Citrullination of myofilament proteins in heart failure

Justyna Fert-Bober, John T. Giles, Ronald J. Holewinski, Jonathan A. Kirk, Helge Uhrigshardt, Erin L. Crowgey, Felipe Andrade, Clifton O. Bingham, Jin Kyun Park, Marc K. Halushka, David A. Kass, Joan M. Bathon, Jennifer E. Van Eyk

Research output: Contribution to journalArticlepeer-review

Abstract

Aims: 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. Methods and 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 Ca2+ sensitivity in skinned cardiomyocytes, with no change in maximal calcium-activated force or hill coefficient. Conclusion: 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.

Original languageEnglish (US)
Pages (from-to)232-242
Number of pages11
JournalCardiovascular research
Volume108
Issue number2
DOIs
StatePublished - Jan 7 2015

Keywords

  • Citrullination
  • Heart failure
  • Myofilament
  • Peptidylarginine deiminases
  • Sarcomere

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine
  • Physiology (medical)

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