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Single Molecule Detection Approach to Muscle Study: Kinetics of a Single Cross-Bridge During Contraction of Muscle

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Book cover Spectroscopic Methods of Analysis

Part of the book series: Methods in Molecular Biology ((MIMB,volume 875))

Abstract

D166V point mutation in the ventricular myosin regulatory light chain (RLC) is one of the causes of familial hypertrophic cardiomyopathy (FHC). We show here that the rates of cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricle of WT and Tg-D166V mice. To avoid averaging over ensembles of molecules composing muscle fibers, the data was collected from a single molecule. Kinetics were derived by tracking the orientation of a single actin molecule by fluorescence anisotropy. Orientation oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The cross-bridge in a wild-type (healthy) heart stayed attached and detached from thin filament on average for 0.7 and 2.7 s, respectively. In FHC heart, these numbers increased to 2.5 and 5.8 s, respectively. These findings suggest that alterations in myosin cross-bridge kinetics associated with D166V mutation of RLC ultimately affect the ability of a heart to efficiently pump the blood.

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Abbreviations

t ON :

The time cross-bridge is strongly attached to actin

t OFF :

The time cross-bridge is detached from actin

Ψ :

Duty cycle of the cross-bridge

AP:

Alexa488-phalloidin

RP:

Rhodamine-phalloidin

UP:

Unlabeled-phalloidin

DA:

Detection area

EDC:

1-Ethyl-3-(3′-dimethylaminopropyl) carbodiimide

ROI:

Region-of-interest

DTT:

Cleland’s reagent

APD:

Avalanche photodiode

FCS:

Fluorescence correlation spectroscopy

SMD:

Single molecule detection

References

  1. Nihei T, Mendelson RA, Botts J (1974) Use of fluorescence polarization to observe changes in attitude of S1 moieties in muscle fibers. Biophys J 14:236–242

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Borejdo J, Assulin O, Ando T, Putnam S (1982) Cross-bridge orientation in skeletal muscle measured by linear dichroism of an extrinsic chromophore. J Mol Biol 158:391–414

    Article  CAS  PubMed  Google Scholar 

  3. Thomas DD, Cooke R (1980) Orientation of spin-labeled myosin heads in glycerinated muscle fibers. Biophys J 32:891–905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Huxley AF, Simmons RM (1971) Proposed mechanism of force generation in striated muscle. Nature 233:533–538

    Article  CAS  PubMed  Google Scholar 

  5. Lymn RW, Taylor EW (1971) Mechanism of adenosine triphosphate hydrolysis by actomyosin. Biochemistry 10:4617–4624

    Article  CAS  PubMed  Google Scholar 

  6. Siemankowski RF, Wiseman MO, White HD (1985) ADP dissociation from actomyosin subfragment 1 is sufficiently slow to limit the unloaded shortening velocity in vertebrate muscle. Proc Natl Acad Sci U S A 82:658–662

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Geeves MA, Holmes KC, Bodis E et al (2005) The molecular mechanism of muscle contraction. Adv Protein Chem 71:161–193

    Article  CAS  PubMed  Google Scholar 

  8. Magde D, Elson EL, Webb WW (1974) Fluorescence correlation spectroscopy. II. An experimental realization. Biopolymers 13:29–61

    Article  CAS  PubMed  Google Scholar 

  9. Qian H, Saffarian S, Elson EL (2002) Concentration fluctuations in a mesoscopic oscillating chemical reaction system. Proc Natl Acad Sci U S A 99:10376–10381

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Bagshaw CR (1982) Muscle contraction. Chapman & Hall, London

    Book  Google Scholar 

  11. Enderlein J, Ambrose WP (1997) Optical collection efficiency function in single-molecule detection experiments. Appl Opt 36:5298–5302

    Article  CAS  PubMed  Google Scholar 

  12. Willets KA, Ostroverkhova O, He M, Twieg RJ, Moerner WE (2003) Novel fluorophores for single-molecule imaging. J Am Chem Soc 125:1174–1175

    Article  CAS  PubMed  Google Scholar 

  13. Wang Y, Qin H, Kudaravalli RD et al (2007) Single-molecule structural dynamics of EF-G-ribosome interaction during translocation. Biochemistry 46:10767–10775

    Article  CAS  PubMed  Google Scholar 

  14. Taniguchi Y, Karagiannis P, Nishiyama M, Ishii Y, Yanagida T (2007) Single molecule thermodynamics in biological motors. Biosystems 88:283–292

    Article  CAS  PubMed  Google Scholar 

  15. Warshaw DM, Hayes E, Gaffney D et al (1998) Myosin conformational states determined by single fluorophore polarization. Proc Natl Acad Sci U S A 95:8034–8039

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Quinlan ME, Forkey JN, Goldman YE (2005) Orientation of the myosin light chain region by single molecule total internal reflection fluorescence polarization microscopy. Biophys J 89:1132–1142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Forkey JN, Quinlan ME, Shaw MA, Corrie JE, Goldman YE (2003) Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization. Nature 422:399–404

    Article  CAS  PubMed  Google Scholar 

  18. Yildiz A, Forkey JN, McKinney SA, Ha T, Goldman YE, Selvin PR (2003) Myosin V walks hand-over-hand: single fluorophore imaging with 1.5-nm localization. Science 300:2061–2065

    Article  CAS  PubMed  Google Scholar 

  19. Toprak E, Enderlein J, Syed S et al (2006) Defocused orientation and position imaging (DOPI) of myosin V. Proc Natl Acad Sci U S A 103:6495–6499

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Minton AP, Wilf J (1981) Effect of macromolecular crowding upon the structure and function of an enzyme: glyceraldehyde-3-phosphate dehydrogenase. Biochemistry 20:4821–4826

    Article  CAS  PubMed  Google Scholar 

  21. Minton AP (1981) Excluded volume as a determinant of macromolecular structure and reactivity. Biopolymers 20:2093–2120

    Article  CAS  Google Scholar 

  22. Eigen M, Rigler R (1994) Sorting single molecules: application to diagnostics and evolutionary biotechnology. Proc Natl Acad Sci U S A 91:5740–5747

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Borejdo J, Shepard AA, Akopova I, Grudzinski W, Malicka J (2004) Rotation of the lever-arm of myosin in contracting skeletal muscle fiber measured by two-photon anisotropy. Biophys J 87:3912–3921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Levene MJ, Korlach J, Turner SW, Foquet M, Craighead HG, Webb WW (2003) Zero-mode waveguides for single-molecule analysis at high concentrations. Science 299:682–686

    Article  CAS  PubMed  Google Scholar 

  25. Lewis A (1991) The optical near-field and cell biology. Semin Cell Biol 2:187–192

    CAS  PubMed  Google Scholar 

  26. de Lange F, Cambi A, Huijbens R et al (2001) Cell biology beyond the diffraction limit: near-field scanning optical microscopy. J Cell Sci 114:4153–4160

    PubMed  Google Scholar 

  27. Borejdo J, Talent J, Akopova I, Burghardt TP (2006) Rotations of a few cross-bridges in muscle by confocal total internal reflection microscopy. Biochim Biophys Acta 1763:137–140

    Article  CAS  PubMed  Google Scholar 

  28. Vecer J, Kowalczyk AA, Davenport L, Dale RE (1993) Reconvolution analysis in time-resolved fluorescence experiments, an alternative approach: reference-to-excitation-to-fluorescence reconvolution. Rev Sci Instrum 64:3413–3424

    Article  CAS  Google Scholar 

  29. Bukatina AE, Fuchs F, Watkins SC (1996) A study on the mechanism of phalloidin-induced tension changes in skinned rabbit psoas muscle fibres. J Muscle Res Cell Motil 17:365–371

    Article  CAS  PubMed  Google Scholar 

  30. Prochniewicz-Nakayama E, Yanagida T, Oosawa F (1983) Studies on conformation of F-actin in muscle fibers in the relaxed state, rigor, and during contraction using fluorescent phalloidin. J Cell Biol 97:1663–1667

    Article  CAS  PubMed  Google Scholar 

  31. Shepard A, Borejdo J (2004) Correlation between mechanical and enzymatic events in contracting skeletal muscle fiber. Biochemistry 43:2804–2811

    Article  CAS  PubMed  Google Scholar 

  32. Szczesna D, Lehrer SS (1993) The binding of fluorescent phallotoxins to actin in myofibrils. J Muscle Res Cell Motil 14:594–597

    Article  CAS  PubMed  Google Scholar 

  33. Yanagida T, Oosawa F (1980) Conformational changes of F-actin-epsilon-ADP in thin filaments in myosin-free muscle fibers induced by Ca2+. J Mol Biol 140:313–320

    Article  CAS  PubMed  Google Scholar 

  34. Yanagida T, Oosawa F (1998) Polarized fluorescence from epsilon-ADP incorporated into F-actin in a myosin-free single fiber: conformation of F-actin and changes induced in it by heavy meromyosin. J Mol Biol 126:507–524

    Article  Google Scholar 

  35. Borovikov YS, Kuleva NV, Khoroshev MI (1991) Polarization microfluorimetry study of interaction between myosin head and F-actin in muscle fibers. Gen Physiol Biophys 10:441–459

    CAS  PubMed  Google Scholar 

  36. Borejdo J, Shepard A, Dumka D et al (2004) Changes in orientation of actin during contraction of muscle. Biophys J 86:2308–2317

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Dos Remedios CG, Millikan RG, Morales MF (1972) Polarization of tryptophan fluorescence from single striated muscle fibers. A molecular probe of contractile state. J Gen Physiol 59:103–120

    Article  PubMed  PubMed Central  Google Scholar 

  38. Dos Remedios CG, Yount RG, Morales MF (1972) Individual states in the cycle of muscle contraction. Proc Natl Acad Sci U S A 69:2542–2546

    Article  PubMed  PubMed Central  Google Scholar 

  39. Tregear RT, Mendelson RA (1975) Polarization from a helix of fluorophores and its relation to that obtained from muscle. Biophys J 15:455–467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Morales MF (1984) Calculation of the polarized fluorescence from a labeled muscle fiber. Proc Nat Acad Sci U S A 81:145–149

    Article  CAS  Google Scholar 

  41. Maron BJ, Olivotto I, Spirito P et al (2000) Epidemiology of hypertrophic cardiomyopathy-related death: revisited in a large non-referral-based patient population. Circulation 102:858–864

    Article  CAS  PubMed  Google Scholar 

  42. Elson EL (2008) Introduction to FCS. Course Cell Mol Fluorescence 2:8

    Google Scholar 

  43. Huxley AF (1957) A hypothesis for the mechanism of contraction of muscle. Prog Biophys Biophys Chem 7:255–318

    CAS  PubMed  Google Scholar 

  44. Cooke R, Crowder MS, Thomas DD (1982) Orientation of spin labels attached to cross-bridges in contracting muscle fibres. Nature 300:776–778

    Article  CAS  PubMed  Google Scholar 

  45. Thomas DD, Ramachandran S, Roopnarine O, Hayden DW, Ostap EM (1995) The mechanism of force generation in myosin: a disorder-to-order transition, coupled to internal structural changes. Biophys J 68:135S–141S

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Reedy MC (2000) Visualizing myosin’s power stroke in muscle contraction. J Cell Sci 113:3551–3562

    CAS  PubMed  Google Scholar 

  47. Sweeney HL, Houdusse A (2004) The motor mechanism of myosin V: insights for muscle contraction. Philos Trans R Soc Lond B Biol Sci 359:1829–1841

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Takagi Y, Shuman H, Goldman YE (2004) Coupling between phosphate release and force generation in muscle actomyosin. Philos Trans R Soc Lond B Biol Sci 359:1913–1920

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Poetter K, Jiang H, Hassanzadeh S et al (1996) Mutations in either the essential or regulatory light chains of myosin are associated with a rare myopathy in human heart and skeletal muscle. Nat Genet 13:63–69

    Article  CAS  PubMed  Google Scholar 

  50. Flavigny J, Richard P, Isnard R et al (1998) Identification of two novel mutations in the ventricular regulatory myosin light chain gene (MYL2) associated with familial and classical forms of hypertrophic cardiomyopathy. J Mol Med 76:208–214

    Article  CAS  PubMed  Google Scholar 

  51. Andersen PS, Havndrup O, Bundgaard H et al (2001) Myosin light chain mutations in familial hypertrophic cardiomyopathy: phenotypic presentation and frequency in Danish and South African populations. J Med Genet 38:E43

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Kabaeva ZT, Perrot A, Wolter B et al (2002) Systematic analysis of the regulatory and essential myosin light chain genes: genetic variants and mutations in hypertrophic cardiomyopathy. Eur J Hum Genet 10:741–748

    Article  CAS  PubMed  Google Scholar 

  53. Richard P, Charron P, Carrier L et al (2003) Hypertrophic cardiomyopathy: distribution of disease genes. Spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation 107:2227–2232

    Article  PubMed  Google Scholar 

  54. Morner S, Richard P, Kazzam E et al (2003) Identification of the genotypes causing hypertrophic cardiomyopathy in northern Sweden. J Mol Cell Cardiol 35:841–849

    Article  CAS  PubMed  Google Scholar 

  55. Hougs L, Havndrup O, Bundgaard H et al (2005) One third of Danish hypertrophic cardiomyopathy patients have mutations in MYH7 rod region. Eur J Hum Genet 13:161–165

    Article  CAS  PubMed  Google Scholar 

  56. Maron BJ (2002) The young competitive athlete with cardiovascular abnormalities: causes of sudden death, detection by preparticipation screening, and standards for disqualification. Card Electrophysiol Rev 6:100–103

    Article  PubMed  Google Scholar 

  57. Ao X, Lehrer SS (1995) Phalloidin unzips nebulin from thin filaments in skeletal myofibrils. J Cell Sci 108:3397–3403

    CAS  PubMed  Google Scholar 

  58. Borovikov YS, Chernogriadskaia NA (1979) Studies on conformational changes in F-actin of glycerinated muscle fibers during relaxation by means of polarized ultraviolet fluorescence microscopy. Microsc Acta 81:383–392

    CAS  PubMed  Google Scholar 

  59. Houdusse A, Sweeney HL (2001) Myosin motors: missing structures and hidden springs. Curr Opin Struct Biol 11:182–194

    Article  CAS  PubMed  Google Scholar 

  60. Pesce AJ, Rosen CG, Pasby TL (1971) Fluorescence spectroscopy. Marcel Dekker, New York

    Google Scholar 

  61. Mettikolla P, Luchowski R, Gryczynski I, Gryczynski Z, Szczesna-Cordary D, Borejdo J (2009) Fluorescence lifetime of actin in the FHC transgenic heart. Biochemistry 48(6):1264–1271

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Cooper WC, Chrin LR, Berger CL (2000) Detection of fluorescently labeled actin-bound cross-bridges in actively contracting myofibrils. Biophys J 78:1449–1457

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Duong AM, Reisler E (1989) Binding of myosin to actin in myofibrils during ATP hydrolysis. Biochemistry 28:1307–1313

    Article  CAS  PubMed  Google Scholar 

  64. Hilber K, Sun YB, Irving M (2001) Effects of sarcomere length and temperature on the rate of ATP utilisation by rabbit psoas muscle fibres. J Physiol 531:771–780

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Kerrick WG, Kazmierczak K, Xu Y, Wang Y, Szczesna-Cordary D (2009) Malignant familial hypertrophic cardiomyopathy D166V mutation in the ventricular myosin regulatory light chain causes profound effects in skinned and intact papillary muscle fibers from transgenic mice. FASEB J 23:855–865

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Ando T (1987) Propagation of Acto-S-1 ATPase reaction-coupled conformational change in actin along the filament. J Biochem (Tokyo) 105:818–822

    Google Scholar 

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Acknowledgments

Supported by NIH grant R01AR048622 to J.B. and by Texas ETF grant (CCFT). R.L. is the recipient of the Research Mobility program from the Polish Ministry of Science and Higher Education.

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Authors and Affiliations

  1. Department of Molecular Biology and Immunology, Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX, USA

    Julian Borejdo, Priya Muthu & Prasad Metticolla

  2. University of Miami Miller School of Medicine, Miami, FL, USA

    Danuta Szczesna-Cordary

  3. Department of Biophysics, Institute of Physics, Maria Curie-Sklodowska University, Lublin, Poland

    Rafal Luchowski

  4. Center for Commercialization of Fluorescence Technologies, University of North Texas Health Science Center, Fort Worth, TX, USA

    Zygmunt Gryczynski & Ignacy Gryczynski

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  1. Julian Borejdo
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Correspondence to Julian Borejdo .

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  1. Dept. Biochemistry & Molecular Biology, University of Texas Medical Branch, University Blvd. 301, Galveston, 77555-1053, Texas, USA

    Wlodek M. Bujalowski

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Borejdo, J. et al. (2012). Single Molecule Detection Approach to Muscle Study: Kinetics of a Single Cross-Bridge During Contraction of Muscle. In: Bujalowski, W. (eds) Spectroscopic Methods of Analysis. Methods in Molecular Biology, vol 875. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-806-1_17

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  • DOI: https://doi.org/10.1007/978-1-61779-806-1_17

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  • Publisher Name: Humana Press, Totowa, NJ

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