Oxidized CaMKII and O-GlcNAcylation cause increased atrial fibrillation in diabetic mice by distinct mechanisms

Abstract

Diabetes mellitus (DM) and atrial fibrillation (AF) are major unsolved public health problems, and diabetes is an independent risk factor for AF. However, the mechanism(s) underlying this clinical association is unknown. ROS and protein O-GlcNAcylation (OGN) are increased in diabetic hearts, and calmodulin kinase II (CaMKII) is a proarrhythmic signal that may be activated by ROS (oxidized CaMKII, ox-CaMKII) and OGN (OGN-CaMKII). We induced type 1 (T1D) and type 2 DM (T2D) in a portfolio of genetic mouse models capable of dissecting the role of ROS and OGN at CaMKII and global OGN in diabetic AF. Here, we showed that T1D and T2D significantly increased AF, and this increase required CaMKII and OGN. T1D and T2D both required ox-CaMKII to increase AF; however, we did not detect OGN-CaMKII or a role for OGN-CaMKII in diabetic AF. Collectively, our data affirm CaMKII as a critical proarrhythmic signal in diabetic AF and suggest ROS primarily promotes AF by ox-CaMKII, while OGN promotes AF by a CaMKII-independent mechanism(s). These results provide insights into the mechanisms for increased AF in DM and suggest potential benefits for future CaMKII and OGN targeted therapies.

Keywords: Arrhythmias; Cardiology; Diabetes; Protein kinases.

Figure 1. Proposed CaMKII posttranslational modifications and type 1 and type 2 diabetic models.

(A) Oxidation at methionines 281/282 and OGN at serine 280 are posttranslational modifications hypothesized to promote diabetic heart disease and arrhythmias. (B) Schematic representation of proposed hypothesis that excessive ROS and OGN in diabetes mellitus (DM) promotes AF through CaMKII-dependent signaling. (C) Schematic of diabetes induction and experimental protocol for T1D. Summary data for (D) blood glucose and (E) body weight in nondiabetic and T1D mice 2 weeks after STZ injection. (F) Schematic of DM induction and experimental protocol for T2D. Summary data for (G) blood glucose and (H) body weight in nondiabetic and T2D mice 2 weeks after LD-STZ injection. T1D, type 1 DM; T2D, type 2 DM; HFD, high-fat diet; LD-STZ, low-dose STZ; NCD, normal chow diet; OGN, O-GlcNAcylation; RyR2, ryanodine receptor type 2; STZ, streptozocin. Data are represented as mean ± SEM. Statistical comparisons were performed using 1-way ANOVA with Tukey’s multiple-comparison test (D, E, G, and H) (^#P < 0.0001 vs. WT non-DM, *P < 0.05 vs. WT non-DM).

Figure 2. ROS and O-GlcNAcylation are elevated in type 1 and type 2 diabetic hearts.

(A) Representative confocal images (original magnification, ×40) and summary data for DHE fluorescence in mouse atrial tissue show increased ROS in T1D (top) and T2D (bottom). Scale bars: 10μm (n = 6 WT non-DM, n = 8 WT T1D, n = 4 WT non-DM, n = 4 WT T2D) (B) Representative Western blots and summary data for total OGN modified protein levels (OGN monoclonal antibody – RL2) normalized to tubulin and competition assay (RL2 + 500 mM GlcNAc) from heart lysates, from T1D (left) and T2D (right) (n = 3–7/group). OGN quantification excluded the noncompeted bands. DHE, dihydroethidium; DM, diabetes mellitus; T1D, type 1 DM; T2D, type 2 DM; OGN, O-GlcNAcylation. Data are represented as mean ± SEM. Statistical comparisons were performed using 2-tailed Student’s t test (A and B) (*P < 0.05 vs. WT non-DM).

Figure 3. CaMKII promotes enhanced atrial fibrillation susceptibility in type 1 and type 2 diabetic mice.

(A) Representative tracings of intracardiac (atrial, A-EGM; ventricular, V-EGM) and lead II surface electrocardiograms recorded immediately after rapid atrial burst pacing demonstrating normal sinus rhythm in a control non-DM WT mouse and irregular atrial and ventricular electrical impulses marking AF in a diabetic WT T1D mouse. (B) Marked AF susceptibility in WT T1D mice compared with WT non-DM mice. This is reversed in AC3-I and MMVV T1D mice, but not in S280A T1D mice. (C) Pre- and post-insulin pump (LinBit) implantation blood glucose levels 1 week after STZ treatment (pre-insulin pump) and 1 week after insulin pump implantation (post-insulin) (n = 22). (D) Insulin treatment prevents enhanced AF in WT T1D mice with blood glucose (BG) level less than 300 mg/dL on insulin treatment. (E) Increased AF susceptibility is present in WT T2D mice compared with nondiabetic controls; AC3-I and MMVV T2D mice are protected from enhanced AF, but there is no protection in S280A T2D mice. AF, atrial fibrillation; DM, diabetes mellitus; T1D, type 1 DM; T2D, type 2 DM; EGM, electrogram. Data are represented as percentage frequency distribution (B, D, and E) and mean ± SEM (C). The numerals in the bars represent the sample size in each group (B, D, and E). Statistical comparisons were performed using 2-tailed Fischer’s exact test with Holm-Bonferroni correction for multiple comparisons (B, D, and E) and 2-tailed Student’s t test (C) (*P < 0.05).

Figure 4. MM281/282 but not S280 is critical for CaMKII activation in response to hyperglycemia.

(A) Schematic of CaMKII kinase translocation reporter (CaMKII-KTR) assay. CaMKII-KTR traffics between the nucleus and cytoplasm and phosphorylation by CaMKII results in net translocation of the KTR to the cytosol. Cytosolic to nuclear fluorescent signal ratio is a measure of CaMKII activity. (B) Representative fluorescent micrographs of KTR transfected neonatal mouse cardiomyocytes at baseline and time, t = 18 hours after treatment. Cells from WT pups were incubated as indicated with 5.5 mM glucose (low glucose, n = 18 cells), 5.5 mM glucose + 24.5 mM mannitol (mannitol, n = 19 cells), 30 mM glucose (high glucose, n = 26 cells), high glucose + 2 mM N-acetyl cysteine (high glucose + NAC, n = 11 cells), or high glucose + 1 μM AS105 (a CaMKII inhibitor, n = 13 cells). Cells from MMVV (n = 15 cells) and S280A (n = 15 cells) pups were incubated with high glucose. Cells from WT pups at baseline and time, t = 20 minutes after treatment with 10 mM caffeine (n = 15 cells). Arrowheads indicate nuclei. (C) Summary data of the change in KTR cytosolic/nuclear ratio before and after treatments. Data are represented as mean ± SEM, and statistical comparisons were performed using 1-way ANOVA with Dunnett’s multiple-comparison test (*P < 0.05).

Figure 5. Targeted ROS inhibition and MsrA overexpression protects against atrial fibrillation in diabetes.

(A) Inhibition of mitochondrial ROS by MitoTEMPO treatment or inhibition of cytoplasmic ROS by loss of the p47 subunit of NADPH oxidase (p47^–/– mice) protect against AF in T1D mice. Mice with myocardial targeted transgenic overexpression of methionine sulfoxide reductase A (MsrA TG) were protected from T1D primed AF; nondiabetic mice lacking MsrA (MrsA^–/– non-DM) showed increased AF susceptibility in the absence of diabetes. (B) Summary data of blood glucose measurements at the time of electrophysiology study. (C) Increased mitochondrial ROS in isolated atrial myocytes from WT T1D (bottom) compared with WT non-DM (top) mice detected by MitoSOX fluorescence. Representative confocal fluorescent images (original magnification, ×40) show MitoSOX (red, left), MitoTracker (green, middle), and merged images (right). Scale bars: 10 μm. (n = 41–47 cells in each group from 2 mice per group). Data are represented as percentage frequency distribution (A) and mean ± SEM (B and C). The numerals in the bars represent the sample size in each group (A). Statistical comparisons were performed using 2-tailed Fischer’s exact test with Holm-Bonferroni correction for multiple comparisons (A), 1-way ANOVA with Tukey’s multiple-comparison test (B) and 2-tailed Student’s t test (C). (*P < 0.05). WT T1D data set (A and B), control data previously presented. AF, atrial fibrillation; DM, diabetes mellitus; T1D, type 1 DM; T2D, type 2 DM.

Figure 6. Targeted OGN inhibition is protective against atrial fibrillation susceptibility in type 1 and type 2 diabetes.

(A) DON pretreatment (5 mg/kg i.p.) protected from AF in T1D WT and S280A mice, with no additional protection in T1D MMVV mice. (B) DON pretreatment did not protect from AF in T2D WT mice. (C) Schematic of the OGA (O-GlcNAcase) transgene construct with the α-myosin heavy chain (α-MHC) promoter, HA epitope marker, and human growth hormone polyA signal (HGH1) (top). PCR product validation of OGA transgene expression in 2 founder pups (OGA-transgenic mice, OGA-TG). The line with the higher OGA expression was chosen for further experiments (bottom). (D) Western blot for OGA transgene and HA epitope expression in heart (H), gastrocnemius muscle (G), liver (L), and kidney (K) from WT and OGA-TG mice. (E) OGA-TG mice were protected from enhanced AF in T1D. (F) OGA-TG mice had similar blood glucose levels as WT littermates under T1D and nondiabetic conditions. (G) OGA-TG were protected from enhanced AF in T2D. (H) OGA-TG mice had similar blood glucose levels as WT T2D mice. DON, 6-diazo-5-oxo-L-norleucine; AF, atrial fibrillation; DM, diabetes mellitus; T1D, type 1 DM; T2D, type 2 DM. Data are represented as percentage frequency distribution (A, B, E, and G) and mean ± SEM (F and H). The numerals in the bars represent the sample size in each group (A, B, E, and G). Statistical comparisons were performed using 2-tailed Fischer’s exact test with Holm-Bonferroni correction for multiple comparisons (A, B, E, and G), 1-way ANOVA with Tukey’s multiple-comparison test (F and H). (*P < 0.05). WT T1D (A) and T2D (B, G, and H) data sets are control data previously presented.