Recently, we have identified specific sites of
O
-GlcNAcylation in major cardiac myofilament proteins. We used a methodology based on GalNaz-Biotin labeling followed by DTT switch and LC-MS/MS site mapping. As a result, 42
O-
GlcNAc peptides from cardiac myofilaments were identified, corresponding to 32 of cardiac myosin heavy chain (MHC), 6 of α-sarcomeric actin, 2 of myosin light chain 1 (MLC1), 1 of MLC2, and 1 of troponin I (cTnI). Most of the identified
O
-GlcNAcylation sites are novel post-translational modification sites. To assess the potential physiological role of myofilament GlcNAcylation, Force-Calcium relationships studies were performed on skinned rat trabeculae. We have previously reported that exposure to GlcNAc but not Glycerol significantly decreases calcium sensitivity, further investigation confirm these preliminary findings (pCa 50 1.81 ± 0.13 μM for Control vs. 3.83 ± 0.44 μM for GlcNAc,
n
=7, P <=.001), and in addition demonstrate that maximal force (F
max
) and Hill coefficient (
n
) are not significantly changed. Troponin I phosphorylation at Ser23, 24 was determined in three pooled trabeculea by WB using a specific antibody phosphor-TnI (Cell Signaling) normalized to actin signal (GlcNAc 0.722 vs Glycerol 0.667 A.U., n=3, p=NS). Phosphorylation at this PKA sites was ruled out as responsible for myofilament desensitization. Acute exposure of cardiac myofilaments to GlcNAc significantly increased α-sarcomeric actin
O
-GlcNAcylation from 27.6±4.2% to 35.1±2.36% (
n
= 4, p<0.05) whereas total GlcNAcylation levels were unchanged. Additionally, we showed by immunofluoresence that O-GlcNAc transferase (OGT) and O-GlcNAcase (OGase) are abundant in rat hearts, OGT localizes predominantly in cardiac nuclei and less in cytoplasm, whereas OGase shows the opposite pattern. These studies provides the first site mapping of
O
-GlcNAcylation sites in cardiac myofilament proteins, and demonstrates their potential role in regulating myocardial contractile function. Regulation of myofilament
O
-GlcNAcylation may represent a novel and useful therapeutic target in heart failure, especially in diabetic cardiomyopathy.