title: Evolutionarily conserved intercalated disc protein Tmem65 regulates cardiac conduction and connexin 43 function.
abstract: Membrane proteins are crucial to heart function and development. Here we combine cationic silica-bead coating with shotgun proteomics to enrich for and identify plasma membrane-associated proteins from primary mouse neonatal and human fetal ventricular cardiomyocytes. We identify Tmem65 as a cardiac-enriched, intercalated disc protein that increases during development in both mouse and human hearts. Functional analysis of Tmem65 both in vitro using lentiviral shRNA-mediated knockdown in mouse cardiomyocytes and in vivo using morpholino-based knockdown in zebrafish show marked alterations in gap junction function and cardiac morphology. Molecular analyses suggest that Tmem65 interaction with connexin 43 (Cx43) is required for correct localization of Cx43 to the intercalated disc, since Tmem65 deletion results in marked internalization of Cx43, a shorter half-life through increased degradation, and loss of Cx43 function. Our data demonstrate that the membrane protein Tmem65 is an intercalated disc protein that interacts with and functionally regulates ventricular Cx43.
authors.affiliation: Department of Physiology, University of Toronto, Toronto General Hospital Research Institute, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Parveen
authors.initials: P
authors.lastname: Sharma
authors.affiliation: Department of Physiology, University of Toronto, Toronto General Hospital Research Institute, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Cynthia
authors.initials: C
authors.lastname: Abbasi
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.
authors.forename: Savo
authors.initials: S
authors.lastname: Lazic
authors.affiliation: Department of Physiology, University of Toronto, Toronto General Hospital Research Institute, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Allen C T
authors.initials: ACT
authors.lastname: Teng
authors.affiliation: Department of Physiology, University of Toronto, Toronto General Hospital Research Institute, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Dingyan
authors.initials: D
authors.lastname: Wang
authors.affiliation: McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Nicole
authors.initials: N
authors.lastname: Dubois
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Ignatchenko
authors.affiliation: Departments of Molecular Genetics and Biochemistry, Donnelly Centre,, University of Toronto, Toronto, Ontario, Canada M5S 3E1.
authors.forename: Victoria
authors.initials: V
authors.lastname: Wong
authors.affiliation: Department of Mechanical and Industrial Engineering, Advanced Micro and Nanosystems Laboratory, University of Toronto, Toronto, Ontario, Canada M5S 3G8.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Araki
authors.affiliation: Institute of Pharmacology, University Medical Center Göttingen and DZHK (German Center for Cardiovascular Research) partner site Göttingen, Göttingen 37075, Germany.
authors.forename: Malte
authors.initials: M
authors.lastname: Tiburcy
authors.affiliation: The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.
authors.forename: Cameron
authors.initials: C
authors.lastname: Ackerley
authors.affiliation: Institute of Pharmacology, University Medical Center Göttingen and DZHK (German Center for Cardiovascular Research) partner site Göttingen, Göttingen 37075, Germany.
authors.forename: Wolfram H
authors.initials: WH
authors.lastname: Zimmermann
authors.affiliation: The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.|Ted Rogers Centre for Heart Research, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Robert
authors.initials: R
authors.lastname: Hamilton
authors.affiliation: Department of Mechanical and Industrial Engineering, Advanced Micro and Nanosystems Laboratory, University of Toronto, Toronto, Ontario, Canada M5S 3G8.
authors.forename: Yu
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Toronto General Hospital, University Health Network, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Peter P
authors.initials: PP
authors.lastname: Liu
authors.affiliation: McEwen Centre for Regenerative Medicine, University Health Network, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Gordon
authors.initials: G
authors.lastname: Keller
authors.affiliation: Departments of Molecular Genetics and Biochemistry, Donnelly Centre,, University of Toronto, Toronto, Ontario, Canada M5S 3E1.
authors.forename: Igor
authors.initials: I
authors.lastname: Stagljar
authors.affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada M5S 1A8.|The Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8.|Ted Rogers Centre for Heart Research, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Ian C
authors.initials: IC
authors.lastname: Scott
authors.affiliation: Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada M5G 1L7.|Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada M5G 2M9.
authors.forename: Thomas
authors.initials: T
authors.lastname: Kislinger
authors.affiliation: Department of Physiology, University of Toronto, Toronto General Hospital Research Institute, Toronto, Ontario, Canada M5G 1L7.|Ted Rogers Centre for Heart Research, Toronto, Ontario, Canada M5G 1L7.
authors.forename: Anthony O
authors.initials: AO
authors.lastname: Gramolini
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D018031:Connexin 43 / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D055785:Gene Knockdown Techniques; D006329:Heart Conduction System / Q000378:metabolism* / Q000502:physiology; D006352:Heart Ventricles / Q000378:metabolism*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008854:Microscopy, Electron; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D040901:Proteomics; D012822:Silicon Dioxide; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism
chemical_list: D018031:Connexin 43; D008565:Membrane Proteins; C000607207:Tmem65 protein, mouse; D029961:Zebrafish Proteins; C000607209:tmem65 protein, zebrafish; D012822:Silicon Dioxide