Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation

Abstract

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is an enzyme with important regulatory functions in the heart and brain, and its chronic activation can be pathological. CaMKII activation is seen in heart failure, and can directly induce pathological changes in ion channels, Ca(2+) handling and gene transcription. Here, in human, rat and mouse, we identify a novel mechanism linking CaMKII and hyperglycaemic signalling in diabetes mellitus, which is a key risk factor for heart and neurodegenerative diseases. Acute hyperglycaemia causes covalent modification of CaMKII by O-linked N-acetylglucosamine (O-GlcNAc). O-GlcNAc modification of CaMKII at Ser 279 activates CaMKII autonomously, creating molecular memory even after Ca(2+) concentration declines. O-GlcNAc-modified CaMKII is increased in the heart and brain of diabetic humans and rats. In cardiomyocytes, increased glucose concentration significantly enhances CaMKII-dependent activation of spontaneous sarcoplasmic reticulum Ca(2+) release events that can contribute to cardiac mechanical dysfunction and arrhythmias. These effects were prevented by pharmacological inhibition of O-GlcNAc signalling or genetic ablation of CaMKIIδ. In intact perfused hearts, arrhythmias were aggravated by increased glucose concentration through O-GlcNAc- and CaMKII-dependent pathways. In diabetic animals, acute blockade of O-GlcNAc inhibited arrhythmogenesis. Thus, O-GlcNAc modification of CaMKII is a novel signalling event in pathways that may contribute critically to cardiac and neuronal pathophysiology in diabetes and other diseases.

Figure 1. Glucose induced CaMKII activity is O-GlcNAc dependent

a, Schematic of Camui sensor. b, Direct (Ca²⁺/CaM) or autonomous (+EGTA) activation of Camui measured in lysates after HEK cell exposure to indicated [glucose]. c, S279 mutation does not affect Ca²⁺/CaM, phosphorylation, or oxidation-dependent activity. d, Glucose-dependent Camui activation is ablated by KN93 or Ca²⁺ chelation (+EGTA). e, Increased [glucose] enhances pacing- or Iso-induced CaMKII activity. f, Glucose-dependent CaMKII activity measured by ³²P incorporation. g, Glucose-dependent Camui activation is enhanced by Thm-G and ablated by O-GlcNAc inhibition (+DON). Mean±s.e.m. 3 preparations × triplicates, unless indicated. * p<0.05 vs. control.

Figure 2. O-GlcNAcylation of CaMKII occurs in vivo

a, Immunoblot with O-GlcNAc- and P-T286-specific antibodies shows that high [glucose] increases O-GlcNAcylation and activation of CaMKII, but not in S279A mutant or after KN93 (n=3 myocyte preparations). b, The ratio of O-GlcNAc modified to total CaMKII is increased in heart (n=6 hearts/group) and brain (n=3 brains/group) from diabetic vs. control non-diabetic human patients. c, O-GlcNAc modification of CaMKII is also increased in heart and brain from diabetic rats compared to wild type controls (number of rats indicated). Mean±s.e.m. * p<0.05, ** p<0.01 vs. control.

Figure 3. Glucose-induced cardiac Ca²⁺ sparks are O-GlcNAc- and CaMKII-dependent

Ca²⁺ sparks and waves a, are increased by GlcNAcase inhibitor Thm-G. b, Thm-G-induced Ca²⁺ sparks are prevented by CaMKII inhibitor KN93 (not inactive analog KN92). c, Glucose induced Ca²⁺ sparks are ablated by CaMKII inhibitor KN93 and the O-GlcNAc inhibitor DON. # p<0.05 vs. 350 mg/dL glucose. d, Thm-G induces Ca²⁺ sparks in WT mice, but not mice lacking CaMKIIδ (baseline Ca²⁺ spark frequency differs in rat vs. mouse myocytes). Data are mean±s.e.m. number of myocytes indicated in panels, * p<0.05, ** p<0.01 vs. control.

Figure 4. Glucose-induced PVCs are suppressed by DON and KN-93

a, ECGs during baseline (top), high [glucose] (HG), and DON pretreatment +HG. b, PVCs (/15 min) after HG increased (vs. baseline), while DON or KN93 reduced PVCs only in HG. c, Activation maps for normal activation (top) and HG-induced PVC (bottom). d, Action potentials (black) and Ca²⁺ transients (red) from area indicated by white boxes in (c). e,f, Caff/DOB-induced PVCs were inhibited by DON in diabetic rats in vivo. g, Working model of O-GlcNAc-induced and CaMKII-dependent arrhythmic events. Mean±s.e.m. n=3 hearts/animals, * p<0.05 vs. control.