Skip to main content
NCBI home page
The Journal of General Physiology logoLink to The Journal of General Physiology
. 1989 May 1;93(5):855–883. doi: 10.1085/jgp.93.5.855

Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. Implications for twitch potentiation in intact muscle

PMCID: PMC2216237  PMID: 2661721

Abstract

The Ca2+ sensitivities of the rate constant of tension redevelopment (ktr; Brenner, B., and E. Eisenberg. 1986. Proceedings of the National Academy of Sciences. 83:3542-3546) and isometric force during steady- state activation were examined as functions of myosin light chain 2 (LC2) phosphorylation in skinned single fibers from rabbit and rat fast- twitch skeletal muscles. To measure ktr the fiber was activated with Ca2+ and steady isometric tension was allowed to develop; subsequently, the fiber was rapidly (less than 1 ms) released to a shorter length and then reextended by approximately 200 nm per half sarcomere. This maneuver resulted in the complete dissociation of cross-bridges from actin, so that the subsequent redevelopment of tension was related to the rate of cross-bridge reattachment. The time course of tension redevelopment, which was recorded under sarcomere length control, was best fit by a first-order exponential equation (i.e., tension = C(1 - e- kt) to obtain the value of ktr. In control fibers, ktr increased sigmoidally with increases in [Ca2+]; maximum values of ktr were obtained at pCa 4.5 and were significantly greater in rat superficial vastus lateralis fibers (26.1 +/- 1.2 s-1 at 15 degrees C) than in rabbit psoas fibers (18.7 +/- 1.0 s-1). Phosphorylation of LC2 was accomplished by repeated Ca2+ activations (pCa 4.5) of the fibers in solutions containing 6 microM calmodulin and 0.5 microM myosin light chain kinase, a protocol that resulted in an increase in LC2 phosphorylation from approximately 10% in the control fibers to greater than 80% after treatment. After phosphorylation, ktr was unchanged at maximum or very low levels of Ca2+ activation. However, at intermediate levels of Ca2+ activation, between pCa 5.5 and 6.2, there was a significant increase in ktr such that this portion of the ktr-pCa relationship was shifted to the left. The steady-state isometric tension-pCa relationship, which in control fibers was left shifted with respect to the ktr-pCa relationship, was further left-shifted after LC2 phosphorylation. Phosphorylation of LC2 had no effect upon steady-state tension during maximum Ca2+ activation. In fibers from which troponin C was partially extracted to disrupt molecular cooperativity within the thin filament (Moss et al. 1985. Journal of General Physiology. 86:585- 600), the effect of LC2 phosphorylation to increase the Ca2+ sensitivity of steady-state isometric force was no longer evident, although the effect of phosphorylation to increase ktr was unaffected by this maneuver.(ABSTRACT TRUNCATED AT 400 WORDS)

Full Text

The Full Text of this article is available as a PDF (1.9 MB).

Selected References

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

  1. Alexis M. N., Gratzer W. B. Interaction of skeletal myosin light chains with calcium ions. Biochemistry. 1978 Jun 13;17(12):2319–2325. doi: 10.1021/bi00605a010. [DOI] [PubMed] [Google Scholar]
  2. Bagshaw C. R. Divalent metal ion binding and subunit interactions in myosins: a critical review. J Muscle Res Cell Motil. 1980 Sep;1(3):255–277. doi: 10.1007/BF00711931. [DOI] [PubMed] [Google Scholar]
  3. Baldwin K. M., Klinkerfuss G. H., Terjung R. L., Molé P. A., Holloszy J. O. Respiratory capacity of white, red, and intermediate muscle: adaptative response to exercise. Am J Physiol. 1972 Feb;222(2):373–378. doi: 10.1152/ajplegacy.1972.222.2.373. [DOI] [PubMed] [Google Scholar]
  4. Blinks J. R., Rüdel R., Taylor S. R. Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin. J Physiol. 1978 Apr;277:291–323. doi: 10.1113/jphysiol.1978.sp012273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. 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]
  7. Brenner B., Eisenberg E. Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution. Proc Natl Acad Sci U S A. 1986 May;83(10):3542–3546. doi: 10.1073/pnas.83.10.3542. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Brenner B. The cross-bridge cycle in muscle. Mechanical, biochemical, and structural studies on single skinned rabbit psoas fibers to characterize cross-bridge kinetics in muscle for correlation with the actomyosin-ATPase in solution. Basic Res Cardiol. 1986;81 (Suppl 1):1–15. doi: 10.1007/978-3-662-11374-5_1. [DOI] [PubMed] [Google Scholar]
  9. Brown G. L., von Euler U. S. The after effects of a tetanus on mammalian muscle. J Physiol. 1938 Jun 14;93(1):39–60. doi: 10.1113/jphysiol.1938.sp003623. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Butler T. M., Siegman M. J., Mooers S. U., Barsotti R. J. Myosin light chain phosphorylation does not modulate cross-bridge cycling rate in mouse skeletal muscle. Science. 1983 Jun 10;220(4602):1167–1169. doi: 10.1126/science.6857239. [DOI] [PubMed] [Google Scholar]
  11. Bárány K., Bárány M., Gillis J. M., Kushmerick M. J. Myosin light chain phosphorylation during the contraction cycle of frog muscle. Fed Proc. 1980 Apr;39(5):1547–1551. [PubMed] [Google Scholar]
  12. Bárány K., Bárány M. Phosphorylation of the 18,000-dalton light chain of myosin during a single tetanus of frog muscle. J Biol Chem. 1977 Jul 25;252(14):4752–4754. [PubMed] [Google Scholar]
  13. Cannell M. B., Allen D. G. Model of calcium movements during activation in the sarcomere of frog skeletal muscle. Biophys J. 1984 May;45(5):913–925. doi: 10.1016/S0006-3495(84)84238-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Close R., Hoh J. F. The after-effects of repetitive stimulation on the isometric twitch contraction of rat fast skeletal muscle. J Physiol. 1968 Jul;197(2):461–477. doi: 10.1113/jphysiol.1968.sp008570. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cooke R., Franks K., Stull J. T. Myosin phosphorylation regulates the ATPase activity of permeable skeletal muscle fibers. FEBS Lett. 1982 Jul 19;144(1):33–37. doi: 10.1016/0014-5793(82)80563-2. [DOI] [PubMed] [Google Scholar]
  16. Crow M. T., Kushmerick M. J. Phosphorylation of myosin light chains in mouse fast-twitch muscle associated with reduced actomyosin turnover rate. Science. 1982 Aug 27;217(4562):835–837. doi: 10.1126/science.6285472. [DOI] [PubMed] [Google Scholar]
  17. Dedman J. R., Potter J. D., Jackson R. L., Johnson J. D., Means A. R. Physicochemical properties of rat testis Ca2+-dependent regulator protein of cyclic nucleotide phosphodiesterase. Relationship of Ca2+-binding, conformational changes, and phosphodiesterase activity. J Biol Chem. 1977 Dec 10;252(23):8415–8422. [PubMed] [Google Scholar]
  18. Fabiato A., Fabiato F. Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol (Paris) 1979;75(5):463–505. [PubMed] [Google Scholar]
  19. Ford L. E., Huxley A. F., Simmons R. M. Tension responses to sudden length change in stimulated frog muscle fibres near slack length. J Physiol. 1977 Jul;269(2):441–515. doi: 10.1113/jphysiol.1977.sp011911. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Giulian G. G., Moss R. L., Greaser M. Analytical isoelectric focusing using a high-voltage vertical slab polyacrylamide gel system. Anal Biochem. 1984 Nov 1;142(2):421–436. doi: 10.1016/0003-2697(84)90486-x. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. 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]
  23. 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]
  24. HUXLEY A. F. Muscle structure and theories of contraction. Prog Biophys Biophys Chem. 1957;7:255–318. [PubMed] [Google Scholar]
  25. Holroyde M. J., Potter J. D., Solaro R. J. The calcium binding properties of phosphorylated and unphosphorylated cardiac and skeletal myosins. J Biol Chem. 1979 Jul 25;254(14):6478–6482. [PubMed] [Google Scholar]
  26. Kakol I., Kasman K., Michnicka M. The phosphorylation-dephosphorylation process as a myosin-linked regulation of superprecipitation of fast skeletal muscle actomyosin. Biochim Biophys Acta. 1982 Jun 24;704(3):437–443. doi: 10.1016/0167-4838(82)90065-6. [DOI] [PubMed] [Google Scholar]
  27. Kendrick-Jones J. Role of myosin light chains in calcium regulation. Nature. 1974 Jun 14;249(458):631–634. doi: 10.1038/249631a0. [DOI] [PubMed] [Google Scholar]
  28. Klug G. A., Botterman B. R., Stull J. T. The effect of low frequency stimulation on myosin light chain phosphorylation in skeletal muscle. J Biol Chem. 1982 May 10;257(9):4688–4690. [PubMed] [Google Scholar]
  29. Lehman W. Thick-filament-linked calcium regulation in vertebrate striated muscle. Nature. 1978 Jul 6;274(5666):80–81. doi: 10.1038/274080a0. [DOI] [PubMed] [Google Scholar]
  30. Manning D. R., Stull J. T. Myosin light chain phosphorylation and phosphorylase A activity in rat extensor digitorum longus muscle. Biochem Biophys Res Commun. 1979 Sep 12;90(1):164–170. doi: 10.1016/0006-291x(79)91604-8. [DOI] [PubMed] [Google Scholar]
  31. Manning D. R., Stull J. T. Myosin light chain phosphorylation-dephosphorylation in mammalian skeletal muscle. Am J Physiol. 1982 Mar;242(3):C234–C241. doi: 10.1152/ajpcell.1982.242.3.C234. [DOI] [PubMed] [Google Scholar]
  32. Metzger J. M., Moss R. L. Greater hydrogen ion-induced depression of tension and velocity in skinned single fibres of rat fast than slow muscles. J Physiol. 1987 Dec;393:727–742. doi: 10.1113/jphysiol.1987.sp016850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Metzger J. M., Moss R. L. Thin filament regulation of shortening velocity in rat skinned skeletal muscle: effects of osmotic compression. J Physiol. 1988 Apr;398:165–175. doi: 10.1113/jphysiol.1988.sp017036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Michnicka M., Kasman K., Kakol I. The binding of actin to phosphorylated and dephosphorylated myosin. Biochim Biophys Acta. 1982 Jun 24;704(3):470–475. doi: 10.1016/0167-4838(82)90069-3. [DOI] [PubMed] [Google Scholar]
  35. Moore R. L., Stull J. T. Myosin light chain phosphorylation in fast and slow skeletal muscles in situ. Am J Physiol. 1984 Nov;247(5 Pt 1):C462–C471. doi: 10.1152/ajpcell.1984.247.5.C462. [DOI] [PubMed] [Google Scholar]
  36. Morgan M., Perry S. V., Ottaway J. Myosin light-chain phosphatase. Biochem J. 1976 Sep 1;157(3):687–697. doi: 10.1042/bj1570687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Moss R. L., Allen J. D., Greaser M. L. Effects of partial extraction of troponin complex upon the tension-pCa relation in rabbit skeletal muscle. Further evidence that tension development involves cooperative effects within the thin filament. J Gen Physiol. 1986 May;87(5):761–774. doi: 10.1085/jgp.87.5.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Moss R. L. Effects on shortening velocity of rabbit skeletal muscle due to variations in the level of thin-filament activation. J Physiol. 1986 Aug;377:487–505. doi: 10.1113/jphysiol.1986.sp016199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. 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]
  40. 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]
  41. 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]
  42. Mrakovcić-Zenic A., Reisler E. Light-chain phosphorylation and cross-bridge conformation in myosin from vertebrate skeletal muscle. Biochemistry. 1983 Feb 1;22(3):525–530. doi: 10.1021/bi00272a001. [DOI] [PubMed] [Google Scholar]
  43. Nagamoto H., Yagi K. Properties of myosin light chain kinase prepared from rabbit skeletal muscle by an improved method. J Biochem. 1984 Apr;95(4):1119–1130. doi: 10.1093/oxfordjournals.jbchem.a134700. [DOI] [PubMed] [Google Scholar]
  44. Pemrick S. M. The phosphorylated L2 light chain of skeletal myosin is a modifier of the actomyosin ATPase. J Biol Chem. 1980 Sep 25;255(18):8836–8841. [PubMed] [Google Scholar]
  45. Perrie W. T., Smillie L. B., Perry S. B. A phosphorylated light-chain component of myosin from skeletal muscle. Biochem J. 1973 Sep;135(1):151–164. doi: 10.1042/bj1350151. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Persechini A., Stull J. T., Cooke R. The effect of myosin phosphorylation on the contractile properties of skinned rabbit skeletal muscle fibers. J Biol Chem. 1985 Jul 5;260(13):7951–7954. [PubMed] [Google Scholar]
  47. Persechini A., Stull J. T. Phosphorylation kinetics of skeletal muscle myosin and the effect of phosphorylation on actomyosin adenosinetriphosphatase activity. Biochemistry. 1984 Aug 28;23(18):4144–4150. doi: 10.1021/bi00313a021. [DOI] [PubMed] [Google Scholar]
  48. Pires E. M., Perry S. V. Purification and properties of myosin light-chain kinase from fast skeletal muscle. Biochem J. 1977 Oct 1;167(1):137–146. doi: 10.1042/bj1670137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Pollack G. H. Quantal mechanisms in cardiac contraction. Circ Res. 1986 Jul;59(1):1–8. doi: 10.1161/01.res.59.1.1. [DOI] [PubMed] [Google Scholar]
  50. Pulliam D. L., Sawyna V., Levine R. J. Calcium sensitivity of vertebrate skeletal muscle myosin. Biochemistry. 1983 May 10;22(10):2324–2331. doi: 10.1021/bi00279a004. [DOI] [PubMed] [Google Scholar]
  51. Shenolikar S., Cohen P. T., Cohen P., Nairn A. C., Perry S. V. The role of calmodulin in the structure and regulation of phosphorylase kinase from rabbit skeletal muscle. Eur J Biochem. 1979 Oct 15;100(2):329–337. doi: 10.1111/j.1432-1033.1979.tb04175.x. [DOI] [PubMed] [Google Scholar]
  52. Sweeney H. L., Kushmerick M. J. Myosin phosphorylation in permeabilized rabbit psoas fibers. Am J Physiol. 1985 Sep;249(3 Pt 1):C362–C365. doi: 10.1152/ajpcell.1985.249.3.C362. [DOI] [PubMed] [Google Scholar]
  53. Sweeney H. L., Stull J. T. Phosphorylation of myosin in permeabilized mammalian cardiac and skeletal muscle cells. Am J Physiol. 1986 Apr;250(4 Pt 1):C657–C660. doi: 10.1152/ajpcell.1986.250.4.C657. [DOI] [PubMed] [Google Scholar]
  54. Ueno H., Harrington W. F. Cross-bridge movement and the conformational state of the myosin hinge in skeletal muscle. J Mol Biol. 1981 Jul 15;149(4):619–640. doi: 10.1016/0022-2836(81)90350-8. [DOI] [PubMed] [Google Scholar]
  55. Watterson D. M., Sharief F., Vanaman T. C. The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem. 1980 Feb 10;255(3):962–975. [PubMed] [Google Scholar]
  56. Winkelmann D. A., Lowey S., Press J. L. Monoclonal antibodies localize changes on myosin heavy chain isozymes during avian myogenesis. Cell. 1983 Aug;34(1):295–306. doi: 10.1016/0092-8674(83)90160-5. [DOI] [PubMed] [Google Scholar]

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

RESOURCES