title: Zebrafish cardiac muscle thick filaments: isolation technique and three-dimensional structure.
abstract: To understand how mutations in thick filament proteins such as cardiac myosin binding protein-C or titin, cause familial hypertrophic cardiomyopathies, it is important to determine the structure of the cardiac thick filament. Techniques for the genetic manipulation of the zebrafish are well established and it has become a major model for the study of the cardiovascular system. Our goal is to develop zebrafish as an alternative system to the mammalian heart model for the study of the structure of the cardiac thick filaments and the proteins that form it. We have successfully isolated thick filaments from zebrafish cardiac muscle, using a procedure similar to those for mammalian heart, and analyzed their structure by negative-staining and electron microscopy. The isolated filaments appear well ordered with the characteristic 42.9 nm quasi-helical repeat of the myosin heads expected from x-ray diffraction. We have performed single particle image analysis on the collected electron microscopy images for the C-zone region of these filaments and obtained a three-dimensional reconstruction at 3.5 nm resolution. This reconstruction reveals structure similar to the mammalian thick filament, and demonstrates that zebrafish may provide a useful model for the study of the changes in the cardiac thick filament associated with disease processes.
authors.affiliation: Department of Anatomy and Neurobiology, University of Puerto Rico Medical School, San Juan, Puerto Rico. Electronic address: maryvi.gonzalez@upr.edu.
authors.forename: Maryví
authors.initials: M
authors.lastname: González-Solá
authors.affiliation: National Heart and Lung Institute, Faculty of Medicine, Imperial College London, London, United Kingdom.
authors.forename: Hind A
authors.initials: HA
authors.lastname: Al-Khayat
authors.affiliation: Department of Anatomy and Neurobiology, University of Puerto Rico Medical School, San Juan, Puerto Rico.
authors.forename: Martine
authors.initials: M
authors.lastname: Behra
authors.affiliation: Department of Anatomy and Neurobiology, University of Puerto Rico Medical School, San Juan, Puerto Rico.
authors.forename: Robert W
authors.initials: RW
authors.lastname: Kensler
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000648:ultrastructure; D064211:Connectin / Q000648:ultrastructure; D005583:Fourier Analysis; D021621:Imaging, Three-Dimensional; D008958:Models, Molecular; D009206:Myocardium / Q000648:ultrastructure*; D009218:Myosins / Q000302:isolation & purification* / Q000648:ultrastructure*; D015027:Zebrafish / Q000378:metabolism*; D000099163:Myosin Binding Protein C
chemical_list: D002352:Carrier Proteins; D064211:Connectin; D009218:Myosins; D000099163:Myosin Binding Protein C
references.citation: Curr Opin Pharmacol. 2012 Oct;12(5):608-14
references.pmid: 22858242
references.citation: Circ Res. 2003 Oct 17;93(8):752-8
references.pmid: 14500336
references.citation: J Struct Biol. 2008 Aug;163(2):117-26
references.pmid: 18472277
references.citation: J Mol Biol. 1997 Aug 22;271(3):417-37
references.pmid: 9268669
references.citation: J Mol Biol. 1974 Feb 5;82(4):527-36
references.pmid: 4817795
references.citation: Semin Cell Dev Biol. 2002 Dec;13(6):507-13
references.pmid: 12468254
references.citation: Proc Natl Acad Sci U S A. 1979 Aug;76(8):3698-702
references.pmid: 291034
references.citation: Dev Biol. 2005 Jan 1;277(1):1-15
references.pmid: 15572135
references.citation: J Struct Biol. 2005 Mar;149(3):303-12
references.pmid: 15721584
references.citation: J Mol Biol. 1999 Feb 26;286(3):933-49
references.pmid: 10024460
references.citation: Biophys J. 2008 Mar 1;94(5):1707-18
references.pmid: 17993479
references.citation: J Biol Chem. 1991 Feb 15;266(5):2811-7
references.pmid: 1671569
references.citation: FEBS Lett. 1980 Dec 15;122(1):129-32
references.pmid: 6260526
references.citation: Mol Cell Biochem. 2004 Aug;263(1):73-80
references.pmid: 27520666
references.citation: J Physiol. 2000 Oct 1;528 Pt 1:151-6
references.pmid: 11018113
references.citation: Proc Natl Acad Sci U S A. 2013 Jan 2;110(1):318-23
references.pmid: 23251030
references.citation: J Mol Biol. 1967 Dec 14;30(2):383-434
references.pmid: 5586931
references.citation: J Biol Chem. 2009 May 1;284(18):12318-27
references.pmid: 19269976
references.citation: Circ Res. 1999 May 28;84(10):1117-26
references.pmid: 10347086
references.citation: J Muscle Res Cell Motil. 1994 Dec;15(6):633-45
references.pmid: 7706420
references.citation: Physiol Genomics. 2010 Aug;42(3):406-19
references.pmid: 20484158
references.citation: Proc Natl Acad Sci U S A. 2001 Apr 10;98(8):4361-6
references.pmid: 11287639
references.citation: Proc Natl Acad Sci U S A. 2008 Feb 19;105(7):2386-90
references.pmid: 18252826
references.citation: Biophys J. 2002 Mar;82(3):1497-508
references.pmid: 11867464
references.citation: FEBS Lett. 2007 Apr 3;581(7):1501-4
references.pmid: 17376443
references.citation: Nature. 2005 Aug 25;436(7054):1195-9
references.pmid: 16121187
references.citation: FEBS Lett. 1971 Jun 2;15(1):40-44
references.pmid: 11945810
references.citation: Circ Res. 2004 May 28;94(10):1279-89
references.pmid: 15166115
references.citation: Biochem J. 1988 Feb 1;249(3):709-14
references.pmid: 2895634
references.citation: Circ Res. 2004 May 14;94(9):1249-55
references.pmid: 15059932
references.citation: J Muscle Res Cell Motil. 2004;25(8):635-44
references.pmid: 15750848
references.citation: J Cell Biol. 1993 Nov;123(3):619-26
references.pmid: 8227129
references.citation: Biophys J. 1993 Apr;64(4):1161-77
references.pmid: 8494977
references.citation: Circ Res. 2002 Oct 18;91(8):704-11
references.pmid: 12386147
references.citation: J Struct Biol. 2005 Sep;151(3):250-62
references.pmid: 16125414
references.citation: Biochem J. 1978 Jun 1;171(3):813-6
references.pmid: 352343
references.citation: Biophys J. 1996 Aug;71(2):898-907
references.pmid: 8842229
references.citation: PLoS One. 2009 Aug 12;4(8):e6596
references.pmid: 19672293
references.citation: J Mol Biol. 1981 Oct 5;151(4):703-30
references.pmid: 6977036
references.citation: BMC Biotechnol. 2008 Feb 27;8:21
references.pmid: 18304347
references.citation: J Mol Cell Cardiol. 2001 Apr;33(4):655-70
references.pmid: 11273720
references.citation: J Struct Biol. 2011 Apr;174(1):44-51
references.pmid: 21163356
references.citation: J Cell Sci. 1989 Nov;94 ( Pt 3):391-401
references.pmid: 2632577
references.citation: J Mol Biol. 2008 Dec 26;384(4):780-97
references.pmid: 18951904
references.citation: Int Rev Cytol. 1986;104:81-114
references.pmid: 3531066
references.citation: J Mol Biol. 2007 Mar 16;367(1):36-41
references.pmid: 17254601
references.citation: J Struct Biol. 2005 Mar;149(3):313-24
references.pmid: 15721585
references.citation: Science. 1995 Oct 13;270(5234):293-6
references.pmid: 7569978
references.citation: Circ Res. 2006 Aug 4;99(3):323-31
references.pmid: 16809551
references.citation: J Gen Physiol. 2003 Dec;122(6):761-74
references.pmid: 14638934
references.citation: Circ Res. 2011 Mar 18;108(6):743-50
references.pmid: 21415408
references.citation: J Mol Cell Cardiol. 1997 Dec;29(12):3267-74
references.pmid: 9441832
references.citation: J Mol Biol. 2011 Jul 8;410(2):214-25
references.pmid: 21601575
references.citation: J Biol Chem. 1982 Feb 25;257(4):2111-20
references.pmid: 6276407
references.citation: Eur J Biochem. 1996 Jan 15;235(1-2):317-23
references.pmid: 8631348
references.citation: J Mol Biol. 1986 Dec 20;192(4):831-51
references.pmid: 3495665
references.citation: J Struct Biol. 2006 Aug;155(2):202-17
references.pmid: 16731006
references.citation: Proc Natl Acad Sci U S A. 2011 Jul 12;108(28):11423-8
references.pmid: 21705660
references.citation: J Cell Sci. 1993 Jul;105 ( Pt 3):841-8
references.pmid: 7691850
references.citation: Proc R Soc Lond B Biol Sci. 1976 Mar 16;192(1109):451-61
references.pmid: 4802
references.citation: Biophys J. 2006 Jun 1;90(11):4119-27
references.pmid: 16513777
references.citation: Proc Natl Acad Sci U S A. 1996 Aug 20;93(17):8999-9003
references.pmid: 8799143
references.citation: J Cell Biol. 1983 Jun;96(6):1797-802
references.pmid: 6602135
references.citation: Circ Res. 1998 Jul 13;83(1):60-72
references.pmid: 9670919
references.citation: Biophys J. 1986 Jul;50(1):145-55
references.pmid: 3730499