Skip to main content
PMC home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1991 Jun 1;97(6):1141–1163. doi: 10.1085/jgp.97.6.1141

Alterations in Ca2+ sensitive tension due to partial extraction of C- protein from rat skinned cardiac myocytes and rabbit skeletal muscle fibers

PMCID: PMC2216516  PMID: 1678777

Abstract

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 Ca(2+)-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 readdition of C-protein. The solution for extracting C-protein contained 10 mM EDTA, 31 mM Na2HPO2, 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 degrees C; a similar amount was extracted from psoas fibers during a 3- h soak at 25 degrees C. For both cardiac myocytes and skeletal muscle fibers, partial extraction of C-protein resulted in increased active tension at submaximal concentrations of Ca2+, but had little effect upon maximum tension. C-protein extraction also reduced the slope of the tension-pCa relationships, suggesting that the cooperativity of Ca2+ 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 microns 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 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.

Full Text

The Full Text of this article is available as a PDF (2.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Babu A., Scordilis S. P., Sonnenblick E. H., Gulati J. The control of myocardial contraction with skeletal fast muscle troponin C. J Biol Chem. 1987 Apr 25;262(12):5815–5822. [PubMed] [Google Scholar]
  2. Bennett P., Craig R., Starr R., Offer G. The ultrastructural location of C-protein, X-protein and H-protein in rabbit muscle. J Muscle Res Cell Motil. 1986 Dec;7(6):550–567. doi: 10.1007/BF01753571. [DOI] [PubMed] [Google Scholar]
  3. Brandt P. W., Cox R. N., Kawai M. Can the binding of Ca2+ to two regulatory sites on troponin C determine the steep pCa/tension relationship of skeletal muscle? Proc Natl Acad Sci U S A. 1980 Aug;77(8):4717–4720. doi: 10.1073/pnas.77.8.4717. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brandt P. W., Diamond M. S., Schachat F. H. The thin filament of vertebrate skeletal muscle co-operatively activates as a unit. J Mol Biol. 1984 Dec 5;180(2):379–384. doi: 10.1016/s0022-2836(84)80010-8. [DOI] [PubMed] [Google Scholar]
  5. Bremel R. D., Weber A. Cooperation within actin filament in vertebrate skeletal muscle. Nat New Biol. 1972 Jul 26;238(82):97–101. doi: 10.1038/newbio238097a0. [DOI] [PubMed] [Google Scholar]
  6. Cornish-Bowden A., Koshland D. E., Jr Diagnostic uses of the Hill (Logit and Nernst) plots. J Mol Biol. 1975 Jun 25;95(2):201–212. doi: 10.1016/0022-2836(75)90390-3. [DOI] [PubMed] [Google Scholar]
  7. Cox J. A., Comte M., Stein E. A. Calmodulin-free skeletal-muscle troponin C prepared in the absence of urea. Biochem J. 1981 Apr 1;195(1):205–211. doi: 10.1042/bj1950205. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Craig R., Offer G. The location of C-protein in rabbit skeletal muscle. Proc R Soc Lond B Biol Sci. 1976 Mar 16;192(1109):451–461. doi: 10.1098/rspb.1976.0023. [DOI] [PubMed] [Google Scholar]
  9. Dahlquist F. W. The meaning of Scatchard and Hill plots. Methods Enzymol. 1978;48:270–299. doi: 10.1016/s0076-6879(78)48015-2. [DOI] [PubMed] [Google Scholar]
  10. Dennis J. E., Shimizu T., Reinach F. C., Fischman D. A. Localization of C-protein isoforms in chicken skeletal muscle: ultrastructural detection using monoclonal antibodies. J Cell Biol. 1984 Apr;98(4):1514–1522. doi: 10.1083/jcb.98.4.1514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Endo M. Stretch-induced increase in activation of skinned muscle fibres by calcium. Nat New Biol. 1972 Jun 14;237(76):211–213. doi: 10.1038/newbio237211a0. [DOI] [PubMed] [Google Scholar]
  12. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378–417. doi: 10.1016/0076-6879(88)57093-3. [DOI] [PubMed] [Google Scholar]
  13. Fabiato A., Fabiato F. Effects of magnesium on contractile activation of skinned cardiac cells. J Physiol. 1975 Aug;249(3):497–517. doi: 10.1113/jphysiol.1975.sp011027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Giulian G. G., Moss R. L., Greaser M. Improved methodology for analysis and quantitation of proteins on one-dimensional silver-stained slab gels. Anal Biochem. 1983 Mar;129(2):277–287. doi: 10.1016/0003-2697(83)90551-1. [DOI] [PubMed] [Google Scholar]
  15. Godt R. E., Lindley B. D. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol. 1982 Aug;80(2):279–297. doi: 10.1085/jgp.80.2.279. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Grabarek Z., Grabarek J., Leavis P. C., Gergely J. Cooperative binding to the Ca2+-specific sites of troponin C in regulated actin and actomyosin. J Biol Chem. 1983 Dec 10;258(23):14098–14102. [PubMed] [Google Scholar]
  17. Greaser M. L., Gergely J. Reconstitution of troponin activity from three protein components. J Biol Chem. 1971 Jul 10;246(13):4226–4233. [PubMed] [Google Scholar]
  18. Gulati J., Scordilis S., Babu A. Effect of troponin C on the cooperativity in Ca2+ activation of cardiac muscle. FEBS Lett. 1988 Aug 29;236(2):441–444. doi: 10.1016/0014-5793(88)80073-5. [DOI] [PubMed] [Google Scholar]
  19. Güth K., Potter J. D. Effect of rigor and cycling cross-bridges on the structure of troponin C and on the Ca2+ affinity of the Ca2+-specific regulatory sites in skinned rabbit psoas fibers. J Biol Chem. 1987 Oct 5;262(28):13627–13635. [PubMed] [Google Scholar]
  20. Hartzell H. C. Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase. J Mol Biol. 1985 Nov 5;186(1):185–195. doi: 10.1016/0022-2836(85)90268-2. [DOI] [PubMed] [Google Scholar]
  21. Hartzell H. C., Glass D. B. Phosphorylation of purified cardiac muscle C-protein by purified cAMP-dependent and endogenous Ca2+-calmodulin-dependent protein kinases. J Biol Chem. 1984 Dec 25;259(24):15587–15596. [PubMed] [Google Scholar]
  22. Hartzell H. C., Sale W. S. Structure of C protein purified from cardiac muscle. J Cell Biol. 1985 Jan;100(1):208–215. doi: 10.1083/jcb.100.1.208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hofmann P. A., Fuchs F. Bound calcium and force development in skinned cardiac muscle bundles: effect of sarcomere length. J Mol Cell Cardiol. 1988 Aug;20(8):667–677. doi: 10.1016/s0022-2828(88)80012-9. [DOI] [PubMed] [Google Scholar]
  24. Hofmann P. A., Metzger J. M., Greaser M. L., Moss R. L. Effects of partial extraction of light chain 2 on the Ca2+ sensitivities of isometric tension, stiffness, and velocity of shortening in skinned skeletal muscle fibers. J Gen Physiol. 1990 Mar;95(3):477–498. doi: 10.1085/jgp.95.3.477. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koretz J. F., Coluccio L. M., Bertasso A. M. The aggregation characteristics of column-purified rabbit skeletal myosin in the presence and absence of C-protein at pH 7.0. Biophys J. 1982 Feb;37(2):433–440. doi: 10.1016/S0006-3495(82)84689-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lim M. S., Walsh M. P. Phosphorylation of skeletal and cardiac muscle C-proteins by the catalytic subunit of cAMP-dependent protein kinase. Biochem Cell Biol. 1986 Jul;64(7):622–630. doi: 10.1139/o86-086. [DOI] [PubMed] [Google Scholar]
  27. Margossian S. S. Reversible dissociation of dog cardiac myosin regulatory light chain 2 and its influence on ATP hydrolysis. J Biol Chem. 1985 Nov 5;260(25):13747–13754. [PubMed] [Google Scholar]
  28. Moos C., Feng I. N. Effect of C-protein on actomyosin ATPase. Biochim Biophys Acta. 1980 Oct 1;632(2):141–149. doi: 10.1016/0304-4165(80)90071-9. [DOI] [PubMed] [Google Scholar]
  29. Moos C. Fluorescence microscope study of the binding of added C protein to skeletal muscle myofibrils. J Cell Biol. 1981 Jul;90(1):25–31. doi: 10.1083/jcb.90.1.25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Moos C., Mason C. M., Besterman J. M., Feng I. N., Dubin J. H. The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1. J Mol Biol. 1978 Oct 5;124(4):571–586. doi: 10.1016/0022-2836(78)90172-9. [DOI] [PubMed] [Google Scholar]
  31. Moss R. L., Giulian G. G., Greaser M. L. Effects of EDTA treatment upon the protein subunit composition and mechanical properties of mammalian single skeletal muscle fibers. J Cell Biol. 1983 Apr;96(4):970–978. doi: 10.1083/jcb.96.4.970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Moss R. L., Giulian G. G., Greaser M. L. The effects of partial extraction of TnC upon the tension-pCa relationship in rabbit skinned skeletal muscle fibers. J Gen Physiol. 1985 Oct;86(4):585–600. doi: 10.1085/jgp.86.4.585. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moss R. L., Lauer M. R., Giulian G. G., Greaser M. L. Altered Ca2+ dependence of tension development in skinned skeletal muscle fibers following modification of troponin by partial substitution with cardiac troponin C. J Biol Chem. 1986 May 5;261(13):6096–6099. [PubMed] [Google Scholar]
  34. Moss R. L. Sarcomere length-tension relations of frog skinned muscle fibres during calcium activation at short lengths. J Physiol. 1979 Jul;292:177–192. doi: 10.1113/jphysiol.1979.sp012845. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Moss R. L., Swinford A. E., Greaser M. L. Alterations in the Ca2+ sensitivity of tension development by single skeletal muscle fibers at stretched lengths. Biophys J. 1983 Jul;43(1):115–119. doi: 10.1016/S0006-3495(83)84329-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Offer G., Moos C., Starr R. A new protein of the thick filaments of vertebrate skeletal myofibrils. Extractions, purification and characterization. J Mol Biol. 1973 Mar 15;74(4):653–676. doi: 10.1016/0022-2836(73)90055-7. [DOI] [PubMed] [Google Scholar]
  37. Reiser P. J., Greaser M. L., Moss R. L. Myosin heavy chain composition of single cells from avian slow skeletal muscle is strongly correlated with velocity of shortening during development. Dev Biol. 1988 Oct;129(2):400–407. doi: 10.1016/0012-1606(88)90387-9. [DOI] [PubMed] [Google Scholar]
  38. Rome E., Offer G., Pepe F. A. X-ray diffraction of muscle labelled with antibody to C-protein. Nat New Biol. 1973 Aug 1;244(135):152–154. doi: 10.1038/newbio244152a0. [DOI] [PubMed] [Google Scholar]
  39. Starr R., Offer G. The interaction of C-protein with heavy meromyosin and subfragment-2. Biochem J. 1978 Jun 1;171(3):813–816. doi: 10.1042/bj1710813. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sweitzer N. K., Moss R. L. The effect of altered temperature on Ca2(+)-sensitive force in permeabilized myocardium and skeletal muscle. Evidence for force dependence of thin filament activation. J Gen Physiol. 1990 Dec;96(6):1221–1245. doi: 10.1085/jgp.96.6.1221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wagner P. D. Preparation and fractionation of myosin light chains and exchange of the essential light chains. Methods Enzymol. 1982;85(Pt B):72–81. doi: 10.1016/0076-6879(82)85010-6. [DOI] [PubMed] [Google Scholar]
  42. Yamamoto K., Moos C. The C-proteins of rabbit red, white, and cardiac muscles. J Biol Chem. 1983 Jul 10;258(13):8395–8401. [PubMed] [Google Scholar]
  43. Yamamoto K. The binding of skeletal muscle C-protein to regulated actin. FEBS Lett. 1986 Nov 10;208(1):123–127. doi: 10.1016/0014-5793(86)81545-9. [DOI] [PubMed] [Google Scholar]
  44. Zot H. G., Potter J. D. A structural role for the Ca2+-Mg2+ sites on troponin C in the regulation of muscle contraction. Preparation and properties of troponin C depleted myofibrils. J Biol Chem. 1982 Jul 10;257(13):7678–7683. [PubMed] [Google Scholar]
  45. el-Saleh S. C., Warber K. D., Potter J. D. The role of tropomyosin-troponin in the regulation of skeletal muscle contraction. J Muscle Res Cell Motil. 1986 Oct;7(5):387–404. doi: 10.1007/BF01753582. [DOI] [PubMed] [Google Scholar]

Articles from The Journal of General Physiology are provided here courtesy of The Rockefeller University Press

RESOURCES