title: Citrullination of myofilament proteins in heart failure.
abstract: OBJECTIVE
  Citrullination, the post-translational conversion of arginine to citrulline by the enzyme family of peptidylarginine deiminases (PADs), is associated with several diseases, and specific citrullinated proteins have been shown to alter function while others act as auto-antigens. In this study, we identified citrullinated proteins in human myocardial samples, from healthy and heart failure patients, and determined several potential functional consequences. Further we investigated PAD isoform cell-specific expression in the heart.
  
  
  RESULTS
  A citrullination-targeted proteomic strategy using data-independent (SWATH) acquisition method was used to identify the modified cardiac proteins. Citrullinated-induced sarcomeric proteins were validated using two-dimensional gel electrophoresis and investigated using biochemical and functional assays. Myocardial PAD isoforms were confirmed by RT-PCR with PAD2 being the major isoform in myocytes. In total, 304 citrullinated sites were identified that map to 145 proteins among the three study groups: normal, ischaemia, and dilated cardiomyopathy. Citrullination of myosin (using HMM fragment) decreased its intrinsic ATPase activity and inhibited the acto-HMM-ATPase activity. Citrullinated TM resulted in stronger F-actin binding and inhibited the acto-HMM-ATPase activity. Citrullinated TnI did not alter the binding to F-actin or acto-HMM-ATPase activity. Overall, citrullination of sarcomeric proteins caused a decrease in Ca(2+) sensitivity in skinned cardiomyocytes, with no change in maximal calcium-activated force or hill coefficient.
  
  
  CONCLUSIONS
  Citrullination unique to the cardiac proteome was identified. Our data indicate important structural and functional alterations to the cardiac sarcomere and the contribution of protein citrullination to this process.
authors.affiliation: The Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Advanced Clinical BioSystems Research Institute, Advanced Health Science Building, 9229, Los Angeles, CA, USA Bayview Proteomics Center, Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA Justyna.Fert-Bober@cshs.org.
authors.forename: Justyna
authors.initials: J
authors.lastname: Fert-Bober
authors.affiliation: Division of Rheumatology, Department of Medicine, Columbia University, New York, NY, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Giles
authors.affiliation: The Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Advanced Clinical BioSystems Research Institute, Advanced Health Science Building, 9229, Los Angeles, CA, USA.
authors.forename: Ronald J
authors.initials: RJ
authors.lastname: Holewinski
authors.affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins University Medical Institutions, Baltimore, MD, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Kirk
authors.affiliation: Bayview Proteomics Center, Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Helge
authors.initials: H
authors.lastname: Uhrigshardt
authors.affiliation: The Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Advanced Clinical BioSystems Research Institute, Advanced Health Science Building, 9229, Los Angeles, CA, USA.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Crowgey
authors.affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins University Medical Institutions, Baltimore, MD, USA.
authors.forename: Felipe
authors.initials: F
authors.lastname: Andrade
authors.affiliation: Division of Rheumatology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA Division of Rheumatology, Department of Medicine, Seoul National University Hospital, Seoul, Korea.
authors.forename: Clifton O
authors.initials: CO
authors.lastname: Bingham
authors.affiliation: Division of Rheumatology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA Division of Rheumatology, Department of Medicine, Seoul National University Hospital, Seoul, Korea.
authors.forename: Jin Kyun
authors.initials: JK
authors.lastname: Park
authors.affiliation: Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Marc K
authors.initials: MK
authors.lastname: Halushka
authors.affiliation: Division of Cardiology, Department of Medicine, The Johns Hopkins University Medical Institutions, Baltimore, MD, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Kass
authors.affiliation: Division of Rheumatology, Department of Medicine, Columbia University, New York, NY, USA.
authors.forename: Joan M
authors.initials: JM
authors.lastname: Bathon
authors.affiliation: The Heart Institute and Department of Medicine, Cedars-Sinai Medical Center, Advanced Clinical BioSystems Research Institute, Advanced Health Science Building, 9229, Los Angeles, CA, USA Bayview Proteomics Center, Division of Cardiology, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA.
authors.forename: Jennifer E
authors.initials: JE
authors.lastname: Van Eyk
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D000831:Animals, Newborn; D002956:Citrulline / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D006333:Heart Failure / Q000378:metabolism*; D006801:Humans; D006867:Hydrolases; D008297:Male; D013058:Mass Spectrometry / Q000379:methods; D008810:Mice, Inbred C57BL; D009206:Myocardium / Q000378:metabolism*; D009210:Myofibrils / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D020543:Proteome
chemical_list: D003598:Cytoskeletal Proteins; D015879:Myosin Subfragments; D020543:Proteome; D002956:Citrulline; D006867:Hydrolases; C000620352:PADI2 protein, human; D000080064:Protein-Arginine Deiminase Type 2; D000076342:Protein-Arginine Deiminases; D000251:Adenosine Triphosphatases
keywords: Citrullination; Heart failure; Myofilament; Peptidylarginine deiminases; Sarcomere