibers of the superficial VL muscle were 2D/X fibers (not shown). When HM 1.2 was reacted with sections of diaphragm muscles, type l fibers were negative, but all type 2 fibers were positive (Fig. 2). It has been shown previously that diaphragm of adult rat contains about 14% type 1 MHC, 32% 2A MHC, and 53% 2D/X,“I6 with true 2B MHC being virtually absent in this muscle.7314Furthermore, no coexpression of 2A MHC in 2D/X fibers of the diaphragm has been demonstrated.’6 It follows that MAb HM 1.2 reacts with 2D/X MHC in the rat diaphragm as well as in appendicular muscles. N o type 2D/X fibers nor any HM 1.2 reactive fibers were seen in the LA muscle (Fig. S ) , which is considered to be a pure true type 2B muscle. All of the fibers of the LA, however, reacted strongly
with ALD 19, a MAb which reacts with all subtypes of fast fibers (data not shown). In summary, HM 1.2 reacts with type 2A fibers, type 2D/X fibers (both in the rat diaphragm as well as appendages), and with a subset of “2B” fibers in the SLG and EDL, but not in the VL. Immunoblots of SDS-polyacrylamide gradient gels containing MHCs prepared from the lateral gastrocnemius, EDL, LA, and soleus muscles show that there is antigenic heterogeneity in the 2B MHC from these muscles (Fig. 4). HM 1.2 reacts with the 2B MHC band derived from the lateral gastrocnemius and EDL muscles but not with that from the LA muscle in the adjacent lane (Fig. 4B). Despite the fact that, in our study, the gradient gel system did not distinguish the bands of 2A MHC from the 2D/X MHC (Fig. 4A),7 the bands corresponding to type 1, 2D/X, and 2B MHCs were well-separated, hence, our results clearly support the concept of antigenic heterogeneity of MHC in rat type 2B fibers as suggested by the immunocytochemical studies.
DISCUSSION
It has been suggested previously l 4 that the heterogeneity of reactivity of SLG fibers seen by immunocytochemical staining with HM 1.2 might be due to the presence of 2D/X fibers. We confirm that the SLG does have a significant number of 2D/X fibers which react positively with HM 1.2, thus establishing that a part of the HM 1.2 de-
FIGURE 2. Serial frozen cross sections of rat diaphragm muscle treated with myosin ATPase reaction with acid (pH 4.6) at preincubation (A), indirect immunofluorescencewith monoclonal antibody (MAb) HM 1.2 (B), and ALD 47 (C). Note that ALD 47 reacts with the type 1 fibers and the remainder are type 2 fibers which all react with HM 1.2. Bar = 50 pm.
New lsornyosin in Fast Muscle
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FIGURE 3.Serial frozen cross sections of LA muscle of a male rat treated as described for Figure 1. Although all the fibers are type 2B (A), there is no reactivity with HM 1.2 (B), nor for SDH (C).
tected heterogeneity of the SLG 2B fibers is due to the presence of 2D/X fibers. However, the presence of 2D/X MHC doe5 not account for all of the heterogeneity, since there are a number of bona fide 2B (i.e., n o n 3 D / X ) fi-
A
SOL
2A-2DI
LA B
--
GAS
EDL
Mm-
bers in the S I S which are also reactive with HM 1.2. HM 1.2 reactivity in a subset of true 2B fibers of SLG may represent coexpression of 2A and/or 2D/X MHC in such fibers. Although coexpression of myosin isoforms has been found in diseased“ and functionally altered muscles’ and in normal eye muscles,6 Gorza5 did not find coexpression of 2A or 2D/X and 2B MHC isoform in any myofibers of tibialis anterior, soleus, peroneus, gastrocnemius, or EDL muscles of normal rats. Furthermore, we have found “non-2D/X HM 1.2 positive” 2B fibers in EDL muscle in which biochemical analysis of microdissected and histochemically identified single fibers16 did not show coexpression of MHC isoforms, thus making the heterogeneity of 2B fibers in EDL (and by comparison SLG) of normal rats unlikely to be due t; coexpression of multiple MHC isoforms. Therefore, we postulate that a previously undetected type of fast MHC is present in certain 2B fibers of SLG, EDL, and possibly other skeletal muscles of the rat. T h e possibility that the hitherto identified “true 2B” fibers may contain two distinct myosin isoforms is reinforced by comparison of SLG and EDL muscles with the LA muscles. The latter, ‘Onsidered to be Of 2B fibers sively, shows no reactivity with HM 1.2 either on cryosection or in Western blots. Further comparison of immunoblots of myosin from LA and other muscles reveal presence of two antigenically distinct MHC isoforms with the Same mobility as that of 2B MHC in gradient gels. Based on the similar electrophoretic mobility of
-
B PA-2D/X+h-,
2B4
1-
m -
FIGURE 4. SDS-polyacrylamide gel electrophoresis Of myosins from rat soleus (Sol), levator ani (LA), lateral gastrocnemius (GAS), and extensor digitorum longus (EDL) muscle on 5% to 8% gradient gel stained with silver (Bio-rad Kit) (A), and a corresponding immunoblot reacted with MAb HM 1.2 and processed for immunoperoxidase staining with an ABC kit (Vector Labs) (B). Only the top Portion of the gel is shown. The MHC bands are labeled 2A-2D/X, 28, and 1 in order of increasing mobility. Note in immunoblot (6) the absence of reactivity of the MHC band of type 1 myosin of soleus and of the 2 8 myosin of LA, but strong reactivity with the MHC band of 28 myosin of gastrocnemius and EDL muscles.
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this distinct MHC isoform with 2B MHC, the divergence between 2B MHC and the new MHC isoform may not involve a significant change in the size (MW) of the isoform. We suspect that the new isoform differs from 2B myosin in the primary structure of the rod portion of the protein, because the epitope for MAb HM 1.2 is located in the rod portion of the molecule (unpublished results, Sawchak, 1990), and the antigenic distinction is present in unfixed frozen sections of muscle used for immunocytochemistry as well as in proteins transferred from denaturing SDS gels used for immunoblots. Presently, it is not known if this fast MHC is a result of a posttranscriptional or posttranslational modification of a known MHC gene or whether it represents the product of a previously unknown MHC gene. Clinically, the results of this study are relevant in that it compels one to consider whether there is a similar undetected heterogeneity of 2B fibers in human muscle. Because it has recently been shown that human disease can be the result of genetically determined abnormalities of a selective MHC i ~ o f o r m it, ~becomes particularly important to identify the full complement of normal myosin isoforms in human muscle.
REFERENCES 1. Ausoni S , Gorza L, Schiaffino S , Gunderson K, Lomo -1: Expression of myosin heavy chain isoforms in stimulated fast and slow rat musc1es.J Neurosci 1990;lO: 153- 160. 2. Bar A, Pette D: Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett 1988;235:153- 155. 3. Brooke MH, Kaiser KK: Muscle fiber types: How many and what kind? Arch Neurol 1970;23:369-379. 4. Geisterfer-Lowrance AAT, Kass S, lanigawa G, Vosberg H-P, McKenna W, Seidman CE, Seidman JGH: A molecular basis for familial hypertrophic cardiomyopathy: A p cardiac myosin heavy chain gene missense mutation. Cell 1990;62:999- 1006. 5. Gorza L: Identification of a novel type 2 fiber population
New lsomyosin in Fast Muscle
in mammalian skeletal muscle by use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J Hi.stochem Cytochem 1990;38:257- 265. 6. Jacoby J, KO K, Weiss C, Rushbrook JI: Systematic variation in myosin expression along extraocular muscle fibres of the adult rat. J Mwcle Res Cell Mold 1989;11:25-40. 7 LaFramboise WA, Daood MJ, Guthrie KD, Moretti P, Schiaffino S , Ontel M: Electrophoretic separation of and inimunolgocial identification of type 2X myosin heavy chain in rat skeletal muscle. Biophyszca Riophys Aclu 1990; 1035: 109- 112. 8 . Leung B, Kula RW, Shafiq SA: Fiber types in normal and neonatally denervated fast muscles of the rat: immunocytochemical study with an antimyosin monoclonal antibody. Exp Neurol 1987;97:429-440. 9. Nachlas MM, Tsou KG, De Sousa E, Cheng, Seligman AM: Cytochemical demonstration of succinate dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J Hislochem Cytochem 1957;5:420. 10. Padykula HA, Herman E: Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques. J Histochem Cylochem 1955;3:161- 169. 11. SawchakJA, Leung B, Shafiq Saiyid A: Characterization of a monoclonal antibody to myosin specific for mammalian and human type 11 muscle fibers. J Neurol Sci 1985;69:247- 254. 12. Sawchak JA, Lewis S, Shafiq SA: Coexpression of myosin isoforms in muscle of patients with neurogenic disease. Mwcle Nerve 1989; 12:679-689. 13. Schiaffino S , Ausoni S, Gorza L, Saggin I,, Gunderson K, Lomo T: Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibers. Actu Physiol Scund 1988; 134:575-576. 14. Schiaffino S, Gorza L, Sartore S , Saggin L, Ausoni S, Vianello M, Gundersen K, Lomo T : Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.1 Muscle Res Cell Mold 1989; 10: 197-205. 15 Shafiq SA, Shimizu T, Fischman DA: Heterogeneity of type 1 skeletal muscle fibers revealed by monoclonal antibodv to slow mvosin. Muscle Nerve 1984:7:380-387. 16. Termin A, Staron RS, Pette D: Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Hzstochernzslry 1989;92:453-457. 17. Towbin H , Stahelin T, Gordon J: Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nut1 Acad Scz USA 1979;76:4350-4354. 18. Zhang Y, Sher J. Leung B, Shafiq SA: An immunocytochemical study of type I muscle fibres in developing human skeletal muscles. J Neurol Sci 1987;80:1 - 12.
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Key words: myosin heavy chain isoforms skeletal muscle monoclonal antibody MUSCLE & NERVE 15~1349-1353 1992
EVIDENCE FOR NEW ISOFORM OF FAST MYOSIN HEAVY CHAIN IN RAT SKELETAL MUSCLE JUDITH A. SAWCHAK, MD, BETTY LEUNG, BA, and SAYID A. SHAFIQ, PhD
T h r e e fast myosin heavy chain (MHC) isoforms, 2A, 2B, and 2D/X, have been distinguished in rat skeletal and assigned to fiber types (of the same names) by analyzing electrophoretically microdissected fragments of histochemically classified fibers.16 Physiologic studies have shown that 2D/X MHC is correlated with a velocity of shortening intermediate between slow (type 1) and 2B M H C S . ’ Subsequently, ~ it has been demonstrated5 that 2D/X fibers can be identified histochemically in rat limb muscles; they, like 2B fibers, are intermediate in reactivity for myosin ATPase with preincubation at p H 4.6 but, unlikd 2B fibers, display moderate to high reactivity for succinate dehydrogenase. We had demonstrated heterogeneity of MHC content in type 2B fibers of the superficial portion of the lateral gastrocnemius muscle (hereafter abbreviated SLG) of the rat,’ and it was suggested that MHC heterogeneity in SLG may be From the Department of Neurology, State University of New York. Health Science Center at Brooklyn, Brooklyn, New York Acknowledgments Thls work was partially funded by NINCDS ClDA Grant #NSO1125 Address reprint requests to Judith A Sawchak, MD, Department of Neurology, Box 1213, SUNY Health Science Center at Brooklyn, 450 Clarkson Avenue, Brooklyn, NY 11203 Accepted for publication May 1, 1992 CCC 0148-639x1921121349-05 0 1992 John Wiley & Sons, Inc
New lsomyosin in Fast Muscle
d u e to presence of type 2DlX fibers.14 Identifying 2D/X fibers by the procedure of Gorza5 allowed us to study whether the heterogeneity of MHC expression in the SLG was d u e to the presence of 2D/X fibers. During this investigation, w e found that some, but not all, of the heterogeneity of rat SLG is correlated with the presence of type 2DlX fibers in this muscle. Moreover, we uncovered immunocytochemical evidence to suggest _that a novel type of fast MHC is expressed in rat skeletal muscle. This MHC isoform is absent in the levator ani and superficial vastus lateralis muscles of rats, but is present in SLG and EDL muscles in a subset of type 2B fibers which is distinct from that o f 2 D l X fibers. Immunoblots of gradient gels of MHC prepared from these muscles corroborate that the electrophoretically localized 2B band derived from rat skeletal muscle contains antigenically distinct isoforms of MHC. AND
Six normal adult Sprague-Dawley rats were maintained according to guidelines of the Animal Care and Use Committee of the State University of N~~ York ~ ~ science ~ center l ~prior hto ;heir use in this study. They were killed by decapitation and the lateral gastrocnemius muscle, and portions of the diaphragm, soleus, vastus lateralis (VL), extensor digitorum longus (EDL), and leva-
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1349
tor ani (LA) muscles were dissected from each animal and frozen in liquid nitrogen-cooled isopentane and stored at -70". Ch-yosections of the muscles were reacted for myosin adenosine triphosphatase (ATPase) with preincubation at pH 9.4, 4.6, and 4.3"z'0and also reacted for succinic dehydrogenase (SDH)." Sections serial to those used for histochemical staining were reacted with anti-MHC monoclonal antibodies (MAbs) and visualized by indirected immunofluorescence as described before." T h e MHC MAbs used in this study included HM 1.2, which is reactive with MHC present in a subset of rat type 2 fibers and with all human type 2 fibers,x ALD 47 (specific for adult slow MHC),I8 and AI,D 19 (specific for all fast MHC).' Controls included (a) omitting MAbs from the prot.oco1 for immuriocytochemistry, and (b) processing sections of the different muscles (mounted on the same slide) together so that all muscle sections receive identical treatment. Myosin was extracted from the aforementioned rat muscles as described previously'5 and electrophoresed on 5% to 8% gradient SDS polyacrylamide gels with a stacking gel of 4% per the method of Bar and Pette.* Part of each gel was stained with either Coomassie blue or silver stain (Rio-rad Kit), and the other part was electrophoretically transferred to nitrocellulose (NC) sheets." The NC sheets were then treated with MAb HM 1.2 to detect reactive MHC bands by the avidin-biotin complex (ABC) peroxidase proce-
dure (Vector Laboratories). Controls included omitting treatment with either HM 1.2 or the second antibody complex in the ABC procedure.
RESULTS
In order to investigate the basis of the MHC heterogeneity in the SLG we first sought to determine if MAb HM 1.2 reacts with the 2D/X fibers in this muscle. Figure 1A shows the uniform 2B fiber type composition in the SLG demonstrated by the moderately strong staining of all myofibers when reacted with myosin ATPase with preincubation at pH 4.6. All o f these fibers in the SLG, in accordance with their 2B characteristics,x reacted strongly with preincubation at pH 9.4 and negatively with preincubation at pH 4.3 (data not shown), Figure lB, however, reveals the heterogeneity of MHC content in these fibers when reacted with MAb HM 1.2. At least three grades of reactivity with HM 1.2 are seen; that is, strongly reactive, moderately to minimally reactive, and nonreactive. When an adjacent section is reacted for SDH one can see that some of the fibers that are very strongly reactive with HM 1.2 are also strongly reactive with SDH (Fig. lC), and hence, are 2D/X fibers.5 Thus, some of the heterogeneity demonstrated with MAb HM 1.2 in the SLG can be attributed to the presence of type 2D/X fibers (approximately 13% of the fibers in the SLAG).
FIGURE 1. Serial frozen sections of rat superficial lateral gastrocnemius (SLG) muscle treated with adenosine triphosphatase (ATPase) with acid (pH 4.6) at preincubation (A), indirect imrnunofluorescence with MAb HM 1.2 (B),and reacted for succinic dehydrogenase (SDH). (C). Note the uniform ATPase reaction of the SLG fibers in (A) as contrasted to the varied reactivity with HM 1.2 (B) and SDH (C).The fibers, which are dark with SDH, are type 2DIX fibers labeled 20. Bar = 50 prn.
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However, the remainder of the fibers in the field which have to be considered as “true 2B” (non2D/X) fibers still demonstrate at least two grades of reactivity with HM 1.2 (Fig. 1). Depending on area sampled, 17% to 36% of these “true 2B” fibers react with HM 1.2. In sections of rat EDL muscle, 2A, 2B, and 2D/X fibers could be identified histochemically. When treated with the same protocol as in Figure 1, all 2A and 2D/X fibers reacted positively with HM 1.2, and the pattern of reactivity of “true 2B” fibers with HM 1.2 in the EDL was similar to that seen in the SLG: HM 1.2 reacted with only a subset of the “true 2B” fibers (not shown). In contrast, the only HM 1.2 positive fibers in the homogenous field of 2B fibers of the superficial VL muscle were 2D/X fibers (not shown). When HM 1.2 was reacted with sections of diaphragm muscles, type l fibers were negative, but all type 2 fibers were positive (Fig. 2). It has been shown previously that diaphragm of adult rat contains about 14% type 1 MHC, 32% 2A MHC, and 53% 2D/X,“I6 with true 2B MHC being virtually absent in this muscle.7314Furthermore, no coexpression of 2A MHC in 2D/X fibers of the diaphragm has been demonstrated.’6 It follows that MAb HM 1.2 reacts with 2D/X MHC in the rat diaphragm as well as in appendicular muscles. N o type 2D/X fibers nor any HM 1.2 reactive fibers were seen in the LA muscle (Fig. S ) , which is considered to be a pure true type 2B muscle. All of the fibers of the LA, however, reacted strongly
with ALD 19, a MAb which reacts with all subtypes of fast fibers (data not shown). In summary, HM 1.2 reacts with type 2A fibers, type 2D/X fibers (both in the rat diaphragm as well as appendages), and with a subset of “2B” fibers in the SLG and EDL, but not in the VL. Immunoblots of SDS-polyacrylamide gradient gels containing MHCs prepared from the lateral gastrocnemius, EDL, LA, and soleus muscles show that there is antigenic heterogeneity in the 2B MHC from these muscles (Fig. 4). HM 1.2 reacts with the 2B MHC band derived from the lateral gastrocnemius and EDL muscles but not with that from the LA muscle in the adjacent lane (Fig. 4B). Despite the fact that, in our study, the gradient gel system did not distinguish the bands of 2A MHC from the 2D/X MHC (Fig. 4A),7 the bands corresponding to type 1, 2D/X, and 2B MHCs were well-separated, hence, our results clearly support the concept of antigenic heterogeneity of MHC in rat type 2B fibers as suggested by the immunocytochemical studies.
DISCUSSION
It has been suggested previously l 4 that the heterogeneity of reactivity of SLG fibers seen by immunocytochemical staining with HM 1.2 might be due to the presence of 2D/X fibers. We confirm that the SLG does have a significant number of 2D/X fibers which react positively with HM 1.2, thus establishing that a part of the HM 1.2 de-
FIGURE 2. Serial frozen cross sections of rat diaphragm muscle treated with myosin ATPase reaction with acid (pH 4.6) at preincubation (A), indirect immunofluorescencewith monoclonal antibody (MAb) HM 1.2 (B), and ALD 47 (C). Note that ALD 47 reacts with the type 1 fibers and the remainder are type 2 fibers which all react with HM 1.2. Bar = 50 pm.
New lsornyosin in Fast Muscle
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FIGURE 3.Serial frozen cross sections of LA muscle of a male rat treated as described for Figure 1. Although all the fibers are type 2B (A), there is no reactivity with HM 1.2 (B), nor for SDH (C).
tected heterogeneity of the SLG 2B fibers is due to the presence of 2D/X fibers. However, the presence of 2D/X MHC doe5 not account for all of the heterogeneity, since there are a number of bona fide 2B (i.e., n o n 3 D / X ) fi-
A
SOL
2A-2DI
LA B
--
GAS
EDL
Mm-
bers in the S I S which are also reactive with HM 1.2. HM 1.2 reactivity in a subset of true 2B fibers of SLG may represent coexpression of 2A and/or 2D/X MHC in such fibers. Although coexpression of myosin isoforms has been found in diseased“ and functionally altered muscles’ and in normal eye muscles,6 Gorza5 did not find coexpression of 2A or 2D/X and 2B MHC isoform in any myofibers of tibialis anterior, soleus, peroneus, gastrocnemius, or EDL muscles of normal rats. Furthermore, we have found “non-2D/X HM 1.2 positive” 2B fibers in EDL muscle in which biochemical analysis of microdissected and histochemically identified single fibers16 did not show coexpression of MHC isoforms, thus making the heterogeneity of 2B fibers in EDL (and by comparison SLG) of normal rats unlikely to be due t; coexpression of multiple MHC isoforms. Therefore, we postulate that a previously undetected type of fast MHC is present in certain 2B fibers of SLG, EDL, and possibly other skeletal muscles of the rat. T h e possibility that the hitherto identified “true 2B” fibers may contain two distinct myosin isoforms is reinforced by comparison of SLG and EDL muscles with the LA muscles. The latter, ‘Onsidered to be Of 2B fibers sively, shows no reactivity with HM 1.2 either on cryosection or in Western blots. Further comparison of immunoblots of myosin from LA and other muscles reveal presence of two antigenically distinct MHC isoforms with the Same mobility as that of 2B MHC in gradient gels. Based on the similar electrophoretic mobility of
-
B PA-2D/X+h-,
2B4
1-
m -
FIGURE 4. SDS-polyacrylamide gel electrophoresis Of myosins from rat soleus (Sol), levator ani (LA), lateral gastrocnemius (GAS), and extensor digitorum longus (EDL) muscle on 5% to 8% gradient gel stained with silver (Bio-rad Kit) (A), and a corresponding immunoblot reacted with MAb HM 1.2 and processed for immunoperoxidase staining with an ABC kit (Vector Labs) (B). Only the top Portion of the gel is shown. The MHC bands are labeled 2A-2D/X, 28, and 1 in order of increasing mobility. Note in immunoblot (6) the absence of reactivity of the MHC band of type 1 myosin of soleus and of the 2 8 myosin of LA, but strong reactivity with the MHC band of 28 myosin of gastrocnemius and EDL muscles.
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this distinct MHC isoform with 2B MHC, the divergence between 2B MHC and the new MHC isoform may not involve a significant change in the size (MW) of the isoform. We suspect that the new isoform differs from 2B myosin in the primary structure of the rod portion of the protein, because the epitope for MAb HM 1.2 is located in the rod portion of the molecule (unpublished results, Sawchak, 1990), and the antigenic distinction is present in unfixed frozen sections of muscle used for immunocytochemistry as well as in proteins transferred from denaturing SDS gels used for immunoblots. Presently, it is not known if this fast MHC is a result of a posttranscriptional or posttranslational modification of a known MHC gene or whether it represents the product of a previously unknown MHC gene. Clinically, the results of this study are relevant in that it compels one to consider whether there is a similar undetected heterogeneity of 2B fibers in human muscle. Because it has recently been shown that human disease can be the result of genetically determined abnormalities of a selective MHC i ~ o f o r m it, ~becomes particularly important to identify the full complement of normal myosin isoforms in human muscle.
REFERENCES 1. Ausoni S , Gorza L, Schiaffino S , Gunderson K, Lomo -1: Expression of myosin heavy chain isoforms in stimulated fast and slow rat musc1es.J Neurosci 1990;lO: 153- 160. 2. Bar A, Pette D: Three fast myosin heavy chains in adult rat skeletal muscle. FEBS Lett 1988;235:153- 155. 3. Brooke MH, Kaiser KK: Muscle fiber types: How many and what kind? Arch Neurol 1970;23:369-379. 4. Geisterfer-Lowrance AAT, Kass S, lanigawa G, Vosberg H-P, McKenna W, Seidman CE, Seidman JGH: A molecular basis for familial hypertrophic cardiomyopathy: A p cardiac myosin heavy chain gene missense mutation. Cell 1990;62:999- 1006. 5. Gorza L: Identification of a novel type 2 fiber population
New lsomyosin in Fast Muscle
in mammalian skeletal muscle by use of histochemical myosin ATPase and anti-myosin monoclonal antibodies. J Hi.stochem Cytochem 1990;38:257- 265. 6. Jacoby J, KO K, Weiss C, Rushbrook JI: Systematic variation in myosin expression along extraocular muscle fibres of the adult rat. J Mwcle Res Cell Mold 1989;11:25-40. 7 LaFramboise WA, Daood MJ, Guthrie KD, Moretti P, Schiaffino S , Ontel M: Electrophoretic separation of and inimunolgocial identification of type 2X myosin heavy chain in rat skeletal muscle. Biophyszca Riophys Aclu 1990; 1035: 109- 112. 8 . Leung B, Kula RW, Shafiq SA: Fiber types in normal and neonatally denervated fast muscles of the rat: immunocytochemical study with an antimyosin monoclonal antibody. Exp Neurol 1987;97:429-440. 9. Nachlas MM, Tsou KG, De Sousa E, Cheng, Seligman AM: Cytochemical demonstration of succinate dehydrogenase by the use of a new p-nitrophenyl substituted ditetrazole. J Hislochem Cytochem 1957;5:420. 10. Padykula HA, Herman E: Factors affecting the activity of adenosine triphosphatase and other phosphatases as measured by histochemical techniques. J Histochem Cylochem 1955;3:161- 169. 11. SawchakJA, Leung B, Shafiq Saiyid A: Characterization of a monoclonal antibody to myosin specific for mammalian and human type 11 muscle fibers. J Neurol Sci 1985;69:247- 254. 12. Sawchak JA, Lewis S, Shafiq SA: Coexpression of myosin isoforms in muscle of patients with neurogenic disease. Mwcle Nerve 1989; 12:679-689. 13. Schiaffino S , Ausoni S, Gorza L, Saggin I,, Gunderson K, Lomo T: Myosin heavy chain isoforms and velocity of shortening of type 2 skeletal muscle fibers. Actu Physiol Scund 1988; 134:575-576. 14. Schiaffino S, Gorza L, Sartore S , Saggin L, Ausoni S, Vianello M, Gundersen K, Lomo T : Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.1 Muscle Res Cell Mold 1989; 10: 197-205. 15 Shafiq SA, Shimizu T, Fischman DA: Heterogeneity of type 1 skeletal muscle fibers revealed by monoclonal antibodv to slow mvosin. Muscle Nerve 1984:7:380-387. 16. Termin A, Staron RS, Pette D: Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle. Hzstochernzslry 1989;92:453-457. 17. Towbin H , Stahelin T, Gordon J: Electrophoretic transfer of protein from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Nut1 Acad Scz USA 1979;76:4350-4354. 18. Zhang Y, Sher J. Leung B, Shafiq SA: An immunocytochemical study of type I muscle fibres in developing human skeletal muscles. J Neurol Sci 1987;80:1 - 12.
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