Abstract 16986: A New Cardiac Muscle Post-Translational Modification: Detection of Protein Arginine Deiminases and Protein Citrullination in Heart Tissue
ABSTRACT: Protein citrullination originates from enzymatic deimination of amino acid arginine in a protein and is involved in various biological processes during health and disease. However, it has not been investigated in heart. Therefore, our goal was to identify novel substrates for peptidylarginine deiminase (PAD), enzyme responsible for this modification and associate it with changes in protein citrullination during heart failure (HF).
METHODS: Proteomics methods were used to identify citrullinated proteins and the site of modification in control mouse and control vs. HF human heart tissue (5 per group). The characterization of deiminated proteins was facilitated by modification of peptide-bound citrullineted Arg residues with antipyrine and butanedione, allowing for the unambiguous identification by mass spectrometry. Verification of citrullinated proteins was carried out by 2DE (modification causes pI shift) and western blot with anti-citrullinated antibody. PAD enzyme expression was determined using RT-PCR reaction and immunohistochemistry.
RESULTS: We detected up to 20 proteins markedly modified by PAD in vitro and in vivo in mouse heart including myofilament and mitochondria proteins. Similar proteins were identified to be citrullinated in control human heart tissue including tropomyosin (R220), myosin heavy and light chain (R1176, 1434 and 1479) and myosin binding protein C (R696). Importantly, we showed a specific residues deimination associated with the HF, e.g. tropomyosin from HF samples has 3 different citrullinated sites (R 125, 133, 238) compared to control. The PAD enzyme is present in mouse isolated cardiomyocytes and cardiac fibroblast (predominantly PAD2 vs. PAD2/PAD4, respectively). Immunohistochemistry of control human hearts had positive PADs staining in cardiomyocytes.
CONCLUSIONS: Our study demonstrates, for the first time that protein citrullination is a viable post-translational modification in heart tissue and corresponds to the cardiac expression of PAD. Furthermore we showed that citrullinatated residues are altered HF suggesting that this novel PTM could have a regulatory role.
- © 2011 by American Heart Association, Inc.