O -Linked GlcNAc Modification of Cardiac Myofilament Proteins

In addition to O -phosphorylation, O -linked modifications of serine and threonine by β- N -acetyl- d -glucosamine (GlcNAc) may regulate muscle contractile function. This study assessed the potential role of O -GlcNAcylation in cardiac muscle contractile activation. To identify specific sites of O -GlcNAcylation in cardiac myofilament proteins, a recently developed methodology based on GalNAz-biotin labeling followed by dithiothreitol replacement and light chromatography/tandem mass spectrometry site mapping was adopted. Thirty-two O -GlcNAcylated peptides from cardiac myofilaments were identified on cardiac myosin heavy chain, actin, myosin light chains, and troponin I. To assess the potential physiological role of the GlcNAc, force–[Ca 2+ ] relationships were studied in skinned rat trabeculae. Exposure to GlcNAc significantly decreased calcium sensitivity (pCa50), whereas maximal force ( F max ) and Hill coefficient ( n ) were not modified. Using a pan-specific O -GlcNAc antibody, it was determined that acute exposure of myofilaments to GlcNAc induced a significant increase in actin O -GlcNAcylation. This study provides the first identification of O -GlcNAcylation sites in cardiac myofilament proteins and demonstrates their potential role in regulating myocardial contractile function.