O -Linked GlcNAc Modification of Cardiac Myofilament Proteins

Abstract

In addition to O-phosphorylation, O-linked modifications of serine and threonine by ␤-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. D iabetes mellitus is a risk factor for the development of heart failure, 1 and abnormal glucose metabolism may contribute directly to depressed cardiac function. Studies in humans and animal models of diabetes mellitus have demonstrated abnormal myofilament function 2 and impaired excitation-contraction coupling, 3,4 which may depress myocardial function. Posttranslational modifications of myofilament proteins regulate cardiac function and phosphorylation of myofilament proteins may result in functional abnormalities in heart failure. [5][6][7] In addition to O-linked phosphorylation of serine (Ser) and threonine (Thr) residues of proteins, dynamic Materials and Methods Mass Spectrometric Identification of O-GlcNAc-Modified ProteinsTo label the specific sites (further details are in the online data supplement, available at http://circres.ahajournals.org), GlcNAcmodified peptides were labeled with GalNAz-biotin and enriched by avidin chromatography, and then dithiothreitol (DTT) was used to replace the GlcNAc-GalNAz-biotin by ␤-elimination and Michael addition (BEMAD) as previously described. 14 Isolated Skinned Fiber StudiesFor skinned fiber studies, cardiac trabeculae were isolated and mounted as previously described. 5 ImmunoblotsMyofilament proteins were isolated as previously described, 15 with minor modifications. To determine the global GlcNAc modifications of myofilament proteins, a pan-GlcNAc antibody (CTD 110.6, Covance) was used as previously described. 16 To assess cardiac troponin I (cTnI) phosphorylation, a phospho-TnI (Ser23/Ser24) antibody (Cell Signaling, Danvers, Mass) was used as previously described. 5 Results and Discussion Myofilament Proteins Are Modified by O-GlcNAcWith the enrichment and BEMAD experiments describ...

O-linked β- N -acetylglucosamine transferase is indispensable in the failing heart

“…Based on such a direct demonstration, future studies will be necessary to identify the specific targets that are O-GlcNAcylated in the failing myocardium and how such targets figure in the pathophysiology of heart failure. Interestingly, Murphy and coworkers (19) recently identified several contractile proteins that are O-GlcNAcylated in the myocyte. It would be interesting to identify what, if any, changes occurred at the contractile protein level in the failing heart.…”

AMP-Activated Protein Kinase Phosphorylates Cardiac Troponin I and Alters Contractility of Murine Ventricular Myocytes

“…46,47 In an analysis of in vivo O-GlcNAcylation of myocardial proteins, it was found that Ser150 of cTnI was modified. 48 This raises the possibility of cross-talk between AMPK phosphorylation and O-GlcNAcylation signals at this residue, possibly fine tuning cTnI-mediated contractile regulation.…”

The roles ofO-linked β-N-acetylglucosamine in cardiovascular physiology and disease

“…Several laboratories have suggested that this is due to both changes in UDP-GlcNAc levels and thus the O-GlcNAc modification (1, 3, 8, 31, 39, 40, 46, 54, 60, 72, 82, 101, 109, 110, 133-136, 155, 167, 169, 185, 187), as well as other metabolites (115,124) and possibly other forms of protein glycosylation (65,173). Key data that support a role for O-GlcNAc in mediating the complications associated with type II diabetes includes 1) overexpression of O-GlcO-GlcOGT in the muscle and adipose of mice results in insulin resistance and hyperleptinemia, two hallmarks of type II diabetes (117); 2) deletion of either OGT or O-GlcNAcase in Caenorhabditis elegans leads to changes in glucose and trehalose metabolism, as well as dauer phenotypes characteristic of disrupted insulin signaling (40,54); 3) PUGNAc treatment, an inhibitor of the O-GlcNAcase and lysosomal hexosaminidases, leads to insulin resistance in cell culture models and tissue explants (3,134,167); 4) in several models of diabetes O-GlcNAc levels are elevated on a subset of proteins (1,2,8,42,64,113,133,138,169); 5) numerous pathways are regulated by NAc, such as insulin signaling via Akt (3, 6, 40, 54, 119, 134 -136, 167, 185); and 6) reducing the levels of O-GlcNAc in models of type II diabetes reverses the some of the complications associated with type II diabetes (26,63,64,125). However, recent data suggests that elevating O-GlcNAc levels alone may not be sufficient to induce diabetes: 1) treating 3T3-L1 adipocytes with PUGNAc but not a more specific inhibitor of OGlcNAcase (Thiamet-G) results in insulin resistance (107), 2) treating mice for long periods of time with Thiamet-G does not alter glucose metabolism (108), and 3) overexpressing OGlcNAcase does not restore glucose metabolism in 3T3-L1 adipocytes (142).…”

“…Heart (138) Akt § O-GlcNAcylation is associated with reduced phosphorylation of Akt and is implicated in reducing angiogenesis.…”