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 (500mg/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 10μM) (Ca2+ sparks frequency [#/s/100μm]: 1.07±0.39 for HG vs. 0.16±0.06 for normal glucose [NG, 100mg/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.