Epac2 O-GlcNAcylation, a new player in high glucose-mediated cardiac calcium mishandling

Abstract

Epidemiological and preclinical studies have pointed out a correlation between hyperglycemia and increasing risk of heart failure. In cardiomyocytes, hyperglycemia has been shown to alter Ca2+ signaling via CaMKII a downstream effector of Epac2, a key player in Ca2+ mishandling. However the role of Epac2 in high-glucose (HG) mediated Ca2+ dysregulation is unclear. To test the involvement of Epac2 in hyperglycemia-mediated cardiac Ca2+ mishandling. Ca2+ signaling was measured by confocal microscopy in Fluo-4 loaded ventricular cardiomyocytes isolated from C57BL6 and Epac2-KO mice and treated with different glucose concentrations. HG (500 mg/dL) enhanced the occurrence of Ca2+ sparks and pro-arrhythmic events in WT mice but not in Epac2 KO. This increase was prevented by Epac2 pharmacological inhibition (ESI-05 10 μM) (Ca2+ sparks frequency [#/s/100 μm]: 1.07 ± 0.39 for HG vs. 0.16 ± 0.06 for normal glucose [NG, 100 mg/dL], P < 0.05; 0.23 ± 0.15 for ESI-05 + HG vs. NG, P = NS; 0.09 ± 0.04 for Epac2-KO at HG vs. 0.11 ± 0.04 at NG, P = N.S.). The Epac2-dependent alterations of Ca2+ signaling upon HG were associated to higher RyR open probability measured by single channel recording incorporated into lipid bilayers (P0 = 0.76 ± 0.07 in HG vs. 0.22 ± 0.07 in NG, P < 0.01). This increase was prevented by ESI-05 or O-GlcNAcylation inhibition by diazo-5-oxonorleucine (DON), suggesting an Epac2 activation through O-GlcNAcylation under HG. This is confirmed by Epac2-based FRET biosensor activity measurements. Moreover, Co-IP experiments indicated an O-GlcNacylation of Epac2. Our work shows an Epac2 O-GlcNAcylation by HG responsible for pro-arrhythmic SR Ca2+ leak in cardiomyocytes, a new mechanism that might be involved in diabetic cardiomyopathy.