Alterations in C a 2 + Sensitive Tension Due to Partial Extraction of C-Protein from Rat Skinned Cardiac Myocytes and Rabbit Skeletal Muscle Fibers

C-protein, a substantial component of muscle thick filaments, has been postulated to have various functions, based mainly on results from biochemical studies. In the present study, effects on Ca2+-activated tension due to partial removal of C-protein were investigated in skinned single myocytes from rat ventricle and rabbit psoas muscle. Isometric tension was measured at pCa values of 7.0 to 4.5: (a) in untreated myocytes, (b) in the same myocytes after partial extraction of C-protein, and (c) in some myocytes, after readdit ion of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO~, 124 mM NaH2PO4, pH 5.9 (Offer et al., 1973; Hartzell and Glass, 1984). In addition, the extracting solution contained 0.2 mg/ml troponin and, for skeletal muscle, 0.2 mg/ml myosin light chain-2 in order to minimize loss of these proteins during the extraction procedure. Between 60 and 70% of endogenous C-protein was extracted from cardiac myocytes by a 1-h soak in extracting solution at 20-23°C; a similar amount was extracted from psoas fibers during a 3-h soak at 25°C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca 2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca 2+ activation of tension was decreased. Readdition of C-protein to previously extracted myocytes resulted in recovery of both tension and slope to near their control values. The effects on tension did not appear to be due to disruption of cooperative activation of the thin filament, since C-protein extraction from cardiac myocytes that were 40-60% troponin-C (TnC) deficient produced effects similar to those observed in cells that were TnC replete. Measurements of the tension-pCa relationship in skeletal muscle fibers were also made at a sarcomere length of 3.5 I~m which, because of the distribution of C-protein on the thick filament, should eliminate any interaction between C-protein and actin. The effects of C-protein extraction were similar at long and short sarcomere lengths. These data are consistent with a model Address reprint requests to Dr. Polly A. Hofmann, Department of Physiology, 1300 University Avenue, Madison, WI 53706. j. GEN. PHYSIOL. © The Rockefeller University Press • 0022-1295/91/06/1141/23 $2.00 Volume 97 June 1991 1141-1163 1141 on F ebuary 0, 2005 w w w . jg.org D ow nladed fom 1142 THE . JOURNAL OF GENERAL PHYSIOLOGY " VOLUME 97" 1991 in which C-protein modulates the range of movement of myosin, such that the probability of myosin binding to actin is increased after its extraction. I N T R O D U C T I O N C-protein is an integral component of the myosin-containing thick filaments of cardiac and skeletal muscles. It is a polypeptide of 130-165 kD molecular mass and occurs in vivo in a 1:8 molar ratio of C-protein to myosin (Offer et al., 1973). With immunocytochemical techniques, C-protein has been localized to discreet regions in each half of the A-band along two 0.3-p~m lengths of the thick filament beginning 0.2 Ixm from its center. Within each 0.3-l~m region, C-protein molecules in different thick filaments lie in parallel register in seven or eight transverse, 10-nm wide strips spaced ~43 nm apart (Offer, 1972; Rome et al., 1973; Craig and Offer, 1976; Dennis et al., 1984; Bennett et al., 1986). It has been suggested that three C-protein molecules wrap around each thick filament and that an arm of the C-protein would be able to span the thick-thin filament gap (Craig and Offer, 1976; Moos et al., 1978; Hartzell and Sale, 1985; Solaro, 1987). Two binding sites for C-protein have been identified on myosin, one on light meromyosin and the other on heavy meromyosin subfragment-2 (S-2) (Moos et at., 1975; Starr and Offer, 1978). Previous investigations of the functional importance of C-protein can be categorized into studies that considered C-protein as an organizer or regulator of thick filament structure and those that considered C-protein as a modulator of contractile function (see Solaro, 1987, for a recent review). Purified myosin can self-assemble into thick filaments in the absence of C-protein; however, the addition of C-protein alters the periodicity and length of the final thick filament product (Moos et al., 1975; Koretz et al., 1982). Several investigators have shown that depending on ionic strength and the molar ratio of actin and myosin in solution, the addition of C-protein can modulate actin-activated ATPase activity of skeletal and cardiac myosins (Offer et al., 1973; Moos and Feng, 1980; Yamamoto and Moos, 1983; Hartzell, 1985; Margossian, 1985; Lim and Walsh, 1986). In the present study, the effects of partial extraction of C-protein on the tension-pCa relationship were investigated in single skinned cardiac myocytes and skeletal muscle fibers. These studies used near-physiological conditions to examine the function of C-protein in isolated muscle cells. The skinned single cell allows the study of functions of myofilament proteins in the context of an intact filament lattice, but without confounding effects due to intercellular connective tissue and protein heterogeneity between cells of multicellular preparations. Our results indicate that partial extraction of C-protein leads to a reversible increase in isometric tension at submaximal concentrations of C a 2+ and a decrease in the cooperative activation of Ca2+-dependent tension, supporting the view that contractile function is affected by C-protein.