The expression of the myofibrillar M-band proteins myomesin and M-protein was studied in chicken pectoral muscle and heart during differentiation using monoclonal antibodies in a double-antibody sandwich enzyme-linked immunosorbent assay, immunoblotting, and immunocytochemistry. In presumptive pectoral muscle, myomesin accumulated first, increasing from 2% of the adult concentration at day 7 to 70% by day 16 in ovo. M-protein accumulation lagged 6-7 d behind that of myomesin attaining only 40% of the adult concentration in ovo. The molecular masses of myomesin (185 kD) and M-protein (165 kD) remained constant during embryogenesis. In cultured myogenic cells the accumulation and M-band localization of myomesin preceded that of M-protein by 1.5 d.Chicken heart was shown, in addition to M-protein, to contain unique isoforms of myomesin. In hearts of 6 d embryos, a 195-kD myomesin isoform was the major species; throughout development, however, a transition to a mixture of 195 and 190 kD was observed, the latter being the major species in the adult tissue. During heart differentiation the initial accumulation of myomesin again preceded that of M-protein, albeit on an earlier time scale than in pectoral muscle with M-protein reaching adult proportions first.Recently we have reported on a new 185-kD protein in the M-band of skeletal muscle myofibrils (1). This brings the number of positively identified M-band proteins to three, the other two being MM-CK (2, 3) and M-protein (4). To avoid multiple nomenclature, it has been agreed to call the 165-kD M-band protein "M-protein" and the recently identified 185-kD M-band protein "myomesin" (1, 5).Several lines of evidence indicate the functional significance of the M-band which transverses the center of the sarcomere. M-band structural components appear to provide a scaffolding for the alignment and orientation of myosin thick fdaments within myofibrils (6-10). Furthermore, there is evidence that the M-band, as well as the Z-disc, are cytoskeletal attachment points and that they assist in maintaining intermyofibril register under the stress of lateral tension imposed on muscle fibers during contraction (11-13).Of the known M-band proteins, the substructural localization and function of only one has been elucidated. MM-CK, associated with the M4, 4' substriations (14,15), is present in sufficient quantity to regenerate in isolated chicken myofibrils the ATP required for contraction (16). Attempts have been made to ascertain the roles of other M-band components in

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Abstract. The expression of neonatal myosin heavy chain (MHC) was examined in developing embryonic chicken muscle cultures using a monoclonal antibody (2E9) that has been shown to be specific for that isoform (Bandman, E., 1985, Science (Wash. DC), 227: 780-782). After 1 wk in vitro some myotubes could be stained with the antibody, and the number of cells that reacted with 2E9 increased with time in culture. All myotubes always stained with a second monoclonal antibody that reacted with all MHC isoforms (AG19) or with a third monoclonal antibody that reacted with the embryonic but not the neonatal MHC (EB165). Quantitation by ELISA of an extract from 2-wk cultures demonstrated that the neonatal MHC represented between 10 and 15 % of the total myosin. The appearance of the neonatal isoform was inhibited by switching young cultures to medium with a higher [K ÷] which has been shown to block spontaneous contractions of myotubes in culture. Furthermore, if mature cultures that reacted with the neonatal antibody were placed into high [K +] medium, neonatal MHC disappeared from virtually all myotubes within 3 d. The effect of high [K ÷] medium was reversible. When cultures maintained in high [K ÷] medium for 2 wk were placed in standard medium, which permitted the resumption of contractile activity, within 24 h cells began to react with the neonatal specific antibody, and by. 72 h many myotubes were strongly positive. Since similar results were also obtained by inhibiting spontaneous contractions with tetrodotoxin, we suggest that the development of contractile activity is not only associated with the maturation of myotubes in culture, but may also be the signal that induces the expression of the neonatal MHC. MVOGENIC cultures provide a model system in which the expression of muscle-specific proteins can be studied. Myotubes formed in vitro from embryonic myoblasts contain similar myofibrillar proteins and a similar ultrastructure as newly differentiated muscle cells in the embryo (18,30,33,52,64). However, while crossstriated, contractile myotubes are readily produced in culture, their ability to undergo the subsequent changes associated with muscle maturation is limited.During late embryonic and postnatal development in vivo muscle fibers undergo a number of changes in muscle protein expression. Some proteins found in adult muscle fibers are not produced in embryonic muscle. For example, phosphorylase, a glycolytic enzyme found in adult avian pectoral muscle fibers, initially appears at hatching and increases during neonatal development (14,19). Many skeletal muscle proteins exist as different isoforms, and maturing muscle fibers often exhibit changes in isoform expression. Skeletal muscle myosin, for example, is represented by many isozymes that are expressed at different stages of development in both avian and mammalian muscle (for review see reference 3). In chicken pectoral muscle (PM), 1 three myosin Abbreviations used in thispaper:MHC, myosin heavy chain; PM, pectoral muscle.heavy chains (MHCs) are expresse...

The functions of two myofibrillar proteins, myomesin (Mr 185,000) and M-protein (Mr 165,000), associated with the M-band are as yet unknown. To extend our knowledge of these proteins, we have examined chicken striated muscles with fast and slow contractile properties, e.g., pectoralis major, PLD, ALD, medial adductor, and lateral adductor, to determine the expression and isoform compositon of myomesin and M-protein in various muscles and fiber types The high molecular weight M-band proteins were characterized and quantitated using monoclonal antibodies in immunoblotting and double-antibody sandwich ELISA. Fiber specificity was determined by immuno-and enzyme histochemistry. In addition to the previously reported Mr 195,000 and 190,000 isoforms ofmyomesin in heart [Grove et aL (1985):

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