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Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves
Cell, Vol. 33, 297-304,
May 1983, CopyrIght
0 1983 by MIT
0092.8674/83/050297-08
$02,00/O
lsoform Variants of Troponin in Skeletal and Cardiac Muscle Cells Cultured with and without Nerves Naoji Toyota* and Yutaka Department of Anatomy School of Medicine Chiba University Chiba 280, Japan
Shimada+
Summary lmmunofluorescence microscopy shows that cultured skeletal and cardiac muscle cells of chicken embryos exhibit the same stainabilities with antibodies against skeletal and cardiac troponin components as do those in embryos. Muscle cells of each type cultured with motor or sympathetic nerves or in medium containing the nerve extract exhibit the same reactivities as do those in adult animals. Cardiac muscle cells incubated in the nerve-conditioned medium also change the form of troponin components to the adult type. It appears that the differentiation of individual muscle fibers to specific types is induced by nerves, and especially by the neurohumoral effect. Introduction It is becoming clear that some of the proteins synthesized in embryonic skeletal and cardiac muscles are qualitatively different from the adult forms and that succession of phenotypes to adult forms occurs as the muscles develop (Masaki and Yoshizaki, 1974; Rubinstein et al., 1977; Gauthier et al., 1978; Whalen et al., 1978; Hoh and Yeoh, 1979; Roy et al., 1979; Dhoot and Perry, 1980; Matsuda et al., 1981; Toyota and Shimada, 1981). In skeletal muscles, these transitions have been attributed to the establishment of the adult pattern of motoneuron activity. This assumption came from experiments with adult animals in which it was found that denervation of muscles causes their reactivities with antibodies to differ from those of normal muscles (Dhoot and Perry, 1982; Gauthier and Hobbs, 1982) and that cross-innervation of fast and slow muscle nerves brings about an interchange of the properties of proteins (Amphlett et al., 1975; Salmons and Sreter, 1976; Dhoot et al., 1981). On the other hand, some reports do not support the mediation of motoneurons for diversification of muscle proteins. Transition of the form of myosin from neonatal to fast has been shown to occur in the denervated fast muscle of the newborn rat; thus this change is presumed to be independent of innervation and programmed endogenously in the muscle fiber (Butler-Browne et al., 1982). Furthermore, in cultures of adult human muscle grown with the ventral part of the fetal rat spinal cord, the pattern of immunoreaction of myosin isoforms was found to be the * Present address: Division of Neurochemistry Psychiatric Research tute of Tokyo, Kamikitazawa, Setagaya, Tokyo 156, Japan. t To whom all correspondence should be addressed.
Insti-
same as that of the same muscle cultured aneurally (Schiaffino et al., 1982); the presence of the neural tissue appears to have no influence on isomyosin gene expression in the cocultures of tissues from heterospecies. Thus neurogenic contributions to the changes of skeletal myofibrillar proteins during development are still somewhat controversial. The neurogenic effects on the changes of cardiac myofibrillar proteins in embryos and adult animals are as yet unexplored. By making use of the advantages of Culture Systems, we have investigated the immunochemical properties of troponin (TN) components (T, I, and C) of embryonic skeletal and cardiac muscles in the presence and absence of nerves or nerve extract. The results indicate that the nerves and nerve extract affect the gene expression and induce the differentiation of individual muscle fibers to specific types. Results The general schema of differentiation of skeletal and cardiac myogenic cells has been reported elsewhere (DeHaan, 1970; Shimada, 1971) and will not be repeated here. When fragments of spinal cords (enriched in cholinergic motoneurons) or sympathetic ganglia (adrenergic neurons) were added to each muscle cell culture, they extended many processes on the skeletal and cardiac muscle cells. Although in such muscle-nerve cocultures not all muscle cells were contacted by nerves, no significant difference in the differentiation was found between the muscle cells that were contacted by nerves and those that were not. Cultures of skeletal muscle cells from embryonic pectoralis were stained with affinity-purified antibodies specific for adult skeletal and cardiac TN components (Figure 1). They began to be stained with antibodies at 2 days in vitro. All fibers reacted not only with the antibodies against all of the three components of skeletal TN (Figures la1 c), but also with the antibodies raised against cardiac TNT and TN-C (Figures Id, If). These reactivities of skeletal muscle cells alone, without nerve influence, remained unchanged during the subsequent culture days. When skeletal muscle cells were cultured with motor (Figure 2) or sympathetic nerves or in a medium containing nerve extract (Figure 3) their reactivities with antibodies to TN components changed after 1 week of culture. That is, skeletal muscle cells lost their stainability with antibodies against the heterologous muscle (anticardiac TN-T and TNC) (Figures 2c, 3d, 3f) and were stained only with antibodies specific for skeletal TN components (Figures 2b and 3a-3c). This change was elicited by the addition of more than six spinal cord fragments (approx. 1.8 X lo4 cells), 13 sympathetic ganglion fragments (approx. 2 X 1O5 cells), or 3.5 mg/ml protein of nerve extract per culture dish. With three cord fragments (approx. 9 X lo3 cells), 12 ganglion fragments (approx. 1.8 x IO5 cells), or 0.7 mg/ml protein of nerve extract, the change in the reactivities of skeletal muscle cells was only partial. In some cultures the stainability with antibody against cardiac TN-T disappeared while
Cell 298
Figure 1. Indirect
lmmunofluorescence
MIcrographs
of Embryonic
Skeletal Muscle Cells Cultured
without Nerves
Stalned with specific antibodies against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardiac TN-T (d). TN-I (e), and TN-C (f). Myotubes antibodles against cardiac TN-T (d) and TN-C (c) as well as skeletal TN components (a-c). Two weeks in vitro. Bar: 50 pm,
that with antibody against cardiac TN-C remained, whereas in other cultures the reverse was observed. With one cord fragment (approx. 3 x IO3 cells), one ganglion fragment (approx. 1.5 x 1 O4 cells), or 0.14 mg/ml protein of nerve extract, the reactivity of skeletal muscle cells was the same as that without nerves. The reactivities of skeletal muscle cells were not affected by nonneuronal tissues, even when those tissues were present at a IOO-fold higher cell concentration than the minimum concentration of nerve cells required for the transitions. With 18 kidney fragments (approx. 5.5 X IO6 cells) or 13 lung fragments (approx. 8 X IO6 cells), the stainabilities of skeletal muscle cells were unchanged. Cardiac muscle cells cultured in the absence of nerves or in medium without nerve extract were stained with the six antibodies (Figure 4). They began to react with antibodies 3 hr after inoculation. Cardiac myocytes reacted with antiskeletal TN-I (Figure 4b) as well as with anticardiac TNT, TN-I, and TN-C (Figures 4d-4f). These reactivities of
were stained with
cardiac myocytes remained unchanged even after a prolonged culture period. However, when these cardiac myocytes were incubated with motoneurons (Figure 5) or sympathetic ganglia, or were cultured in medium containing nerve extract, they reacted only with anticardiac TN components (Figure 5~); the stainability with heterologous antibody (antiskeletal TNI) disappeared after 1 week of culture (Figure 5b). The effect of nerve-conditioned medium was examined; in this medium cardiac myocytes came to exhibit the same stainabilities as those with nerves or nerve extract (Figure 6). These changes were evoked with ten spinal cord fragments (approx. 3 X lo4 cells), three ganglion fragments (approx. 4.5 x IO4 cells), 0.7 mg/ml protein of nerve extract per plate, or nerve-conditioned medium prepared from 1 x 1O7 spinal cord cells per plate. Below these cell numbers or at a lower protein concentration, the reactivities of cardiac muscle cells remained unchanged. When nonneuronal tissues (14 kidney fragments [ap-
Troponrn 299
in Skeletal and Cardiac Muscles
Figure 2. Skeletal Muscle
Cultured
In Vivo
Cells Cultured with Sprnal Cord
(a) General view of cultures. Phase-contrast mrcrograph. SC: spinal cord fragment. (b) Fluorescence mrcrograph of the region correspondrng to the rectangle in (a). Stained with antiskeletal TN-T. (c) Fluorescence micrograph of a portion from the same culture dish, but a different area from that of (a). Stained with antrcardrac TN-T. Myotubes reacted with antibody against skeletal TN (1-5 in a and b), but lost reactivity wrth antibody against cardiac TN (c). Two weeks in vitro. Bar. 100 pm
prox. 4 x IO” cells] or 25 lung fragments [approx. 1.5 X 1 Cl7cells]) were added to cardiac muscle cell cultures, the transition of the form of TN did not occur. The results are summarized in Table 1, Discussion The present immunofluorescence study showed that some of the TN components synthesized by embryonic skeletal and cardiac muscle cells cultured in the absence of nerves, nerve extract, or nerve-conditioned medium react with antibodies raised from both adult skeletal and cardiac muscles. Skeletal muscle cells reacted with antibodies against TN-T and TN-C from the adult skeletal muscles as well as with those from cardiac muscles, This suggests that skeletal muscle cells in vitro synthesize TN-T and TNC, which share the antigenicities for adult skeletal and cardiac muscles. Furthermore, cardiac muscle cells displayed multiple reactivity to antibodies raised against two different TN-Is from adult skeletal and cardiac muscles. This indicates that cardiac muscle cells in vitro synthesize TN-Is with antigenic determinants similar to those in adult skeletal and cardiac muscles. These reactivities of embryonic skeletal and cardiac muscle cells in vitro are the same
as those in the respective muscles in the embryo (Toyota and Shimada, 1981). The present study further showed that in cultures of muscle cells of each type with motoneurons (cholinergic nerves) or sympathetic ganglia (adrenergic nerves), or with the presence of nerve extract, the forms of TN components of skeletal (TN-T and TN-C) and cardiac (TN-I) muscle cells change to the forms characteristic of their adult tissue type (Toyota and Shimada, 1981). Since these changes of TN components from embryonic to adult forms occurred in muscle cells of each type that are contacted by nerve processes as well as in those that are not, and also in muscle cells cultured in the medium containing nerve extract (nerve-conditioned medium is also effective for cardiac myocytes), where no living nerve cells are present, the establishment of functional innervation or even physical contact with nerves does not appear to be required for muscle cells to change their TN component isoforms. Since these changes occurred regardless of the neuron types (cholinergic or adrenergic) added to muscle cultures (expression of transmitters [Furshpan et al., 19761 by these nerves in the mixed cultures was not studied here), and also occurred in nerve extract, which contains homogenates of a variety of nerve cell types (in cardiac myocytes
Cell 300
Figure 3. Skeletal Muscle Cells Cultured with Nerve Extract Stalned with antibodies against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardiac TN-T (d), TN-I (e), and TN-C (f). Myotubes were stained only with antibodies against skeletal TN components (a-c). The reactivitles with antibodies against cardiac TN-T (d) and TN-C (f) were lost. Two weeks in vitro. Bar: 50
m. nerve-conditioned medium is also effective, in which supernatants from various nerve cell types are included), the types of neurons do not seem to matter for these transitions Since muscles do not require physical contact with nerves and are not affected by the nonneuronal tissues so far examined, it can be presumed that these changes are due to the influence of humoral, and above all neurohumoral, factor(s). Elucidation of such factor(s) is intriguing in terms of what other nerve and nonneuronal cell type(s) might also possess such characteristics and, further, in regard to the isolation and characterization of the factor(s). Studies concerning temporal requirements of the factor(s) for the differentiation of TN components and other muscle proteins may provide some clues to assist in the analysis of the neural control mechanism for the muscle gene expression. The cell number required to elicit the changes of TN component antigenicities from embryonic to adult forms
was less for cells from spinal cords than for cells from sympathetic ganglia. This is probably due to the fact that only the former tissue was treated with an inhibitor of DNA synthesis for the purpose of motoneuron enrichment (Masuko et al., 1979) before being added to muscle cultures. This procedure increased the relative number of effective constituents (neurons) and reduced fibroblastic and/or glial cells; the tissue from sympathetic ganglia contained many more of these nonneuronal cells, in addition to adrenergic neurons and other neuron types. The isoforms of TN components were changed only partially in cocultures of skeletal muscle cells with a lower quantity of nerves or a lower concentration of nerve extract. Although why, at the reduced neural influence, only one of TN-T or TN-C changes its form, embryonic muscles appear to require that the neural effect be above a certain level for normal transition of TN component isoforms. In the experiments without nerves, the reactivities of
Troponrn 301
in Skeletal and Cardrac Muscles
Figure 4. Indirect lmmunofluorescence
Cultured In Vivo
Micrographs
of Embryonic
Myocardial
Cells Cultured
wrthout Nerves
Stained with antibodres against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardiac TN-T (d), TN-I (e), and TN-C (f). Cardiac antibodies against skeletal TN-I (b) and cardiac TN components (d-f). Two weeks in vitro. Bar: 50 pm.
Table 1. TN in Skeletal and Cardiac
Cultures Tested
Antibody against Skeletal I cells
Cardiac
Skeletal Cardiac
wrth specific
Muscle Cells In Vitro Addition to Standard
Skeletal muscle
muscle cells reacted
muscle cells
Cardiac I
NE: nerve extract. NCM: nerve-conditioned
medium.
TN-T TN-I TN-C TN-T TN-I TN-C TN-T TN-I TN-C TN-T TN-l TN-C
Cultures
None
Spinal Cord
Wv. Ggl.
NE
+ +
+ +
+ +
+ +
+ -
Kidney
Lung
+ +
-I+
+ -
+ -
-
-
-
+ +
+ +
f +
-
-
-
+ -
+ -
-
-I+ -
+ -
+ -I+
+ + +
+ + +
+ + +
+ + +
+ + +
-I+ +
-
+ -
NCM
-
-
Cell 302
Figure 5. Myocardial
Cells Cocultured
with Spinal Cord
(a) Phase-contrast micrograph. SC: spinal cord Stained with antibody against skeletal TN-I. (c) Cardiac myocytes reacted only with antibodies against the heterologous TN (b). Two weeks rn
fragment. (b) Fluorescence micrograph of a different region from that of (a), but from the same culture dish. Fluorescence micrograph of the region corresponding to the rectangle in (a). Statned with anticardiac TN-I. against TN components from the homologous muscle (1-6 in a and c) and lost the reactivities with antibody vttro. Bar: 100 pm
each muscle with antibodies against TN components from the heterologous muscular tissue (TN-T and TN-C in skeletal muscle cells and TN-I in cardiac muscle cells) existed throughout the culture period (maximum of 1 month). In cultures of muscle from embryonic pectoralis, latissimus dorsi anterior, leg, and ventricle, the isoform variants of myosin, myosin light chains, tropomyosin, and TN-T have also been detected (Rubinstein and Holtzer, 1979; Cantini et al., 1980; Keller and Emerson, 1980; Matsuda et al., 1981; Bandman et al., 1982) but their transition to the adult forms has not been seen. Although the present study does not clarify whether after a much longer culture period they might change to the adult forms without the neural influence, it is a distinct possibility that the presence of the neural influence precociously induces the full expression of the differentiative potential of muscle cells, Schiaffino et al. (1982) noted that fetal rat spinal cord did not influence the isomyosin gene expression of cultured adult human muscle; their result is not in accord with our present observations. The differences may be due to the different culture conditions; i.e., in their cultures muscle and nerve tissues come from different species (human
and rat, respectively) and from different stages of development (adult and fetal, respectively), while in our cultures both tissues are from the same species at the embryonic stage (chicken embryos). However, other explanations may also exist. Experimental
Procedures
Preparation of Cultures Skeletal and Cardiac Muscle Cell Cultures Skeletal muscle cell cultures were prepared according to the method of Shimada (1971). Cell suspensions from breast muscles of II-day-old chrcken embryos were obtained by dissociation of the tissues with trypsin. Cell suspensions prepared by a differential cell adhesion procedure and enriched in skeletal myogenic cells (Yaffe, 1968) were used. Cardiac muscle cell cultures were prepared by the method of DeHaan (1970). Ventricular myocardium from 7-day chicken embryos was dissociated by the multiplecycle trypsinization method. Dissociated cells of each muscular tissue were plated at a concentration of 8 x IO5 cells in 1 ml of culture medium in 35 mm plastic dishes. For skeletal muscle cell cultures, the dishes were coated with gelatin; for cardiac muscle cell cultures, they were uncoated. &cultures of Muscle with Nerve Cocultures of skeletal and cardiac muscle cells with cholinergic or adrenergrc nerves were prepared. The cholinergic nerves used were from spinal cords that had been enriched in a-motoneurons and were devoid of glia
Troponrn 303
In Skeletal and Cardiac Muscles
Figure 6. Cardrac
Cultured In VIVO
Muscle Cells Cultured with Nerve-Condrtroned
Medrum
Stained with antrbodies against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardrac TN-T (d), TN-I (e), and TN-C (f). Cardrac muscle cells reacted only with antibodres against TN components from the heart (d-f). The reactivrty with antibody against skeletal TN-I was lost (b). Two weeks in vitro. Bar: 50 pm, cells they were prepared by cutttng sprnal cords from 60-66.hr chicken embryos into about 16 small pieces and incubating them In a skeletal muscle culture medium containing 10 pM 1-b-D-arabinofuranosyfcytosine for 24 hr (Masuko et al., 1979). The adrenergic nerves used were from superior cewical ganglia from 1 I-13.day chicken embryos. They were cut Into halves. Afler skeletal and cardiac muscle cells were grown for 3-12 hr, small fragments of the neural tissue were added to these cultures. Each spinal cord fragment contained approximately 3 x IO3 cells and each sympathetic nerve fragment 1.5 x 1O4 cells. The cell number was estimated after dissocration of the fragments with trypsin. Cocultures of Muscle with Nonneuronal Tissue Cocultures of skeletal and cardiac muscle cells with kidney and lung were prepared. These nonneuronal tissues were obtarned from 1 l-day chicken embryos, and cut into small pieces (about 0.7 mm drameter). They were added to skeletal and cardiac muscle cell cultures 3-12 hr after muscle cells had been plated. Each fragment of kidney and lung contained approximately 3 x 1O5 and 6 x 1 O5 ceils, respectively. Culture Media The medium for skeletal muscle cell cultures consrsted of Eagle’s minimal essentral medium with glutamine, 15% horse serum, 5% embryo extract, and penicillin/streptomycin in concentrations of 50 U/ml and 50 pg/ml, respectively. The medium for cardiac muscle cell cultures was the growth medium (8188) described by DeHaan (1970).
Embryo extract was prepared from 7-1 i-day chicken embryos from which brains and spinal cords with skulls and vertebrae still attached had been removed. They were homogenized in a glass homogenizer with a Teflon pestle with a double volume of Tyrode’s solution. The homogenate was centrifuged at 10,000 x g for 30 min, and the resultant supernatant was used as embryo extract. Protein concentration of embryo extract was adjusted to 7 mg/ml before dilution with the culture medium (i. e., it was 0.35 mg/ml after dilution). In some cultures of skeletal and cardiac muscle cells without nerves, embryo extract in the media was substituted by a nerve extract. It was prepared from brains and spinal cords with surrounding skulls and vertebrae of 7-I l-day chicken embryos by the same procedure as that for embryo extract. Protein concentration of nerve extract in the culture medium was adjusted to 0.14-3.5 mg/ml. Nerve-conditioned medrum was obtained from ce!l cultures of spinal cords. Trypsrn-dissociated cells from 4-day chicken spinal cords were plated at concentrations of 1 x 106, 5 x 106, or 10 x IO6 cells in 1 ml of fresh 8188 medrum in 35 mm plastic dishes. After Incubation for 3 days, the medium was collected. The cultures were resupplied with 1 ml of the fresh medium, and afler another 3 days the medium was agarn collected. These collected media were cleared of cells and debris by centrifugation at 3,000 X g for 10 min. They were mixed wrth the same volume of a fresh medium and used as nerve-conditioned medium.
Cell 304
Preparation and Characterization of Antibodies The preparation and characterization of antisera to TN components (T, I, and C) of skeletal (m. pectoralis major) and cardiac (ventricle) muscles have been described elsewhere (Toyota and Shimada, 1981). In this study, antisera against skeletal TN-T and TN-C were raised in guinea pigs; those agarnst skeletal TN-I and cardiac TN components were raised in rabbits. The specificities of the antisera to all the proteins were examined by double immunodiffusion, immunoelectrophoresis, and the demonstration of specific staining of isolated myofibrils. The specificities were confirmed by two rounds of absorption with a homogenate from heterologous muscle (for example, anticardiac TN-T antiserum absorbed by skeletal muscle homogenate). After the immunoglobulin (IgG) was fractionated from the absorbed serum, reabsorption was performed by passrng the antibody through CNBractrvated Sepharose 48 conjugated with a heterologous TN component (for example, anticardrac TN-T absorbed by immobilized skeletal TN-T). Indirect immunofluorescence staining of myofibrils from skeletal and cardiac muscles showed that antibodies raised from TN components of skeletal and cardiac muscles stained the thin filament region of myofibrils of the homologous muscle. These results indicate that antibodies raised from TN components of skeletal and cardiac muscles are highly specific for those antigenic components and that there is no cross-reaction between antibodies raised from skeletal and cardiac muscles. Indirect lmmunofluorescence Dishes wrth adhering cultures were immersed in 50% glycerol solution containing KMP buffer (50 mM KCI, 2 mM MgC12, 2 mM EGTA, and 10 mM Na-phosphate buffer, pH 7.0) and stored at -2OOC until use. Cultures ware washed with phosphate-buffered saline (PBS), fixed in ethanol at 0°C for 10 min, and air dried. The dishes were cut into small pieces. Six pieces from each dish were incubated for 30 min at room temperature with antibodies agarnst skeletal TN-T, TN-I, and TN-C, and cardiac TN-T, TN-I, and TN-C, followed by three rinses in PBS for 5 min each. Cultures were then incubated in FITC-coupled sheep anti-rabbit IgG (Miles Laboratories Inc.), tetramethylrhodamine-(TMR)-conjugated goat anti-rabbit IgG, or FiTClabeled goat anti-guinea pig IgG (N. L. Cappel Laboratorres Inc.), for 30 min. Specrmens were observed with a Zeiss microscope equipped with filters BP 450-490, LP 520, and KP 560 for FITC fluorescence and BP 546/12, FT 580, and LP 590 for TMR fluorescence. Acknowledgments This research was supported by grants from the following: the Japanese Ministry of Education, Science and Culture: the National Center for Nervous Mental and Muscular Disorders (NCNMMD #82-03) of the Japanese Ministry of Health and Welfare; the Muscular Dystrophy Assocration of America. The authors wish to thank Mr. N. Nakamura and Mrs. K. Shimizu for their technical assistance. The costs of publication of this article were defrayed rn part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
May 17, 1982; revised February
17, 1983
DeHaan, R. L. (1970). The potassium-sensitivity of isolated embryonic cells Increases with development. Dev. Biol. 23, 226-240.
heart
Dhoot, G. K., and Perry, S. V. (1980). The components of the troponin complex and development in skeletal muscle. Exp. Cell Res. 127, 75-87. Dhoot, G. K., and Perry, S. V. (1982). The effect of denervation on the distribution of the polymorphic forms of troponin components in fast and slow muscles of the adult rat. Cell Tissue Res. 225, 201-215. Dhoot, G. K., Perry, S. V., and Vrbova, G. (1981). Changes in thedistribution of the components of the troponin complex in muscle fibers after crossinnervation. Exp. Neural. 72, 513-530. Furshpan, E. J., MacLeish, P. R., O’Lague, P. H., and Potter, D. D. (1976). Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons. Proc. Nat. Acad. Sci. USA 73, 42254229. Gauthier, G. F., and Hobbs, A. W. (1982). Effects of denervation on the drstribution of myosrn isozymes in skeletal muscle fibers. Exp. Neurol. 76, 331-346. Gauthier, G. F., Lowey, S., and Hobbs, A. W. (1978). Fast and slow myosin In developing muscle fibres. Nature 274, 25-29. Hoh, J. F. Y., and Yeoh, G. P. S. (1979). Rabbit skeletal myosin isoenzymes from fetal, fast-twitch and slow-twitch muscles. Nature 280, 321-323. Keller, L. R., and Emerson, C. P. Jr. (1980). Synthesis of adult myosin light chains by embryonic muscle cultures. Proc. Nat. Acad. Sci. USA 77, 10201024. Masaki, T., and Yoshizaki, C. (1974). Differentiation embryos. J. Biochem. (Tokyo) 76, 123-131.
of myosin
in chick
Masuko, S., Kuromi, H., and Shimada, Y. (1979). Isolation and culture of motoneurons from embryonic chicken spinal cords. Proc. Nat. Acad. Sci. USA 76, 3537-3541. Matsuda, R., Obinata, T., and Shimada, Y. (1981). Types of troponin components during development of chicken skeletal muscle. Dev. Biol. 82, 11-19. Roy, R. K., Sreter, F. A., and Sarkar, S. (1979). Changes in tropomyosin subunits and myosin light chains during development chicken and rabbit striated muscles. Dev. Biol. 69, 15-30. Rubinstein, N. A., and Holtzer, H. (1979). Fast and slow muscles culture synthesise only fast myosin. Nature 280, 323-325.
in tissue
Rubinstern, N. A., Pepe, F. A., and Holtzer, H. (1977). Myosin types during the development of embryonic chicken fast and slow muscles. Proc. Nat. Acad. Sci. USA 74, 4524-4527. Salmons, S., and Sreter, F. A. (1976). Significance of impulse activity in the transformation of skeletal muscle type. Nature 263, 30-34. Schiaffino, S., Askanas, V., Engel, W. K., Vitadello, M., and Sartore, S. (1982). Myosrn isoenzymes in cultured human muscle. Arch. Neurol. 39, 347-349. Shimada, Y. (1971). Electron microscope observations chick myoblasts in vitro. J. Cell Biol. 48, 128-142.
on the fusion of
Toyota, N., and Shimada, Y. (1981). Differentiation of troponrn in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence microscopy. J. Cell Biol. 91, 497-504.
References
Whalen, R. G., Butler-Brown, G. S., and Gros, F. (1978). Identification of a novel form of myosin light chain present in embryonic muscle tissue and cultured muscle cells. J. Mol. Biol. 726, 415-431,
Amphlett, G. W., Perry S. V., Syska, H., Brown, M. D., and Vrbova, G. (1975). Crossinnervation and the regulatory protein system of rabbit soleus muscle. Nature 257, 602-604.
Yaffe, D. (1968). Retention of differentiation potentialities during prolonged cultivatron of myogenic cells, Proc. Nat. Acad. Sci. USA 67, 477-483.
Bandman, E., Matsuda, R., and Strohman, R. C. (1982). Developmental appearance of myosin heavy and light chain isoforms in vivo and in vitro in chicken skeletal muscle. Dev. Biol. 93, 508-518. Butler-Browne, G. S., Bugarsky, L. B., Cuenound, S., Schwartz, K.. and Whalen, R. G. (1982). Denervation of newborn rat muscles does not block the appearance of adult fast myosin heavy chain, Nature 299, 830-833. Cantrni, M., Sartore, S., and Schiaffino, muscle cells. J. Cell Biol. 85, 903-909
S. (1980). Myosin types in cultured
May 1983, CopyrIght
0 1983 by MIT
0092.8674/83/050297-08
$02,00/O
lsoform Variants of Troponin in Skeletal and Cardiac Muscle Cells Cultured with and without Nerves Naoji Toyota* and Yutaka Department of Anatomy School of Medicine Chiba University Chiba 280, Japan
Shimada+
Summary lmmunofluorescence microscopy shows that cultured skeletal and cardiac muscle cells of chicken embryos exhibit the same stainabilities with antibodies against skeletal and cardiac troponin components as do those in embryos. Muscle cells of each type cultured with motor or sympathetic nerves or in medium containing the nerve extract exhibit the same reactivities as do those in adult animals. Cardiac muscle cells incubated in the nerve-conditioned medium also change the form of troponin components to the adult type. It appears that the differentiation of individual muscle fibers to specific types is induced by nerves, and especially by the neurohumoral effect. Introduction It is becoming clear that some of the proteins synthesized in embryonic skeletal and cardiac muscles are qualitatively different from the adult forms and that succession of phenotypes to adult forms occurs as the muscles develop (Masaki and Yoshizaki, 1974; Rubinstein et al., 1977; Gauthier et al., 1978; Whalen et al., 1978; Hoh and Yeoh, 1979; Roy et al., 1979; Dhoot and Perry, 1980; Matsuda et al., 1981; Toyota and Shimada, 1981). In skeletal muscles, these transitions have been attributed to the establishment of the adult pattern of motoneuron activity. This assumption came from experiments with adult animals in which it was found that denervation of muscles causes their reactivities with antibodies to differ from those of normal muscles (Dhoot and Perry, 1982; Gauthier and Hobbs, 1982) and that cross-innervation of fast and slow muscle nerves brings about an interchange of the properties of proteins (Amphlett et al., 1975; Salmons and Sreter, 1976; Dhoot et al., 1981). On the other hand, some reports do not support the mediation of motoneurons for diversification of muscle proteins. Transition of the form of myosin from neonatal to fast has been shown to occur in the denervated fast muscle of the newborn rat; thus this change is presumed to be independent of innervation and programmed endogenously in the muscle fiber (Butler-Browne et al., 1982). Furthermore, in cultures of adult human muscle grown with the ventral part of the fetal rat spinal cord, the pattern of immunoreaction of myosin isoforms was found to be the * Present address: Division of Neurochemistry Psychiatric Research tute of Tokyo, Kamikitazawa, Setagaya, Tokyo 156, Japan. t To whom all correspondence should be addressed.
Insti-
same as that of the same muscle cultured aneurally (Schiaffino et al., 1982); the presence of the neural tissue appears to have no influence on isomyosin gene expression in the cocultures of tissues from heterospecies. Thus neurogenic contributions to the changes of skeletal myofibrillar proteins during development are still somewhat controversial. The neurogenic effects on the changes of cardiac myofibrillar proteins in embryos and adult animals are as yet unexplored. By making use of the advantages of Culture Systems, we have investigated the immunochemical properties of troponin (TN) components (T, I, and C) of embryonic skeletal and cardiac muscles in the presence and absence of nerves or nerve extract. The results indicate that the nerves and nerve extract affect the gene expression and induce the differentiation of individual muscle fibers to specific types. Results The general schema of differentiation of skeletal and cardiac myogenic cells has been reported elsewhere (DeHaan, 1970; Shimada, 1971) and will not be repeated here. When fragments of spinal cords (enriched in cholinergic motoneurons) or sympathetic ganglia (adrenergic neurons) were added to each muscle cell culture, they extended many processes on the skeletal and cardiac muscle cells. Although in such muscle-nerve cocultures not all muscle cells were contacted by nerves, no significant difference in the differentiation was found between the muscle cells that were contacted by nerves and those that were not. Cultures of skeletal muscle cells from embryonic pectoralis were stained with affinity-purified antibodies specific for adult skeletal and cardiac TN components (Figure 1). They began to be stained with antibodies at 2 days in vitro. All fibers reacted not only with the antibodies against all of the three components of skeletal TN (Figures la1 c), but also with the antibodies raised against cardiac TNT and TN-C (Figures Id, If). These reactivities of skeletal muscle cells alone, without nerve influence, remained unchanged during the subsequent culture days. When skeletal muscle cells were cultured with motor (Figure 2) or sympathetic nerves or in a medium containing nerve extract (Figure 3) their reactivities with antibodies to TN components changed after 1 week of culture. That is, skeletal muscle cells lost their stainability with antibodies against the heterologous muscle (anticardiac TN-T and TNC) (Figures 2c, 3d, 3f) and were stained only with antibodies specific for skeletal TN components (Figures 2b and 3a-3c). This change was elicited by the addition of more than six spinal cord fragments (approx. 1.8 X lo4 cells), 13 sympathetic ganglion fragments (approx. 2 X 1O5 cells), or 3.5 mg/ml protein of nerve extract per culture dish. With three cord fragments (approx. 9 X lo3 cells), 12 ganglion fragments (approx. 1.8 x IO5 cells), or 0.7 mg/ml protein of nerve extract, the change in the reactivities of skeletal muscle cells was only partial. In some cultures the stainability with antibody against cardiac TN-T disappeared while
Cell 298
Figure 1. Indirect
lmmunofluorescence
MIcrographs
of Embryonic
Skeletal Muscle Cells Cultured
without Nerves
Stalned with specific antibodies against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardiac TN-T (d). TN-I (e), and TN-C (f). Myotubes antibodles against cardiac TN-T (d) and TN-C (c) as well as skeletal TN components (a-c). Two weeks in vitro. Bar: 50 pm,
that with antibody against cardiac TN-C remained, whereas in other cultures the reverse was observed. With one cord fragment (approx. 3 x IO3 cells), one ganglion fragment (approx. 1.5 x 1 O4 cells), or 0.14 mg/ml protein of nerve extract, the reactivity of skeletal muscle cells was the same as that without nerves. The reactivities of skeletal muscle cells were not affected by nonneuronal tissues, even when those tissues were present at a IOO-fold higher cell concentration than the minimum concentration of nerve cells required for the transitions. With 18 kidney fragments (approx. 5.5 X IO6 cells) or 13 lung fragments (approx. 8 X IO6 cells), the stainabilities of skeletal muscle cells were unchanged. Cardiac muscle cells cultured in the absence of nerves or in medium without nerve extract were stained with the six antibodies (Figure 4). They began to react with antibodies 3 hr after inoculation. Cardiac myocytes reacted with antiskeletal TN-I (Figure 4b) as well as with anticardiac TNT, TN-I, and TN-C (Figures 4d-4f). These reactivities of
were stained with
cardiac myocytes remained unchanged even after a prolonged culture period. However, when these cardiac myocytes were incubated with motoneurons (Figure 5) or sympathetic ganglia, or were cultured in medium containing nerve extract, they reacted only with anticardiac TN components (Figure 5~); the stainability with heterologous antibody (antiskeletal TNI) disappeared after 1 week of culture (Figure 5b). The effect of nerve-conditioned medium was examined; in this medium cardiac myocytes came to exhibit the same stainabilities as those with nerves or nerve extract (Figure 6). These changes were evoked with ten spinal cord fragments (approx. 3 X lo4 cells), three ganglion fragments (approx. 4.5 x IO4 cells), 0.7 mg/ml protein of nerve extract per plate, or nerve-conditioned medium prepared from 1 x 1O7 spinal cord cells per plate. Below these cell numbers or at a lower protein concentration, the reactivities of cardiac muscle cells remained unchanged. When nonneuronal tissues (14 kidney fragments [ap-
Troponrn 299
in Skeletal and Cardiac Muscles
Figure 2. Skeletal Muscle
Cultured
In Vivo
Cells Cultured with Sprnal Cord
(a) General view of cultures. Phase-contrast mrcrograph. SC: spinal cord fragment. (b) Fluorescence mrcrograph of the region correspondrng to the rectangle in (a). Stained with antiskeletal TN-T. (c) Fluorescence micrograph of a portion from the same culture dish, but a different area from that of (a). Stained with antrcardrac TN-T. Myotubes reacted with antibody against skeletal TN (1-5 in a and b), but lost reactivity wrth antibody against cardiac TN (c). Two weeks in vitro. Bar. 100 pm
prox. 4 x IO” cells] or 25 lung fragments [approx. 1.5 X 1 Cl7cells]) were added to cardiac muscle cell cultures, the transition of the form of TN did not occur. The results are summarized in Table 1, Discussion The present immunofluorescence study showed that some of the TN components synthesized by embryonic skeletal and cardiac muscle cells cultured in the absence of nerves, nerve extract, or nerve-conditioned medium react with antibodies raised from both adult skeletal and cardiac muscles. Skeletal muscle cells reacted with antibodies against TN-T and TN-C from the adult skeletal muscles as well as with those from cardiac muscles, This suggests that skeletal muscle cells in vitro synthesize TN-T and TNC, which share the antigenicities for adult skeletal and cardiac muscles. Furthermore, cardiac muscle cells displayed multiple reactivity to antibodies raised against two different TN-Is from adult skeletal and cardiac muscles. This indicates that cardiac muscle cells in vitro synthesize TN-Is with antigenic determinants similar to those in adult skeletal and cardiac muscles. These reactivities of embryonic skeletal and cardiac muscle cells in vitro are the same
as those in the respective muscles in the embryo (Toyota and Shimada, 1981). The present study further showed that in cultures of muscle cells of each type with motoneurons (cholinergic nerves) or sympathetic ganglia (adrenergic nerves), or with the presence of nerve extract, the forms of TN components of skeletal (TN-T and TN-C) and cardiac (TN-I) muscle cells change to the forms characteristic of their adult tissue type (Toyota and Shimada, 1981). Since these changes of TN components from embryonic to adult forms occurred in muscle cells of each type that are contacted by nerve processes as well as in those that are not, and also in muscle cells cultured in the medium containing nerve extract (nerve-conditioned medium is also effective for cardiac myocytes), where no living nerve cells are present, the establishment of functional innervation or even physical contact with nerves does not appear to be required for muscle cells to change their TN component isoforms. Since these changes occurred regardless of the neuron types (cholinergic or adrenergic) added to muscle cultures (expression of transmitters [Furshpan et al., 19761 by these nerves in the mixed cultures was not studied here), and also occurred in nerve extract, which contains homogenates of a variety of nerve cell types (in cardiac myocytes
Cell 300
Figure 3. Skeletal Muscle Cells Cultured with Nerve Extract Stalned with antibodies against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardiac TN-T (d), TN-I (e), and TN-C (f). Myotubes were stained only with antibodies against skeletal TN components (a-c). The reactivitles with antibodies against cardiac TN-T (d) and TN-C (f) were lost. Two weeks in vitro. Bar: 50
m. nerve-conditioned medium is also effective, in which supernatants from various nerve cell types are included), the types of neurons do not seem to matter for these transitions Since muscles do not require physical contact with nerves and are not affected by the nonneuronal tissues so far examined, it can be presumed that these changes are due to the influence of humoral, and above all neurohumoral, factor(s). Elucidation of such factor(s) is intriguing in terms of what other nerve and nonneuronal cell type(s) might also possess such characteristics and, further, in regard to the isolation and characterization of the factor(s). Studies concerning temporal requirements of the factor(s) for the differentiation of TN components and other muscle proteins may provide some clues to assist in the analysis of the neural control mechanism for the muscle gene expression. The cell number required to elicit the changes of TN component antigenicities from embryonic to adult forms
was less for cells from spinal cords than for cells from sympathetic ganglia. This is probably due to the fact that only the former tissue was treated with an inhibitor of DNA synthesis for the purpose of motoneuron enrichment (Masuko et al., 1979) before being added to muscle cultures. This procedure increased the relative number of effective constituents (neurons) and reduced fibroblastic and/or glial cells; the tissue from sympathetic ganglia contained many more of these nonneuronal cells, in addition to adrenergic neurons and other neuron types. The isoforms of TN components were changed only partially in cocultures of skeletal muscle cells with a lower quantity of nerves or a lower concentration of nerve extract. Although why, at the reduced neural influence, only one of TN-T or TN-C changes its form, embryonic muscles appear to require that the neural effect be above a certain level for normal transition of TN component isoforms. In the experiments without nerves, the reactivities of
Troponrn 301
in Skeletal and Cardrac Muscles
Figure 4. Indirect lmmunofluorescence
Cultured In Vivo
Micrographs
of Embryonic
Myocardial
Cells Cultured
wrthout Nerves
Stained with antibodres against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardiac TN-T (d), TN-I (e), and TN-C (f). Cardiac antibodies against skeletal TN-I (b) and cardiac TN components (d-f). Two weeks in vitro. Bar: 50 pm.
Table 1. TN in Skeletal and Cardiac
Cultures Tested
Antibody against Skeletal I cells
Cardiac
Skeletal Cardiac
wrth specific
Muscle Cells In Vitro Addition to Standard
Skeletal muscle
muscle cells reacted
muscle cells
Cardiac I
NE: nerve extract. NCM: nerve-conditioned
medium.
TN-T TN-I TN-C TN-T TN-I TN-C TN-T TN-I TN-C TN-T TN-l TN-C
Cultures
None
Spinal Cord
Wv. Ggl.
NE
+ +
+ +
+ +
+ +
+ -
Kidney
Lung
+ +
-I+
+ -
+ -
-
-
-
+ +
+ +
f +
-
-
-
+ -
+ -
-
-I+ -
+ -
+ -I+
+ + +
+ + +
+ + +
+ + +
+ + +
-I+ +
-
+ -
NCM
-
-
Cell 302
Figure 5. Myocardial
Cells Cocultured
with Spinal Cord
(a) Phase-contrast micrograph. SC: spinal cord Stained with antibody against skeletal TN-I. (c) Cardiac myocytes reacted only with antibodies against the heterologous TN (b). Two weeks rn
fragment. (b) Fluorescence micrograph of a different region from that of (a), but from the same culture dish. Fluorescence micrograph of the region corresponding to the rectangle in (a). Statned with anticardiac TN-I. against TN components from the homologous muscle (1-6 in a and c) and lost the reactivities with antibody vttro. Bar: 100 pm
each muscle with antibodies against TN components from the heterologous muscular tissue (TN-T and TN-C in skeletal muscle cells and TN-I in cardiac muscle cells) existed throughout the culture period (maximum of 1 month). In cultures of muscle from embryonic pectoralis, latissimus dorsi anterior, leg, and ventricle, the isoform variants of myosin, myosin light chains, tropomyosin, and TN-T have also been detected (Rubinstein and Holtzer, 1979; Cantini et al., 1980; Keller and Emerson, 1980; Matsuda et al., 1981; Bandman et al., 1982) but their transition to the adult forms has not been seen. Although the present study does not clarify whether after a much longer culture period they might change to the adult forms without the neural influence, it is a distinct possibility that the presence of the neural influence precociously induces the full expression of the differentiative potential of muscle cells, Schiaffino et al. (1982) noted that fetal rat spinal cord did not influence the isomyosin gene expression of cultured adult human muscle; their result is not in accord with our present observations. The differences may be due to the different culture conditions; i.e., in their cultures muscle and nerve tissues come from different species (human
and rat, respectively) and from different stages of development (adult and fetal, respectively), while in our cultures both tissues are from the same species at the embryonic stage (chicken embryos). However, other explanations may also exist. Experimental
Procedures
Preparation of Cultures Skeletal and Cardiac Muscle Cell Cultures Skeletal muscle cell cultures were prepared according to the method of Shimada (1971). Cell suspensions from breast muscles of II-day-old chrcken embryos were obtained by dissociation of the tissues with trypsin. Cell suspensions prepared by a differential cell adhesion procedure and enriched in skeletal myogenic cells (Yaffe, 1968) were used. Cardiac muscle cell cultures were prepared by the method of DeHaan (1970). Ventricular myocardium from 7-day chicken embryos was dissociated by the multiplecycle trypsinization method. Dissociated cells of each muscular tissue were plated at a concentration of 8 x IO5 cells in 1 ml of culture medium in 35 mm plastic dishes. For skeletal muscle cell cultures, the dishes were coated with gelatin; for cardiac muscle cell cultures, they were uncoated. &cultures of Muscle with Nerve Cocultures of skeletal and cardiac muscle cells with cholinergic or adrenergrc nerves were prepared. The cholinergic nerves used were from spinal cords that had been enriched in a-motoneurons and were devoid of glia
Troponrn 303
In Skeletal and Cardiac Muscles
Figure 6. Cardrac
Cultured In VIVO
Muscle Cells Cultured with Nerve-Condrtroned
Medrum
Stained with antrbodies against skeletal TN-T (a), TN-I (b), and TN-C (c); and cardrac TN-T (d), TN-I (e), and TN-C (f). Cardrac muscle cells reacted only with antibodres against TN components from the heart (d-f). The reactivrty with antibody against skeletal TN-I was lost (b). Two weeks in vitro. Bar: 50 pm, cells they were prepared by cutttng sprnal cords from 60-66.hr chicken embryos into about 16 small pieces and incubating them In a skeletal muscle culture medium containing 10 pM 1-b-D-arabinofuranosyfcytosine for 24 hr (Masuko et al., 1979). The adrenergic nerves used were from superior cewical ganglia from 1 I-13.day chicken embryos. They were cut Into halves. Afler skeletal and cardiac muscle cells were grown for 3-12 hr, small fragments of the neural tissue were added to these cultures. Each spinal cord fragment contained approximately 3 x IO3 cells and each sympathetic nerve fragment 1.5 x 1O4 cells. The cell number was estimated after dissocration of the fragments with trypsin. Cocultures of Muscle with Nonneuronal Tissue Cocultures of skeletal and cardiac muscle cells with kidney and lung were prepared. These nonneuronal tissues were obtarned from 1 l-day chicken embryos, and cut into small pieces (about 0.7 mm drameter). They were added to skeletal and cardiac muscle cell cultures 3-12 hr after muscle cells had been plated. Each fragment of kidney and lung contained approximately 3 x 1O5 and 6 x 1 O5 ceils, respectively. Culture Media The medium for skeletal muscle cell cultures consrsted of Eagle’s minimal essentral medium with glutamine, 15% horse serum, 5% embryo extract, and penicillin/streptomycin in concentrations of 50 U/ml and 50 pg/ml, respectively. The medium for cardiac muscle cell cultures was the growth medium (8188) described by DeHaan (1970).
Embryo extract was prepared from 7-1 i-day chicken embryos from which brains and spinal cords with skulls and vertebrae still attached had been removed. They were homogenized in a glass homogenizer with a Teflon pestle with a double volume of Tyrode’s solution. The homogenate was centrifuged at 10,000 x g for 30 min, and the resultant supernatant was used as embryo extract. Protein concentration of embryo extract was adjusted to 7 mg/ml before dilution with the culture medium (i. e., it was 0.35 mg/ml after dilution). In some cultures of skeletal and cardiac muscle cells without nerves, embryo extract in the media was substituted by a nerve extract. It was prepared from brains and spinal cords with surrounding skulls and vertebrae of 7-I l-day chicken embryos by the same procedure as that for embryo extract. Protein concentration of nerve extract in the culture medium was adjusted to 0.14-3.5 mg/ml. Nerve-conditioned medrum was obtained from ce!l cultures of spinal cords. Trypsrn-dissociated cells from 4-day chicken spinal cords were plated at concentrations of 1 x 106, 5 x 106, or 10 x IO6 cells in 1 ml of fresh 8188 medrum in 35 mm plastic dishes. After Incubation for 3 days, the medium was collected. The cultures were resupplied with 1 ml of the fresh medium, and afler another 3 days the medium was agarn collected. These collected media were cleared of cells and debris by centrifugation at 3,000 X g for 10 min. They were mixed wrth the same volume of a fresh medium and used as nerve-conditioned medium.
Cell 304
Preparation and Characterization of Antibodies The preparation and characterization of antisera to TN components (T, I, and C) of skeletal (m. pectoralis major) and cardiac (ventricle) muscles have been described elsewhere (Toyota and Shimada, 1981). In this study, antisera against skeletal TN-T and TN-C were raised in guinea pigs; those agarnst skeletal TN-I and cardiac TN components were raised in rabbits. The specificities of the antisera to all the proteins were examined by double immunodiffusion, immunoelectrophoresis, and the demonstration of specific staining of isolated myofibrils. The specificities were confirmed by two rounds of absorption with a homogenate from heterologous muscle (for example, anticardiac TN-T antiserum absorbed by skeletal muscle homogenate). After the immunoglobulin (IgG) was fractionated from the absorbed serum, reabsorption was performed by passrng the antibody through CNBractrvated Sepharose 48 conjugated with a heterologous TN component (for example, anticardrac TN-T absorbed by immobilized skeletal TN-T). Indirect immunofluorescence staining of myofibrils from skeletal and cardiac muscles showed that antibodies raised from TN components of skeletal and cardiac muscles stained the thin filament region of myofibrils of the homologous muscle. These results indicate that antibodies raised from TN components of skeletal and cardiac muscles are highly specific for those antigenic components and that there is no cross-reaction between antibodies raised from skeletal and cardiac muscles. Indirect lmmunofluorescence Dishes wrth adhering cultures were immersed in 50% glycerol solution containing KMP buffer (50 mM KCI, 2 mM MgC12, 2 mM EGTA, and 10 mM Na-phosphate buffer, pH 7.0) and stored at -2OOC until use. Cultures ware washed with phosphate-buffered saline (PBS), fixed in ethanol at 0°C for 10 min, and air dried. The dishes were cut into small pieces. Six pieces from each dish were incubated for 30 min at room temperature with antibodies agarnst skeletal TN-T, TN-I, and TN-C, and cardiac TN-T, TN-I, and TN-C, followed by three rinses in PBS for 5 min each. Cultures were then incubated in FITC-coupled sheep anti-rabbit IgG (Miles Laboratories Inc.), tetramethylrhodamine-(TMR)-conjugated goat anti-rabbit IgG, or FiTClabeled goat anti-guinea pig IgG (N. L. Cappel Laboratorres Inc.), for 30 min. Specrmens were observed with a Zeiss microscope equipped with filters BP 450-490, LP 520, and KP 560 for FITC fluorescence and BP 546/12, FT 580, and LP 590 for TMR fluorescence. Acknowledgments This research was supported by grants from the following: the Japanese Ministry of Education, Science and Culture: the National Center for Nervous Mental and Muscular Disorders (NCNMMD #82-03) of the Japanese Ministry of Health and Welfare; the Muscular Dystrophy Assocration of America. The authors wish to thank Mr. N. Nakamura and Mrs. K. Shimizu for their technical assistance. The costs of publication of this article were defrayed rn part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
May 17, 1982; revised February
17, 1983
DeHaan, R. L. (1970). The potassium-sensitivity of isolated embryonic cells Increases with development. Dev. Biol. 23, 226-240.
heart
Dhoot, G. K., and Perry, S. V. (1980). The components of the troponin complex and development in skeletal muscle. Exp. Cell Res. 127, 75-87. Dhoot, G. K., and Perry, S. V. (1982). The effect of denervation on the distribution of the polymorphic forms of troponin components in fast and slow muscles of the adult rat. Cell Tissue Res. 225, 201-215. Dhoot, G. K., Perry, S. V., and Vrbova, G. (1981). Changes in thedistribution of the components of the troponin complex in muscle fibers after crossinnervation. Exp. Neural. 72, 513-530. Furshpan, E. J., MacLeish, P. R., O’Lague, P. H., and Potter, D. D. (1976). Chemical transmission between rat sympathetic neurons and cardiac myocytes developing in microcultures: evidence for cholinergic, adrenergic, and dual-function neurons. Proc. Nat. Acad. Sci. USA 73, 42254229. Gauthier, G. F., and Hobbs, A. W. (1982). Effects of denervation on the drstribution of myosrn isozymes in skeletal muscle fibers. Exp. Neurol. 76, 331-346. Gauthier, G. F., Lowey, S., and Hobbs, A. W. (1978). Fast and slow myosin In developing muscle fibres. Nature 274, 25-29. Hoh, J. F. Y., and Yeoh, G. P. S. (1979). Rabbit skeletal myosin isoenzymes from fetal, fast-twitch and slow-twitch muscles. Nature 280, 321-323. Keller, L. R., and Emerson, C. P. Jr. (1980). Synthesis of adult myosin light chains by embryonic muscle cultures. Proc. Nat. Acad. Sci. USA 77, 10201024. Masaki, T., and Yoshizaki, C. (1974). Differentiation embryos. J. Biochem. (Tokyo) 76, 123-131.
of myosin
in chick
Masuko, S., Kuromi, H., and Shimada, Y. (1979). Isolation and culture of motoneurons from embryonic chicken spinal cords. Proc. Nat. Acad. Sci. USA 76, 3537-3541. Matsuda, R., Obinata, T., and Shimada, Y. (1981). Types of troponin components during development of chicken skeletal muscle. Dev. Biol. 82, 11-19. Roy, R. K., Sreter, F. A., and Sarkar, S. (1979). Changes in tropomyosin subunits and myosin light chains during development chicken and rabbit striated muscles. Dev. Biol. 69, 15-30. Rubinstein, N. A., and Holtzer, H. (1979). Fast and slow muscles culture synthesise only fast myosin. Nature 280, 323-325.
in tissue
Rubinstern, N. A., Pepe, F. A., and Holtzer, H. (1977). Myosin types during the development of embryonic chicken fast and slow muscles. Proc. Nat. Acad. Sci. USA 74, 4524-4527. Salmons, S., and Sreter, F. A. (1976). Significance of impulse activity in the transformation of skeletal muscle type. Nature 263, 30-34. Schiaffino, S., Askanas, V., Engel, W. K., Vitadello, M., and Sartore, S. (1982). Myosrn isoenzymes in cultured human muscle. Arch. Neurol. 39, 347-349. Shimada, Y. (1971). Electron microscope observations chick myoblasts in vitro. J. Cell Biol. 48, 128-142.
on the fusion of
Toyota, N., and Shimada, Y. (1981). Differentiation of troponrn in cardiac and skeletal muscles in chicken embryos as studied by immunofluorescence microscopy. J. Cell Biol. 91, 497-504.
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S. (1980). Myosin types in cultured