Biosynthesis of Titin in Cultured Skeletal Muscle Cells

Although significant progress has been made regarding the structure and function of titin, little data exist on the biosynthesis of this large protein in developing muscle. Using pulse-labeling with [35S]methionine and immunoprecipitation with an antititin mAb, we have examined the biosynthesis of titin in synchronized cultures of skeletal muscle cells derived from day 12 chicken embryos. We find that: (a) titin synthesis increases >4-fold during the first week in culture and during this same time period, synthesis of muscle-specific myosin heavy chain increases >12fold; (b) newly synthesized titin has a tl/2 of ~70 h; (c) titin is resistant to extraction with Triton X-100 both during and immediately after its synthesis. These observations suggest that newly synthesized titin molecules are stable proteins that rapidly associate with the cytoskeleton of developing myotubes. C ARDIAC and skeletal muscle are unique in possessing sarcomeres, the highly ordered and periodic arrays of actin and myosin that generate the contractile force of muscle. Although sarcomeres are unique to striated muscle, most contractile proteins, such as actin and myosin, are not; nonmuscle or smooth muscle isoforms are found in most cells. One of the few proteins preferentially found in striated muscle is titin, a recently described myofibril protein with a molecular mass exceeding 1,000,000 D (reviewed in Wang, 1982, 1984, 1985; Ohtsuki et al., 1986). Titin (also called connectin; Maruyama et al., 1984) is relatively abundant, making up "~10% of myofibril mass (Wang et al., 1984; Trinick et al., 1984). It is usually detected as a doublet of proteins, termed TI and TII,~ on low porosity gels. The presence of titin in all striated muscles (Locker and Wild, 1986; Osborn et al., 1986; Gassner, 1986; Hill and Weber, 1986; Wang and Greaser, 1985; Hu et al., 1986; Wang et al., 1985) has sharpened interest in the function of this protein. Titin has been detected in the myofibril by immunofluorescence (Osborn et al., 1986; Gassner, 1986; Hill and Weber, 1984; Wang and Greaser, 1985; Wang et al., 1985) and immunoelectron (Gassner, 1986; Hill and Weber, 1986; Maruyama et al., 1984, 1985) microscopy, mAbs to different epitopes of titin show that titin lies symmetrically relative to the M line (Wang and Greaser, 1985; Wang and RamirezMitchell, 1983) and that single titin molecules stretch from the Z line to within 0.2/zm of the M line (Furst et al., 1988). This latter study by Furst et al. further demonstrated that the epitopes of titin near the Z line are present on the TI form of titin, but not the smaller TII form. Dr. William B. Isaacs' current address is Johns Hopkins Hospital, Room 105, Marburg Building, 600 N. Wolfe Street, Baltimore, MD 21205. 1. Abbreviations used in this paper: /~ME, ~-mercaptoethanol; MHC, myosin heavy chain; TI, TII, titin. At the ultrastructural level, titin has been detected immunologically on "gap" filaments that are visible in overextended muscle, and associated with purified thick filaments (Gassner, 1986; Hill and Weber, 1986; Maruyama et al., 1984, 1985). Thus, the overall image presented by light microscopy, that titin is a myofibril protein with a specific distribution, has been confirmed. The patterns seen so far led Wang (1984) and Wang et al. (1984) and others (Gassner, 1986; Maruyama et al., 1984) to interpret titin as a major component of the "third filament" or "gap filament" system of the myofibril. The existence of a third filament system in the myofibril that provides elasticity has been proposed in several forms, and titin appears to be one major component of such a third filament system. Direct evidence of an elastic role was detected by using ionizing radiation to ablate preferentially titin and nebulin from isolated myofibrils (Horowits et al., 1986). Such myofibrils are much less elastic and become misaligned axially, providing direct evidence for titin's role in elastic recoil and sarcomere stability. These results are consistent with the demonstration by Trinick et al. (1984) that the secondary structure of titin consists almost entirely of random coil, characteristic of elastic proteins. Further support for a general role in striated muscle elasticity comes from the wide distribution of titin (Locker and Wild, 1986; Osborn et al., 1986; Gassner, 1986; Hu et al., 1986; Maruyama et al., 1984). A mechanical role for titin thus is reasonably well established. A few observations suggest that titin may also participate in the morphogenesis of the myofibril. Titin is present and periodic when the first periodic arrays of myosin are seen, in both skeletal (Hill et al., 1986) and cardiac (Wang et al., 1984) cells. Titin may be required for such periodic arrays because it is one of the few proteins unique to and ubiquitous in striated muscle. At present, there is very little known about either the syn© The Rockefeller University Press, 0021-9525/89/11/2189/7 $2.00 The Journal of Cell Biology, Volume 109, November 1989 2189-2195 2189 on A uust 7, 2017 jcb.rress.org D ow nladed fom thesis or assembly of titin in developing muscle cells. These questions are particularly interesting in view of the molecular dimensions of this protein. Given its tremendous size, the question arises of how titin is properly oriented in the developing sarcomere. Before questions pertaining to the assembly of titin can be properly addressed, however, characterization of the biosynthesis of this protein is necessary. In this study, we describe the immunoprecipitation of newly synthesized titin from cultures of developing myotubes. We have used this immunoprecipitation protocol to quantitate the synthesis, accumulation, and stability of titin during the early stages of myofibrillogenesis. Materials and Methods