Myocardial death and dysfunction after ischemia-reperfusion injury require CaMKIIδ oxidation

Figures

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  Figure 1. MMVV mice are protected against I/R injury. (A) Representative images of transverse cardiac sections from WT (left) and MMVV (right) mice after I/R surgery. Area at risk (AAR) is the sum of red and white areas, and the area of necrosis (AON) is white. The blue area is outside of the AAR. (B) Summary data for AON/AAR. The AON/AAR was significantly reduced in MMVV hearts (n = 7) compared to their littermate WT hearts (n = 6). *p < 0.05 unpaired Student’s t-test. (C) Summary data for AAR measured over the left ventricle (LV). (D) LV ejection fractions (E) LV fractional shortening (F) LVID-S (LV internal diameter in systole) before and after I/R surgery in MMVV (n = 22) and WT (n = 21) mice. One way ANOVA (P < 0.0001). Tukey’s multiple comparisons test compared each group, as indicated by brackets *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (G) Representative images of ventricular myocytes isolated from MMVV (right) and littermate WT (left) hearts under I/R control conditions with all experimental steps except addition of an oil layer (−oil, upper) and simulated cellular I/R (+oil, lower). Scale bar is 100 µm. (H) Summary data for cell viability expressed as the percentage…

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  Figure 2. MMVV ventricular myocytes are resistant to H2O2-induced reduction in IKATP. (A) Representative traces of IKATP in response to a ramp command pulse (inset) from ventricular myocytes isolated from WT and MMVV mice in the presence of pinacidil and DNP with (black line) and without (red line) H2O2. Glibenclamide was added to eliminate IKATP (blue line). Inset schematics indicate patch clamp configurations used to obtain each data set, here and throughout. (B) IKATP recorded at 0 mV, with and without H2O2, from ventricular myocytes isolated from WT (n = 11 cells, 2 mice) and MMVV (n = 13 cells, 3 mice) mice. (C) Representative traces of KATP channel currents recorded from ventricular myocytes in cell attached mode in the presence of the DNP with and without H2O2. The vertical scale bar 5 pA, and the horizontal scale bar 2 s (same scale for panel F and I). (D) Number of KATP channels opening in each membrane patch under conditions with and without H2O2. (E) KATP channel open probability (NPo) calculated from the membrane patches analyzed in (D). WT (6–13 cells, 2 mice), MMVV (6 cells, 2 mice). (F) Representative traces of KATP channel recording from excised cell membrane patches in…

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  Figure 3. Normal exercise capacity, glucose tolerance, and glucose-induced insulin secretion, but reduced and H2O2-resistant IKATP in CaMKII resistant KIR 6.2 T224A mice. (A) Sanger sequencing results of PCR products amplified from genomic DNA of wildtype (WT/WT), heterozygous (WT/T224A) and homozygous mutant mice (T224A/T224A). (B) DNA gel electrophoresis of PCR products before and after NheI digestion. (C) WT and T224A mice were fasted for 16 hours (overnight) and then injected intraperitoneally with 2 g/kg glucose to assess glucose tolerance. No significant difference was found between the WT and T224A mice. (D) Area under the curve (AUC) of the measurements in (C). (E) Serum insulin concentrations were not significantly different between WT and T224A mice either after 16-hour fasting or 15 minutes after 2 g/kg glucose injection. (F) No difference between genotypes in blood glucose concentrations measured during glucose‐induced insulin secretion. (G) Representative IKATP currents recorded from ventricular myocytes isolated from WT and T224A mice using the same protocol and conditions as shown in Fig. 2A. (H) IKATP recorded at 0 mV, with and without H2O2, from ventricular myocytes…

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  Figure 4. Ox-CaMKII resistant MMVV and pinacidil provide non-additive protection against I/R injury in isolated ventricular myocytes. (A–D) Summary data for cell viability in ventricular myocytes isolated from WT littermates of MMVV mice (A, cells from 3–9 mice/group), MMVV mice (B, cells from 4–10 mice/group), WT littermate of T224A mice (C, cells from 3 WT mice) and T224A mice (D, cells from 4 T224A mice) under control conditions (−oil) without drug treatment (left bar), with pinacidil 10 µM (middle bar), or with glibenclamide 2 µM (right bar). One way ANOVA was used for statistical analysis; Tukey’s multiple comparisons test was used for intergroup comparisons (*p < 0.05, ***p < 0.001, ****p < 0.0001). (E–H) Summary data for viability of ventricular myocytes isolated from WT littermate of MMVV mice (E, cells from 3–9 mice/group), MMVV mice (F, cells from 4–10 mice/group), WT littermates of T224A mice (G, cells from 3 WT mice) and T224A mice (H, cells from 4 T224A mice) under simulated I/R condition (+oil) without drug treatment (left bar), with pinacidil 10 µM (middle bar), or with glibenclamide 2 µM (right bar). One way ANOVA was used for statistical analysis; Tukey’s multiple…

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  Figure 5. The proposed pathway for ox-CaMKII causing increased myocardial death after I/R injury by enhancing sequestration of KATP channels. I/R injury leads to reduced ATP and increased ROS. Reduced ATP increases KATP opening to protect cardiomyocytes, but ROS oxidize CaMKII, and ox-CaMKII enhances KATP endocytosis leading to reduced IKATP and increased myocardial death. Mice lacking ox-CaMKIIδ (MMVV) are relatively protected from loss of IKATP and myocardial death after I/R. Dynasore, an endocytosis blocker added to WT ventricular myocytes, and ventricular myocytes isolated from MMVV mice show similar protection against loss of IKATP.