Mdx mouse dystrophy is characterized by the absence in the muscle cytoplasmic membrane of a high molecular weight protein called dystrophin. A possible avenue for treatment of muscular dystrophies is to inject normal myoblasts in a dystrophic muscle to form hybrid muscle fibers. Hybrid myotubes were formed in vitro by the fusion of normal rat and dystrophic mouse (mdx) myoblasts. Staining with Hoechst dye 33258 permitted the clear distinction of mouse and rat nuclei. lmmunostaining demonstrated that dystrophin was present over the entire membrane of all hydrid myotubes even when nuclei ratio normal/dystrophicwas low. Key words: dystrophin mdx mouse muscle cell culture hybridization muscular dystrophy MUSCLE & NERVE 14:178-182 1991
DYSTROPHIN EXPRESSION IN MYOTUBES FORMED BY THE F U S I O N OF NORMAL AND DYSTROPHIC MYOBLASTS JOHNNY HUARD, MSc, CLAUDE LABRECQUE, MSc, GUY DANSEREAU, BSc, LYNDA ROBITAILLE, BSc, and JACQUES P. TREMBLAY, PhD
al, 13 and Karpati et al.6 have recently shown dysDystrophin is present in the membrane of normal trophin to be present in mdx muscle following in muscle fibers and absent in those of Duchenne pa1,12,13,15,16,18 vivo normal myoblast transplantation. Because This absence tient and mdx mice. they were not able to distinguish between the nucould be responsible for damage caused to this clei originating from the normal and dystrophic membrane during muscle contraction and for the myoblasts, they could not clearly establish whether eventual necrosis of the muscle fiber."" A possidystrophin was present only near the normal nuble treatment for Duchenne muscular dystrophy clei. Since Pavlath et al.I4 recently showed the rewould consist of complementing the genome by striction of the expression of sarcomeric myosin injecting normal myoblast in a dystrophic muscle heavy chain to a nuclear domain, addressing that to form hybrid muscle fibers. This has been done with success in the dy" dystrophic m o u ~ e ~ " ~ ) ; question concerning the expression of dystrophin seemed an important step in our experiments. however, this mouse model has dystrophin present in its muscle membrane and has a differMATERIALS AND METHODS ent genetic alteration than the Duchenne patients. Another dystrophic animal model, the mdx Myoblasts were obtained from a normal rat emmouse, has an absence of dystrophin in the muscle bryo (19 days) and from mdx mouse embryo (17 membrane as in Duchenne patients. Patridge et days). After two days in culture medium 199 containing 10% horse serum, these cultures were mixed. After a 2 to 5 day-fusion period allowing apparition of a sufficient number of myotubes, the From the Laboratoire de Neurobiologie, Universite Laval. HBpital de I'Enfant-Jesus, Quebec, Canada cultures were fixed in 70% ethanol (1 min). The Acknowledgments: The authors thank Miss Marlyne Goulet for technical endogeneous peroxidase activity was quenched assistance and Miss Lyse Laroche for secretarial assistance The laborawith H,O, (0.3%, 30 min), nonspecific attachment tory team has been supported by a grant from Muscular Dystrophy Canada We also thank Genica Pharm Co. (Boston, MA) for providing us sites were blocked with normal rabbit serum (5%, with an excellent anti-dystrophin antibody. 30 min). The tissue was then incubated with an Address reprint requests to Jacques P Tremblay, Laboratoire de Neuroanti-dystrophin antibody or with pre-immune sebiologie. HBpital de I'Enfant-Jesus, 1401, 18e Rue, Quebec, Canada G I J rum (1: 1000, 1 h, Genica Pharm. Co. (Boston, 124 MA) followed by a 30 min incubation with a rabbit Accepted May 4, 1990. anti-sheep antibody conjugated with horseradish CCC 0148 - 639X/91/020178 05 $04.00 peroxidase (HRP) (Dako, Denmark). The peroxi0 1991 John Wiley & Sons, Inc. ~
178
Dystrophin Expression
in Myotubes
MUSCLE & NERVE
February 1991
FIGURE 1. lmmunoperoxidase reaction using an antibody against dystrophin on cryostat sections from normal rat (A) and dystrophic mouse (B). Note the positive reaction in normal rat and the absence of reaction in dystrophic mouse.
dase activity was revealed with DAB (0.5 mg/mL). Staining with Hoescht 33258 (0.01 mg/mL) for 10 min allowed us to differentiate granular mouse nuclei from uniform rat nuclei. For the in vivo study, 8 pm cryostat sections of 2-month-old normal rats arid mdx mice were used. RESULTS
The specificity of the antidystrophin antibody was first evaluated by immunostaining cryostat sections from normal rat and dystrophic (mdx) mouse muscle. Positive immunoperoxidase reaction was obtained on the normal rat sections (Fig. 1A) but not in dystrophic mouse sections (Fig. IB), thus showing the specificity of this antibody. This antibody has also been shown to label a high molecular weight ( ~ 4 0 0kd) by Western blot analysis (Genica Pharm. Co.) Cultures containing only myoblasts and myotubes obtained from a biopsy of an mdx mouse were also negative when immunostained with this antibody (Fig. 2B). However myotubes present in a normal rat muscle culture were stained with the
Dystrophin Expression in Myotubes
FIGURE 3. Normal rat myoblast cultures: (A) Hoechst dye 33258. (B) lmmunoperoxidase reaction using an antidystrophin antibody on the same myotubes in figure A. The two normal myotubes present are completely labeled for dystrophin. (C) Immunoperoxidase reaction in another normal rat myotube. Note the labeled myotubes and a few normal myoblasts (white arrow heads) weakly stained with the antidystrophin antibody. However most myoblasts are not stained (black arrow heads).
antidystrophin antibody over their entire surface (Figs. 3B, 3C, and 4A). Although most of the myoblasts in that culture were not stained with this antibody, about 5% showed weak immunostaining (Fig. 3C). No staining was obtained on normal rat myotubes when the primary antibody was replaced by the preimmune serum (Figs. 4B and 4C). Hybrid muscle cultures were then obtained by
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FIGURE 2. Dystrophic (mdx) mouse myoblast culture: (A) Phase contrast. (8) lmmunoperoxidase reaction using antidystrophin. The two dystrophic myotubes are not stained with the antidystrophin antibody.
FIGURE 5. Mixed culture with normal rat and dystrophic mouse myoblasts. (A) Hoechst dye: rat nuclei (smooth nuclei, arrow head) and mouse (dotted) nuclei. (B) Hybrid myotube with 2 nuclei from normal rat and 4 nuclei from dystrophic mice, is uniformly labeled with antidystrophin antibody.
FIGURE 6. Mixed culture containing normal rat and dystrophic mouse myoblasts. (A) Hoechst dye. Note the rat (smooth, white arrowhead) and mouse (dotted) nuclei. ( 8 ) lmmunoperoxidase reaction for dystrophin. Dystrophin is present over the entire myotube containing one normal nuclei and 7 dystrophic nuclei.
FIGURE 7. Mixed culture with normal rat and dystrophic mouse myoblasts. (A) Hoecht dye: rat (smooth, white arrow head) and mouse (dotted) nuclei. (B) Hybrid myotube with one normal nuclei of 12 nuclei. The immunoperoxidase reaction for dystrophin shows its presence over the whole myotube.
nuclei were uniformly labeled with this product. This labeling method permitted us to identify the hybrid myotubes, ie, those containing normal rat and dystrophic mouse nuclei. All hybrid myotubes observed were positively stained with the antidystrophin antibody (Figs. 5, 6, and 7). Although some of the hybrid myotubes contained a low proportion of normal rat nuclei, the dystrophin labeling was present over the entire myotubes. For example, Figure 7 illustrates a myotube containing only 1 normal rat nucleus of 12 nuclei. The dystrophin labeling on hybrid myotubes was similar to that obtained on the normal rat myotubes. DISCUSSION
FIGURE 4. (A) Normal rat myoblast culture. Dystrophin was revealed by an imrnunoperoxidase reaction. In this picture taken without phase contrast, the myotube is clearly labeled while most of the rnyoblasts are unlabeled. (B) Normal rat myoblast culture. During the immunoperoxidase reaction the primary antibody against dystrophin was replaced by the preimmune serum. This picture was taken with phase contrast to show the presence of myotubes. (C) The same field as in (B),observed without phase contrast to show the absence of irnmunoperoxidase reaction.
mixing normal rat and dystrophic mouse myoblasts. Two to 5 days later, following spontaneous fusion, these cultures were fixed. As already described by Pavlath et al.I4 and Blau et a1.,2 a punctuate labeling of the mouse nuclei was obtained with the Hoescht stain. We observed that the rat
Dystrophin Expression in Myotubes
Our results clearly show that normal rat muscle fibers contain dystrophin while mdx muscle fibers are not stained with a dystrophin antibody. This result confirms those obtained previously in mouse and in human normal and dystrophic muscles. 1,12,15 We also report for the first time, the absence of immunolabeling for dystrophin on dystrophic (mdx) myoblasts and myotubes in culture. Dystrophin labeling was observed over the whole myotube even on the smallest normal rat myotubes. Although positive immunostaining was previously reported on normal human myotubes by Klamut et al? and Miranda et a1.,12 the immunological reaction was weak and nonuniform. This could be due to a less rapid formation and incorporation of dystrophin in human myotubes than in rat or mouse myotubes. Indeed it is well known that the human myotubes do not mature as rapidly in culture as chick and mouse myotubes and that aneurally cultured human myotubes do not contract even when cross striations are Most of the normal rat myoblasts were not immunopositive for dystrophin; however, a few normal myoblasts were weakly labeled and a similar observation was also done by Miranda et a1.12 These positive myoblasts were often located near myotubes and it is possible that they were in a more differentiated state. Indeed, Emerson and Beckner3 have observed that myosin was sometimes present in myoblasts. They concluded that myoblast fusion is not necessary for the expression of the muscular phenotype. Our most important result is the presence of dystrophin labeling over the complete hybrid myootubes. Pavlath et al.14 have reported that some proteins (eg, human myosin heavy chain and antigen 5.1 H 1 1) are only expressed near the nucleus that contains the genetic information. They have
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181
called this restricted region of expression (< 100 p,m) a “nuclear domain.” No nuclear domain was observed for dystrophin in our hybrid myotubes, although some containing only one normal nucleus were several hundred microns long. Expression of dystrophin was also obtained in hybrid muscle fibers formed in vivo by Karpati et a1.6 In that case a nuclear domain of about 90 to 130 km was observed. The presence of nuclear domain
for dystrophin may depend on the degree of maturity and the diameter of the muscle fiber. Our future experiments will try to verify this possibility. The expression of dystrophin in hybrid myotube is certainly another encouraging result in favor of myoblast transfer therapy for Duchenne muscular dystrophy as proposed by Law and coworkers.8- l o
REFERENCES 1. Arahata K, Ishiura S, Ishiguro ’1, Tsukahara 1,Suhara Y, Eguchi C, lsliihara T , Nonaka I , Ozawa E, Sugita H: Immunostaining of skeletal and cardiac muscle surface mcmhrane with antibody against Duchenne muscular dystrophy peptide. Nature 1988;333:861-863. 2. Blau HM et al: Plasticity of’ the differentiated state. Sczencr
1985;213:758-766. 3 . Emerson P, Reckner SK: Activation of myosin synthesis in fusing and mononucleatecl myohlasts. J Mol Biol 1975;93:431-447. 4. Iannaccone ST, Nagy B, Samaha JF: Partial biochemical maturation of anrurally cultured human skeletal muscle. Neurology 1982;32:846- 85 1. 5. Karpati G, Carpenter S, Prescott S: Small caliber skeletal muscle fibers d o not sufter necrosis in nidx mouse dystrophy. Muscle Nn-me 1988;11:795-803. 6. Karpati G, Pouliot Y, Zuha7ucka-Gaarn E, Carpenter S, Ray PN, Worton RG, Holland P: Dystropliin is expressed in mdx skeletal muscle hhers after normal myohlast iniplantation. A m ] Pathol 1‘389;135. 7. Klamut H.1, ZubrLycka-Gaarn EE, Bulman DE, Malhotra SB, Bodrug SE,Worton RG, Kay PN: Myogenic regulation of dystrophin gcne expression. R r Med Bull 1989;45:681702. 8. Law PK: Beneficial effects of transplanting normal limhbud mesenchyme into dystrophic niouse muscles. M u a d Nrroe 1982;5,619-627. 9. Law PK, Goodwin TG, Li H J : Ilistoincompatible myohlast injection improves muscle structure and function of dystrophic mice. Tmnsplantalion Proc 1988a;N: 1 1 14- 1119. 10. Law I’K, Goodwin ‘X,Wang MG: Noi-ma1 myohlast injection provide genetic trealnient for murine dystrophy. MILTclr Nrrve 1988b;l1:525-533.
182
Dystrophin Expression
in Myotubes
11. Lev AA, Feener CC, Kunkel LM, Brown RH: Expression of the Duchenne’s muscular dystrophy gene in cultured muscle cells.] Biol Chem 1987;262:15817- 15820. 12. Miranda AF, Bonilla E, Martucci G, Maraes C T , Hays AP, llimauro S: Immunocytochcmical study of dystrophin in muscle cultures from patients with Duchenne muscular dystrophy and unaffected control patients. Am J Pulhol 1988;132:410-416. 13. Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM: Conversion of mdx myofibres from dystrophin-negative to positive by injection of normal myoblasts. Nature 1989;337: 176- 179. 14. Pavlath GK, Rich K, Wehster SG, Blau HM: Localization of muscle gene products in nuclear domains. Nuture 1989;337:570-573. 15. Sugita H, Arahata K, Ishiguro T , Suhara Y, Tsukahara T, Ishiura S, Eguchi C, Nonaka I , Ozawa E: Negative immunostaining of Duchenne muscular dystrophy (DMD) ant1 mdx surface membrane with antibody against synthetic peptide fragment predicted from DMD cDNA. Proc Jpn Acad 1988;64,Ser-B:37- 39. 16. Watkins SC, Hoffman EP, Slayter HS, Kunkel LM: Immunoelectron microscopic localization of dystrophin in myofibres. Nature 1988;333:863-866. 17. Witkowski JA, Durhidge M, Duhowitz V: Growth of human muscle in tissue culture. In vztro 1976;12:98-106. 18. Zubrzycka-Gaarn EE, Bulman DE, Karpati G, Burghes HHM, Belfall B, Klamut HI, Talbot J , Hodges RS, Ray PN, Worton RG: T h e Duchenne muscular dystrophy gene product is localized in sarcolenima of human skeletal muscle. Nature 1988:333:466-469.
MUSCLE & NERVE
February 1991
DYSTROPHIN EXPRESSION IN MYOTUBES FORMED BY THE F U S I O N OF NORMAL AND DYSTROPHIC MYOBLASTS JOHNNY HUARD, MSc, CLAUDE LABRECQUE, MSc, GUY DANSEREAU, BSc, LYNDA ROBITAILLE, BSc, and JACQUES P. TREMBLAY, PhD
al, 13 and Karpati et al.6 have recently shown dysDystrophin is present in the membrane of normal trophin to be present in mdx muscle following in muscle fibers and absent in those of Duchenne pa1,12,13,15,16,18 vivo normal myoblast transplantation. Because This absence tient and mdx mice. they were not able to distinguish between the nucould be responsible for damage caused to this clei originating from the normal and dystrophic membrane during muscle contraction and for the myoblasts, they could not clearly establish whether eventual necrosis of the muscle fiber."" A possidystrophin was present only near the normal nuble treatment for Duchenne muscular dystrophy clei. Since Pavlath et al.I4 recently showed the rewould consist of complementing the genome by striction of the expression of sarcomeric myosin injecting normal myoblast in a dystrophic muscle heavy chain to a nuclear domain, addressing that to form hybrid muscle fibers. This has been done with success in the dy" dystrophic m o u ~ e ~ " ~ ) ; question concerning the expression of dystrophin seemed an important step in our experiments. however, this mouse model has dystrophin present in its muscle membrane and has a differMATERIALS AND METHODS ent genetic alteration than the Duchenne patients. Another dystrophic animal model, the mdx Myoblasts were obtained from a normal rat emmouse, has an absence of dystrophin in the muscle bryo (19 days) and from mdx mouse embryo (17 membrane as in Duchenne patients. Patridge et days). After two days in culture medium 199 containing 10% horse serum, these cultures were mixed. After a 2 to 5 day-fusion period allowing apparition of a sufficient number of myotubes, the From the Laboratoire de Neurobiologie, Universite Laval. HBpital de I'Enfant-Jesus, Quebec, Canada cultures were fixed in 70% ethanol (1 min). The Acknowledgments: The authors thank Miss Marlyne Goulet for technical endogeneous peroxidase activity was quenched assistance and Miss Lyse Laroche for secretarial assistance The laborawith H,O, (0.3%, 30 min), nonspecific attachment tory team has been supported by a grant from Muscular Dystrophy Canada We also thank Genica Pharm Co. (Boston, MA) for providing us sites were blocked with normal rabbit serum (5%, with an excellent anti-dystrophin antibody. 30 min). The tissue was then incubated with an Address reprint requests to Jacques P Tremblay, Laboratoire de Neuroanti-dystrophin antibody or with pre-immune sebiologie. HBpital de I'Enfant-Jesus, 1401, 18e Rue, Quebec, Canada G I J rum (1: 1000, 1 h, Genica Pharm. Co. (Boston, 124 MA) followed by a 30 min incubation with a rabbit Accepted May 4, 1990. anti-sheep antibody conjugated with horseradish CCC 0148 - 639X/91/020178 05 $04.00 peroxidase (HRP) (Dako, Denmark). The peroxi0 1991 John Wiley & Sons, Inc. ~
178
Dystrophin Expression
in Myotubes
MUSCLE & NERVE
February 1991
FIGURE 1. lmmunoperoxidase reaction using an antibody against dystrophin on cryostat sections from normal rat (A) and dystrophic mouse (B). Note the positive reaction in normal rat and the absence of reaction in dystrophic mouse.
dase activity was revealed with DAB (0.5 mg/mL). Staining with Hoescht 33258 (0.01 mg/mL) for 10 min allowed us to differentiate granular mouse nuclei from uniform rat nuclei. For the in vivo study, 8 pm cryostat sections of 2-month-old normal rats arid mdx mice were used. RESULTS
The specificity of the antidystrophin antibody was first evaluated by immunostaining cryostat sections from normal rat and dystrophic (mdx) mouse muscle. Positive immunoperoxidase reaction was obtained on the normal rat sections (Fig. 1A) but not in dystrophic mouse sections (Fig. IB), thus showing the specificity of this antibody. This antibody has also been shown to label a high molecular weight ( ~ 4 0 0kd) by Western blot analysis (Genica Pharm. Co.) Cultures containing only myoblasts and myotubes obtained from a biopsy of an mdx mouse were also negative when immunostained with this antibody (Fig. 2B). However myotubes present in a normal rat muscle culture were stained with the
Dystrophin Expression in Myotubes
FIGURE 3. Normal rat myoblast cultures: (A) Hoechst dye 33258. (B) lmmunoperoxidase reaction using an antidystrophin antibody on the same myotubes in figure A. The two normal myotubes present are completely labeled for dystrophin. (C) Immunoperoxidase reaction in another normal rat myotube. Note the labeled myotubes and a few normal myoblasts (white arrow heads) weakly stained with the antidystrophin antibody. However most myoblasts are not stained (black arrow heads).
antidystrophin antibody over their entire surface (Figs. 3B, 3C, and 4A). Although most of the myoblasts in that culture were not stained with this antibody, about 5% showed weak immunostaining (Fig. 3C). No staining was obtained on normal rat myotubes when the primary antibody was replaced by the preimmune serum (Figs. 4B and 4C). Hybrid muscle cultures were then obtained by
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FIGURE 2. Dystrophic (mdx) mouse myoblast culture: (A) Phase contrast. (8) lmmunoperoxidase reaction using antidystrophin. The two dystrophic myotubes are not stained with the antidystrophin antibody.
FIGURE 5. Mixed culture with normal rat and dystrophic mouse myoblasts. (A) Hoechst dye: rat nuclei (smooth nuclei, arrow head) and mouse (dotted) nuclei. (B) Hybrid myotube with 2 nuclei from normal rat and 4 nuclei from dystrophic mice, is uniformly labeled with antidystrophin antibody.
FIGURE 6. Mixed culture containing normal rat and dystrophic mouse myoblasts. (A) Hoechst dye. Note the rat (smooth, white arrowhead) and mouse (dotted) nuclei. ( 8 ) lmmunoperoxidase reaction for dystrophin. Dystrophin is present over the entire myotube containing one normal nuclei and 7 dystrophic nuclei.
FIGURE 7. Mixed culture with normal rat and dystrophic mouse myoblasts. (A) Hoecht dye: rat (smooth, white arrow head) and mouse (dotted) nuclei. (B) Hybrid myotube with one normal nuclei of 12 nuclei. The immunoperoxidase reaction for dystrophin shows its presence over the whole myotube.
nuclei were uniformly labeled with this product. This labeling method permitted us to identify the hybrid myotubes, ie, those containing normal rat and dystrophic mouse nuclei. All hybrid myotubes observed were positively stained with the antidystrophin antibody (Figs. 5, 6, and 7). Although some of the hybrid myotubes contained a low proportion of normal rat nuclei, the dystrophin labeling was present over the entire myotubes. For example, Figure 7 illustrates a myotube containing only 1 normal rat nucleus of 12 nuclei. The dystrophin labeling on hybrid myotubes was similar to that obtained on the normal rat myotubes. DISCUSSION
FIGURE 4. (A) Normal rat myoblast culture. Dystrophin was revealed by an imrnunoperoxidase reaction. In this picture taken without phase contrast, the myotube is clearly labeled while most of the rnyoblasts are unlabeled. (B) Normal rat myoblast culture. During the immunoperoxidase reaction the primary antibody against dystrophin was replaced by the preimmune serum. This picture was taken with phase contrast to show the presence of myotubes. (C) The same field as in (B),observed without phase contrast to show the absence of irnmunoperoxidase reaction.
mixing normal rat and dystrophic mouse myoblasts. Two to 5 days later, following spontaneous fusion, these cultures were fixed. As already described by Pavlath et al.I4 and Blau et a1.,2 a punctuate labeling of the mouse nuclei was obtained with the Hoescht stain. We observed that the rat
Dystrophin Expression in Myotubes
Our results clearly show that normal rat muscle fibers contain dystrophin while mdx muscle fibers are not stained with a dystrophin antibody. This result confirms those obtained previously in mouse and in human normal and dystrophic muscles. 1,12,15 We also report for the first time, the absence of immunolabeling for dystrophin on dystrophic (mdx) myoblasts and myotubes in culture. Dystrophin labeling was observed over the whole myotube even on the smallest normal rat myotubes. Although positive immunostaining was previously reported on normal human myotubes by Klamut et al? and Miranda et a1.,12 the immunological reaction was weak and nonuniform. This could be due to a less rapid formation and incorporation of dystrophin in human myotubes than in rat or mouse myotubes. Indeed it is well known that the human myotubes do not mature as rapidly in culture as chick and mouse myotubes and that aneurally cultured human myotubes do not contract even when cross striations are Most of the normal rat myoblasts were not immunopositive for dystrophin; however, a few normal myoblasts were weakly labeled and a similar observation was also done by Miranda et a1.12 These positive myoblasts were often located near myotubes and it is possible that they were in a more differentiated state. Indeed, Emerson and Beckner3 have observed that myosin was sometimes present in myoblasts. They concluded that myoblast fusion is not necessary for the expression of the muscular phenotype. Our most important result is the presence of dystrophin labeling over the complete hybrid myootubes. Pavlath et al.14 have reported that some proteins (eg, human myosin heavy chain and antigen 5.1 H 1 1) are only expressed near the nucleus that contains the genetic information. They have
MUSCLE & NERVE
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181
called this restricted region of expression (< 100 p,m) a “nuclear domain.” No nuclear domain was observed for dystrophin in our hybrid myotubes, although some containing only one normal nucleus were several hundred microns long. Expression of dystrophin was also obtained in hybrid muscle fibers formed in vivo by Karpati et a1.6 In that case a nuclear domain of about 90 to 130 km was observed. The presence of nuclear domain
for dystrophin may depend on the degree of maturity and the diameter of the muscle fiber. Our future experiments will try to verify this possibility. The expression of dystrophin in hybrid myotube is certainly another encouraging result in favor of myoblast transfer therapy for Duchenne muscular dystrophy as proposed by Law and coworkers.8- l o
REFERENCES 1. Arahata K, Ishiura S, Ishiguro ’1, Tsukahara 1,Suhara Y, Eguchi C, lsliihara T , Nonaka I , Ozawa E, Sugita H: Immunostaining of skeletal and cardiac muscle surface mcmhrane with antibody against Duchenne muscular dystrophy peptide. Nature 1988;333:861-863. 2. Blau HM et al: Plasticity of’ the differentiated state. Sczencr
1985;213:758-766. 3 . Emerson P, Reckner SK: Activation of myosin synthesis in fusing and mononucleatecl myohlasts. J Mol Biol 1975;93:431-447. 4. Iannaccone ST, Nagy B, Samaha JF: Partial biochemical maturation of anrurally cultured human skeletal muscle. Neurology 1982;32:846- 85 1. 5. Karpati G, Carpenter S, Prescott S: Small caliber skeletal muscle fibers d o not sufter necrosis in nidx mouse dystrophy. Muscle Nn-me 1988;11:795-803. 6. Karpati G, Pouliot Y, Zuha7ucka-Gaarn E, Carpenter S, Ray PN, Worton RG, Holland P: Dystropliin is expressed in mdx skeletal muscle hhers after normal myohlast iniplantation. A m ] Pathol 1‘389;135. 7. Klamut H.1, ZubrLycka-Gaarn EE, Bulman DE, Malhotra SB, Bodrug SE,Worton RG, Kay PN: Myogenic regulation of dystrophin gcne expression. R r Med Bull 1989;45:681702. 8. Law PK: Beneficial effects of transplanting normal limhbud mesenchyme into dystrophic niouse muscles. M u a d Nrroe 1982;5,619-627. 9. Law PK, Goodwin TG, Li H J : Ilistoincompatible myohlast injection improves muscle structure and function of dystrophic mice. Tmnsplantalion Proc 1988a;N: 1 1 14- 1119. 10. Law I’K, Goodwin ‘X,Wang MG: Noi-ma1 myohlast injection provide genetic trealnient for murine dystrophy. MILTclr Nrrve 1988b;l1:525-533.
182
Dystrophin Expression
in Myotubes
11. Lev AA, Feener CC, Kunkel LM, Brown RH: Expression of the Duchenne’s muscular dystrophy gene in cultured muscle cells.] Biol Chem 1987;262:15817- 15820. 12. Miranda AF, Bonilla E, Martucci G, Maraes C T , Hays AP, llimauro S: Immunocytochcmical study of dystrophin in muscle cultures from patients with Duchenne muscular dystrophy and unaffected control patients. Am J Pulhol 1988;132:410-416. 13. Partridge TA, Morgan JE, Coulton GR, Hoffman EP, Kunkel LM: Conversion of mdx myofibres from dystrophin-negative to positive by injection of normal myoblasts. Nature 1989;337: 176- 179. 14. Pavlath GK, Rich K, Wehster SG, Blau HM: Localization of muscle gene products in nuclear domains. Nuture 1989;337:570-573. 15. Sugita H, Arahata K, Ishiguro T , Suhara Y, Tsukahara T, Ishiura S, Eguchi C, Nonaka I , Ozawa E: Negative immunostaining of Duchenne muscular dystrophy (DMD) ant1 mdx surface membrane with antibody against synthetic peptide fragment predicted from DMD cDNA. Proc Jpn Acad 1988;64,Ser-B:37- 39. 16. Watkins SC, Hoffman EP, Slayter HS, Kunkel LM: Immunoelectron microscopic localization of dystrophin in myofibres. Nature 1988;333:863-866. 17. Witkowski JA, Durhidge M, Duhowitz V: Growth of human muscle in tissue culture. In vztro 1976;12:98-106. 18. Zubrzycka-Gaarn EE, Bulman DE, Karpati G, Burghes HHM, Belfall B, Klamut HI, Talbot J , Hodges RS, Ray PN, Worton RG: T h e Duchenne muscular dystrophy gene product is localized in sarcolenima of human skeletal muscle. Nature 1988:333:466-469.
MUSCLE & NERVE
February 1991
