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

Abstract

Reactive oxygen species (ROS) contribute to myocardial death during ischemia-reperfusion (I/R) injury, but detailed knowledge of molecular pathways connecting ROS to cardiac injury is lacking. Activation of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKIIδ) is implicated in myocardial death, and CaMKII can be activated by ROS (ox-CaMKII) through oxidation of regulatory domain methionines (Met281/282). We examined I/R injury in mice where CaMKIIδ was made resistant to ROS activation by knock-in replacement of regulatory domain methionines with valines (MMVV). We found reduced myocardial death, and improved left ventricular function 24 hours after I/R injury in MMVV in vivo and in vitro compared to WT controls. Loss of ATP sensitive K⁺ channel (KATP) current contributes to I/R injury, and CaMKII promotes sequestration of KATP from myocardial cell membranes. KATP current density was significantly reduced by H₂O₂ in WT ventricular myocytes, but not in MMVV, showing ox-CaMKII decreases KATP availability. Taken together, these findings support a view that ox-CaMKII and KATP are components of a signaling axis promoting I/R injury by ROS.

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 of live cells. Each point was a summary of 7–10 fields of cell counting (~50–100 cells in each field). Cells from 9 WT mice and 10 MMVV 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. (I) Representative images of ventricular myocytes isolated from MMVV (right) and littermate WT (left) hearts under control conditions (−H₂O₂, upper) and after addition of 1 mM H₂O₂ (+H₂O₂, lower). The scale bar is 100 µm. (J) Summary data for cell viability based on percentage of live cells, under different concentrations of H₂O₂. Cells are from 2 mice from each group. Data were analyzed with a one way ANOVA test (P < 0.0001). Sidak’s multiple comparisons test was used for comparisons between groups. All comparisons between the 1 mM H₂O₂ and other groups were significant (P < 0.0001). Significantly more MMVV compared to WT ventricular myocytes were viable after 1 mM H₂O₂ (*p < 0.05).

Figure 2

MMVV ventricular myocytes are resistant to H₂O₂-induced reduction in I_KATP. (A) Representative traces of I_KATP 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 I_KATP (blue line). Inset schematics indicate patch clamp configurations used to obtain each data set, here and throughout. (B) I_KATP 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 inside-out mode at −60 mV membrane potential and H₂O₂ treatment, ATP, and after washout. (G) Number of KATP channels opening in each membrane patch under conditions with and without H₂O₂. (H) KATP NPo calculated from the membrane patches analyzed in (G), 5–6 cells from 3 WT mice and 12 cells from 3 MMVV mice. (I) Representative KATP channel currents recorded from WT isolated ventricular myocytes in cell attached mode in the presence of DNP with and without H₂O₂. The lower panel shows cells pretreated with Dynasore. (J) Number of KATP channels opening in each membrane patch under conditions with and without H₂O₂ and with and without pretreatment of Dynasore. (K) KATP NPo calculated from the membrane patches analyzed in (J), 5–12 cells in each group, 3 WT mice. One way ANOVA and Tukey’s multiple comparisons test were used for comparing each group in all the bar graph panels,*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3

Normal exercise capacity, glucose tolerance, and glucose-induced insulin secretion, but reduced and H₂O₂-resistant I_KATP 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 I_KATP currents recorded from ventricular myocytes isolated from WT and T224A mice using the same protocol and conditions as shown in Fig. 2A. (H) I_KATP recorded at 0 mV, with and without H₂O₂, from ventricular myocytes isolated from WT (n = 9–13 cells, 3 mice) and T224A (n = 8 cells, 3 mice) mice. One way ANOVA and Tukey’s multiple comparisons test were used for comparisons between groups as marked by brackets (*p < 0.05, **p < 0.01). (I) Representative traces of KATP channel currents recorded from isolated ventricular myocytes in cell attached mode in the presence of the KATP channel opener DNP with and without H₂O₂. The vertical scale bar 5 pA, and the horizontal scale bar 2 s. (J) Number of KATP channel openings in each membrane patch under conditions with and without H₂O₂, as in Fig. 2D. One way ANOVA and Tukey’s multiple comparisons test were used for intergroup comparisons (*p < 0.05, **p < 0.01). (K) Summary data of open probability (NPo) in KATP channels analyzed from cell membrane patches shown in (J). One way ANOVA was used for comparison between all groups (P > 0.05). WT (4–7 cells, 2 mice), T224A (13–23 cells, 4 mice). (L) Expression of Kir6.2 in the heart was measured by RT-qPCR (Normalized against Gapdh, n = 5 for each genotype).

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 comparisons test was used for intergroup comparisons (**p < 0.01, ***p < 0.001, ****p < 0.0001).

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 I_KATP and increased myocardial death. Mice lacking ox-CaMKIIδ (MMVV) are relatively protected from loss of I_KATP 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 I_KATP.