Interplay Between Phosphorylation and O-GlcNAcylation of Sarcomeric Proteins in Ischemic Heart Failure

Abstract

Post-translational modifications (PTMs) of sarcomeric proteins could participate to left ventricular (LV) remodeling and contractile dysfunction leading in advanced heart failure (HF) with altered ejection fraction. Using an experimental rat model of HF (ligation of left coronary artery) and phosphoproteomic analysis, we identified an increase of desmin phosphorylation and a decrease of desmin O-N-acetylglucosaminylation (O-GlcNAcylation). We aim to characterize interplay between phosphorylation and O-GlcNAcylation for desmin in primary cultures of cardiomyocyte by specific O-GlcNAcase (OGA) inhibition with thiamet G and silencing O-GlcNAc transferase (OGT) and, in perfused heart perfused with thiamet G in sham- and HF-rats. In each model, we found an efficiency of O-GlcNAcylation modulation characterized by the levels of O-GlcNAcylated proteins and OGT expression (for silencing experiments in cells). In perfused heart, we found an improvement of cardiac function under OGA inhibition. But none of the treatments either in in vitro or ex vivo cardiac models, induced a modulation of desmin, phosphorylated and O-GlcNAcylated desmin expression, despite the presence of O-GlcNAc moities in cardiac desmin. Our data suggests no interplay between phosphorylation and O-GlcNAcylation of desmin in HF post-myocardial infarction. The future requires finding the targets in heart involved in cardiac improvement under thiamet G treatment.

Keywords: O-GlcNAcylation; desmin; heart failure; interplay; phosphorylation; rat models; systolic.

Figure 1

Post-translational modifications of desmin of LV from HF-rats. Quantification of Ser-phosphorylated desmin (A), total desmin levels (B), and O-GlcNAcylated desmin levels (C) in LV (50 μg) of sham- (n = 11) (white box) and HF- (n = 11) (black box) rats at 2 months after MI. The positions of molecular weight are indicated as kilodalton (kDa) on the left. An internal standard was loaded in each gel for the standardization and quantification. Graphs show mean ± SEM values expressed in arbitrary units (A.U.). *P < 0.05; **P < 0.01.

Figure 2

Impact of OGA inhibition by thiamet G in primary cultures of NCM. (A) Description of the protocol designed for Thiamet G (TG) treatment of primary culture of neonate cardiomyocytes (NCM). (B) Representative western blots (left panel) and quantification (right panel) of O-GlcNAcylated proteins levels in control (Ctrl) and NCM treated with 100 nM of thiamet G (TG) during 24 h (n = 12). (C) Western blots (upper panel) and quantification (lower panel) of total desmin levels in the same samples. (D) Phosphorylation profiles of desmin were analyzed in the same samples by Phos-tag™ gel. (E) Representative immunoprecipation (IP) with desmin antibody before western blot with RL2 antibody (left panel) and quantification (right panel) of O-GlcNAcylated desmin levels in the same samples. Inp, input; IP, immunoprecipitated proteins; S, IP supernatant; Des, desmin; IgG, Immunoglobulin. The arrow indicates the O-GlcNAcylated desmin. Data are expressed as means of an arbitrary unit (A.U.) ± SEM. The positions of molecular weight are indicated as kilodalton (kDa) on the left. **P < 0.01.

Figure 3

Effect of OGT silencing in primary cultures of NCM. (A) Description of the protocol designed for OGT silencing of primary cultures of NCM. (B) Western blots (left panel) and quantification of O-GlcNAcylated proteins levels (upper and right panel) and OGT (lower and right panel) in primary cultures of NCM transfected with non-targeting (NT) siRNA control and OGT1 and OGT2 siRNA (n = 12). (C) Western blots (upper panel) and quantification (lower panel) of total desmin levels in the same samples. (D) Phosphorylation profiles of desmin was analyzed in the same samples by Phos-tag™ gel. (E) Representative Immunoprecipitation (IP) with desmin antibody before western blot with RL2 antibody (left panel) and quantification (right panel) of O-GlcNAcylated desmin levels in the same samples. Inp, input; IP, immunoprecipitated proteins; S, IP supernatant; B, beads alone; S*, IP supernatant of beads. The arrow indicates the O-GlcNAcylated desmin. Graphs show mean ± SEM values expressed in arbitrary units (A.U.). The positions of molecular weight are indicated as kilodalton (kDa) on the left. *P < 0.005; **P < 0.01.

Figure 4

Effect of OGA inhibition by thiamet G in isolated perfused heart. (A) Description of the protocol designed for thiamet G (TG) perfusion in sham- (n = 6) and HF- (n = 7) rats 6 weeks post-MI. (B) Western blot (left panel) and quantification (right panel) of O-GlcNAcylated proteins levels measured in proteins extracted from LVs of isolated perfused sham- and HF-rat hearts treated or not with 100 μM thiamet G for 2 h (n = 7 in each group). (C) Western blots (upper panel) and quantification (lower panel) of total desmin levels in the same samples. (D) Phosphorylation profiles of desmin were analyzed in the same samples by Phos-tag™ gel. Graphs show mean ± SEM values expressed in arbitrary units (A.U.). The positions of molecular weight are indicated as kilodalton (kDa) on the left. *P < 0.05; ** < 0.01.

Figure 5

Analysis of O-GlcNAcylated LV proteins by Western blot and WGA-SDS-PAGE gel electrophoresis. (A) Red ponceau staining (left panel) and western blot (right panel) of O-GlcNAcylated proteins (50 μg) extracted from sham- and HF-rats treated or not with thiamet G. The positions of molecular weight are indicated as kilodalton (kDa) on the left. (B) Red ponceau staining (left panel) and WGA-SDS-PAGE of O-GlcNAcylated proteins levels (middle panel) of O-GlcNAcylated desmin levels (right panel) from the same samples. The arrow in desmin WGA gels indicates the non-O-GlcNAcylated form.