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Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation

Abstract

Ca2+/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, Ca2+ handling and gene transcription1. 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 heart2 and neurodegenerative diseases3,4. 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 Ca2+ 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 Ca2+ release events that can contribute to cardiac mechanical dysfunction and arrhythmias1. 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.

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Figure 1: Glucose-induced CaMKII activity is O-GlcNAc dependent.
Figure 2: O-GlcNAcylation of CaMKII occurs in vivo.
Figure 3: Glucose-induced cardiac Ca2+ sparks are O-GlcNAc and CaMKII dependent.
Figure 4: Glucose-induced arrhythmias are suppressed by DON and KN-93.

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Acknowledgements

We thank Y. Hayashi for providing initial Camui samples, H. Schulman for helpful discussions, K. Margulies for human heart samples, L.-W. Jin, M. Melara and the University of California, Davis Alzheimer’s Disease Center (NIH-P30AG010129) for human brain samples, and J. H. Brown for providing CaMKIIδ-knockout mice. We thank Pfizer, Inc. for the gift of a breeding pair of HIP rats to F.D. This work was supported by American Heart Association 13SDG14680072 and National Institutes of Health (NIH) T32HL86350 (J.R.E.); NIH 1R01HL118474-01A1, NSF CBET 1133339, ADA 1-13-IN-70 and AHA 13GRNT16470034 (F.D.); NIH R01DK61671 and P01HL107153 (G.W.H.); NIH R01HL111600 (C.M.R.); NIH P01HL080101, R37HL30077 and Fondation Leducq Transatlantic CaMKII Alliance (D.M.B.). G.W.H. receives a share of royalty on sales of the CTD 110.6 antibody, which are managed by Johns Hopkins University.

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Authors

Contributions

J.R.E. and D.M.B. conceived the project. J.R.E. and L.P. carried out most of the experiments. L.W. and C.M.R. conducted optical mapping and in vivo ECG experiments and analysis. G.H., R.J.C. and G.W.H. conducted ETD-MS analysis. A.F. and K.D. generated constructs, performed animal surgeries, and participated in data analysis. F.D. contributed diabetic rats and some analysis therewith. J.R.E. and D.M.B. wrote the manuscript, with assistance from the other authors.

Corresponding author

Correspondence to Donald M. Bers.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 O-GlcNAc effect is not abolished by T286A or CM280/1VV mutation and CaMKII regulatory domain contains consensus O-GlcNAc modification sites.

a, Increased glucose concentration, but not osmolarity-matched mannitol, activates CaMKII in HEK cells (n = 9). b, O-GlcNAc-dependent CaMKII activation is reduced but still present in T286A-mutant Camui (n = 9). WT, wild type. c, Glucose-dependent CaMKII activation is preserved in CM280/281VV-mutant Camui expressed in HEK-cell lysates (n = 9). d, Activation of Camui by increased glucose is blunted in the S279A mutant and ablated entirely by DON (n values: wild type = 100, wild type + DON = 72, S279A = 57, S279A + DON = 44 cells). e, These sites are conserved in all known isoforms of CaMKII and in a wide variety of mammalian species. Data are mean ± s.e.m. * P < 0.05, **P < 0.01 versus control.

Extended Data Figure 2 O-GlcNAc modification of CaMKII is enhanced in hyperglycaemic conditions.

a, Immunoblot with an O-GlcNAc-specific antibody shows O-GlcNAc modification of CaMKII is inducible by increased glucose availability and is enhanced by Iso treatment (n values indicated). b, O-GlcNAc modification of CaMKII is reversed by β-elimination reaction before immunoblot. c, O-GlcNAc modification of CaMKII is ablated by DON and enhanced by Thm-G. d, Autophosphorylation of cardiac CaMKII is significantly increased in a rat model of diabetes. n = 3 for all immunoblots except where indicated. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 versus control.

Extended Data Figure 3 ETD-MS analysis confirms O-GlcNAc modification at S279A.

A synthetic peptide encoding the regulatory domain of CaMKII was subjected to in vitro O-GlcNAc labelling followed by ETD-MS analysis. Examination of the 507.25 m/z peptide fragment (top right inset) indicates the presence of an O-GlcNAc modification at S279 (c6 to c7 fragmentation).

Extended Data Figure 4 Sarcoplasmic reticulum Ca2+ content, sparks and twitch Ca2+ transients.

a, b, Sarcoplasmic reticulum (SR) content is unaffected by Thm-G (a) or DON (b) in isolated rat myocytes (n values indicated). c, Mannitol does not enhance calcium spark frequency in isolated rat myocytes. d, e, Ca2+ transient amplitude (d, n = 13) and SR content (e, n = 13) are unaffected by Thm-G treatment in isolated myocytes from wild-type (WT) or CaMKIIδ-knockout mice. Data are mean ± s.e.m. NS, no significant difference.

Extended Data Figure 5 Ca2+ sparks induced by glucose and Thm-G.

a, b, Simultaneous treatment with 3,500 mg dl−1 glucose and Thm-G greatly enhances spark frequency (a) and SR Ca2+ depletion (b) in isolated rat myocytes (n = 6). Data are mean ± s.e.m.

Extended Data Figure 6 Diastolic calcium elevation under high glucose is suppressed by pre-treatment with 50 mM DON.

a, Average diastolic calcium elevation at baseline and following treatment with either high glucose (HG) or DON plus high glucose. Calcium elevation was measured as the percentage increase in the diastolic calcium signal relative to the amplitude of the following transient (n = 3). b, Example transients during baseline conditions (black) and after treatment with either high glucose or DON plus high glucose (grey). Minimal diastolic calcium elevation occurs after pre-treatment with DON. n = 3–4 rats for all data points. c, CaMKII activity is enhanced in heart lysate from diabetic rats (n = 3), and this effect is blunted by treatment with DON. Data are mean ± s.e.m. *P < 0.05 versus control.

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Erickson, J., Pereira, L., Wang, L. et al. Diabetic hyperglycaemia activates CaMKII and arrhythmias by O-linked glycosylation. Nature 502, 372–376 (2013). https://doi.org/10.1038/nature12537

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