CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia

Abstract

[Figure: see text].

Keywords: acetylglucosamine; action potential; electrophysiology; hyperglycemia; phosphorylation.

Figure 1.

CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II) O-GlcNAcylation is increased in human diabetic hearts. A, Schematic of CaMKIIδ structure and sequence of the regulatory hub domain and the O-GlcNAcylation pathway. B, CaMKII activity measured as ³²P incorporation in HEK293 cell lysates expressing wild type (WT), S280A, and T287A CaMKIIδ (n, basal=6, Ca/CaM [calmodulin]=6, EGTA=6, OGT [O-GlcNAc transferase]/UDP-GlcNAc=6, UDP-GlcNAc=3, ATP=3 in all 3 CaMKIIδ forms). Kruskal-Wallis 1-way ANOVA, followed by Dunn multiple comparisons test. C, Western blot data showing enhanced CaMKII O-GlcNAcylation in human atrial and ventricular samples from diabetic (DM) vs nondiabetic (nDM) patients (n, atrial-DM=11, atrial-nDM=11, ventricular-DM=6, ventricular-nDM=6). Mann-Whitney test. D, CaMKII oxidation was not increased in atrial and ventricular samples from patients with diabetes (n, atrial-DM=12, atrial-nDM=12, ventricular-DM=6, ventricular-nDM=6). Mann-Whitney test. MW indicates molecular weight; OGA, O-GlcNAcase; and OSMI-1, αR-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide.

Figure 2.

Glucose-induced diastolic sarcoplasmic reticulum (SR) Ca²⁺ leak is CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II)-S280 O-GlcNAcylation dependent. A, Experimental protocol and representative intracellular Ca²⁺ signals (quantified as changes in Fluo-4 fluorescence). B, High-glucose treatment (540 mg/dL, 6 min) did not change the amplitude and decay of intracellular Ca²⁺ transient (CaT) but reduced SR Ca²⁺ load similarly in CaMKIIδ wild type (WT), cardiac-specific knockout (cKO), and O-GlcNAc-resistant CaMKIIδ-S280A knock-in (n[CaT]=total number of cells/animals, WT/normal glucose=24/8, WT/high-glucose=13/8, cKO/normal glucose=15/7, cKO/high-glucose=10/7, S280A/normal glucose=17/6, S280A/high-glucose=11/6; n[SR load]= total number of cells/animals, WT=20/11, cKO=13/9, S280A=18/9). Nested t test. C, Representative diastolic Ca²⁺ sparks were increased by high-glucose treatment in WT which was prevented in cKO and S280A. D, Increased Ca²⁺ spark frequency normalized to SR load indicates sensitized, leaky ryanodine receptors (n=total number of cells/animals, WT=20/11, cKO=13/9, S280A=18/9). Nested t test. E, Enhanced fluorescence recovery after photobleaching (FRAP) by high-glucose treatment in cardiomyocytes expressing GFP (green fluorescent protein)-tagged WT-CaMKIIδ (n=16 cells from 6 animals in normal glucose and n=13 cells from 6 animals in high-glucose) but not in GFP-CaMKIIδ-S280A (n=16 cells from 6 animals in both normal and high-glucose) indicates increased activation-dependent mobility of CaMKIIδ. Nested t test.

Figure 3.

Glucose-induced arrhythmogenic action potentials are CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II)-S280 O-GlcNAcylation dependent. A, Action potential duration (APD) prolongation and alternans (S, short; L, long) were increased by acute high-glucose treatment in wild-type (WT) myocytes (n=16 cells from 9 animals). B, Arrhythmogenic APD responses were prevented by CaMKIIδ-cardiac-specific knockout (cKO; n=21 cells from 8 animals). C and D, CaMKIIδ-S280A knock-in (n=15 ells from 8 animals) but not mutated Met281Val and Met282Val (MMVV; n=17 cells from 9 animals) was resistant to glucose-induced acute APD changes. Nested t test. E and F, Glucose-induced APD changes were prevented by CaMKIIδ-cKO, S280A, intracellular Ca²⁺ buffering (EGTA) or the O-GlcNAc transferase inhibitor OSMI-1 (αR-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide) but enhanced by the O-GlcNAcase inhibitor Thiamet-G (Thm-G) in WT. Ang II (angiotensin II) further enhanced arrhythmogenic APD changes, which was prevented by CaMKIIδ-cKO, MMVV, or in NOX2^−/− (NADPH oxidase 2; n=total number of cells/animals is reported in the figure). Nested 1-way ANOVA, followed by Dunnett multiple comparisons test was used to compare 3 groups. Nested t test was used to compare 2 groups.

Figure 4.

Glucose-induced delayed afterdepolarizations are CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II)-S280 O-GlcNAcylation dependent. A and B, Delayed afterdepolarizations (DADs) and spontaneous action potentials (sAPs) were increased by high-glucose treatment in control (n=21 cells from 13 animals), the increases were prevented in CaMKIIδ-S280A (n=23 cells from 12 animals). Nested t test. C and D, Glucose-induced spontaneous diastolic activities were prevented by EGTA, OSMI-1 (αR-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide), and in CaMKIIδ cardiac-specific knockout (cKO) and S280A. Additional Ang II (angiotensin II) treatment further enhanced the arrhythmogenic activities, which were prevented in NOX2^−/− (NADPH oxidase 2) and CaMKIIδ-cKO and mutated Met281Val and Met282Val (MMVV; n=total number of cells/animals is reported in the figure). Nested 1-way ANOVA, followed by Dunnett multiple comparisons test was used to compare 3 groups. Nested t test was used to compare 2 groups (EGTA, OSMI, Thm-G). WT indicates wild type.

Figure 5.

Diabetes-induced cardiac remodeling and arrhythmias are dependent on CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II) activation. A, Study protocol of streptozotocin (STZ)-induced diabetes and assessment of cardiac function. B, Blood glucose levels were highly elevated, whereas cardiac ejection fraction was preserved in STZ. Wilcoxon matched-pairs signed-rank test. C, Morphological parameters in either vehicle-treated or STZ-treated CaMKIIδ-wild type (WT), S280A, and mutated Met281Val and Met282Val (MMVV) mice. One-way ANOVA, followed by Dunnett multiple comparisons test. D, Preserved systolic heart function in STZ. E, Diastolic heart function (enlarged left atria, reduced E/A, increased E/e′) in 4-week diabetes. One-way ANOVA, followed by Dunnett multiple comparisons test. F, Incidence of premature ventricular complexes (PVCs) following isoproterenol+caffeine stress test was increased in diabetic WT animals, whereas CaMKIIδ-cardiac-specific knockout (cKO) was protective. Mann-Whitney test. G, Increased RyR (ryanodine receptor) S2814 phosphorylation and PLB (phospholamban) O-GlcNAcylation in STZ. Two technical replicates (blots) were performed for each protein sample. Mann-Whitney test. The number of biological replicates (n) is shown. HW/BW indicates heart weight to body weight ratio; IVS, interventricular septum; LA, left atrial; and LV, left ventricular.

Figure 6.

Diabetes-induced diastolic sarcoplasmic reticulum (SR) Ca2+ leak is CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II)-S280 O-GlcNAcylation-dependent. A, Experimental protocol and representative intracellular Ca²⁺ signals (as changes in Fluo-4 fluorescence) showing spontaneous Ca²⁺ waves in wild type (WT) following 4-week of streptozotocin (STZ)-induced diabetes. B, Diabetes slightly reduced the amplitude of the intracellular Ca²⁺ transient and prolonged the Ca²⁺ transient decay without significantly altering SR Ca²⁺ load (n[CaT]=total number of cells/animals, WT+vehicle/normal glucose=16/7, WT+vehicle/high-glucose=9/7, WT+STZ/normal glucose=20/6, WT+STZ/high-glucose=11/6, S280A+STZ/normal glucose=13/5, S280A+STZ/high-glucose=11/5, mutated Met281Val and Met282Val [MMVV]+STZ/normal glucose=13/5, MMVV+STZ/high-glucose=10/5; n[SR load]=total number of cells/animals, WT+Vehicle=12/6, WT+STZ=12/7, S280A+STZ=9/5, MMVV+STZ=10/5). Acute effect of high-glucose within the same genotype/treatment was tested using nested t test. Effect of STZ-induced diabetes (in normal glucose) vs WT+Vehicle was tested using nested 1-way ANOVA, followed by Dunnett multiple comparisons test. C, Ca²⁺ sparks were increased in diabetic WT and further increased by acute high-glucose treatment. D, The increase in Ca²⁺ spark rate was prevented in CaMKIIδ-S280A and was attenuated in the CaMKIIδ-MMVV (n=total number of cells/animals, WT+Vehicle=12/6, WT+STZ=10/6, S280A+STZ=9/5, MMVV+STZ=10/5). Nested t test. CaSpF indicates Ca²⁺ spark frequency.

Figure 7.

Diabetes-induced arrhythmogenic action potential remodeling is dependent predominantly on CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II)-S280 O-GlcNAcylation. A, Action potential duration (APD) prolongation and alternans in streptozotocin (STZ)-treated wild-type (WT) murine ventricular myocytes (n=16 cells from 7 animals). B, Arrhythmogenic action potential (AP) remodeling in STZ was abolished by AIP (autocamtide-2-related inhibitory peptide; n=11 cells from 4 animals). C, APD prolongation was prevented in STZ-treated CaMKIIδ-S280A (n=22 cells from 6 animals). D, APD prolongation and alternans in STZ-treated CaMKIIδ-mutated Met281Val and Met282Val (MMVV; n=17 cells from 6 animals). Nested t test. E and F, AP remodeling was CaMKII-dependent and involved both hyperglycemia-dependent S280 O-GlcNAcylation (predominant mechanism) and Ang II-dependent 281/2MM oxidation (n=total number of cells/animals is reported in the figure). Nested 1 way ANOVA, followed by Dunnett multiple comparisons test. CaSpF indicates Ca²⁺ spark frequency.

Figure 8.

Diabetes-induced delayed afterdepolarizations are dependent on CaMKIIδ (Ca²⁺/calmodulin-dependent protein kinase II)-S280 O-GlcNAcylation. A and B, delayed afterdepolarizations (DADs) and spontaneous action potentials (sAPs) were increased by high-glucose in streptozotocin (STZ)-treated wild type (WT; n=12 cells from 6 animals), but it was prevented in STZ-treated CaMKIIδ-S280A (n=18 cells from 6 animals). Nested t test. C and D, Diabetic hyperglycemia largely enhanced spontaneous diastolic activities, which were prevented by AIP (autocamtide-2-related inhibitory peptide), in CaMKIIδ-cKO and S280A but not in mutated Met281Val and Met282Val (MMVV). Additional Ang II (angiotensin II) treatment further enhanced the arrhythmogenic activities, which were prevented in CaMKIIδ-cKO and MMVV but not in S280A (n=total number of cells/animals is reported in the figure). Nested 1-way ANOVA, followed by Dunnett multiple comparisons test.