AKT and ERK1/2 activation via remote ischemic preconditioning prevents Kcne2 -dependent sudden cardiac death

Figures

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  Figure 1. Experimental protocols. Remote ischemic preconditioning (RIPC) of liver or limb was induced by four cycles of 5 min of ischemia with 5 min intermittent reperfusions. Pharmacological inhibitors were administered as a bolus 30 min prior to myocardial ischemia. Mice of either genotype were subjected to 10‐min left main coronary artery occlusion followed by 20‐min reperfusion.

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  Figure 2. Remote ischemic preconditioning (RIPC) protects against Kcne2‐dependent ventricular arrhythmias. Exemplar surface ECGs of Kcne2 +/+ and Kcne2 ‐/‐ mice in the presence or absence of liver or limb preconditioning (RIPC) during the 20 min of cardiac reperfusion period (n = 8–15). AVB, AV block, VT, ventricular tachycardia, PVT indicates polymorphic VT.

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  Figure 3. Remote ischemic preconditioning (RIPC) prevents lethal Kcne2‐dependent ventricular arrhythmias. (A, B) Quantification of cardiac arrhythmia incidence in Kcne2 +/+ and Kcne2 ‐/‐ mice with or without RIPC (Liver or Limb) treatment (n = 9–15). Numbers of mice per category are indicated in parentheses. (A) Arrhythmia, all arrhythmia classes pooled; AVB, AV block; SCD, sudden cardiac death; VT, ventricular tachycardia; PVT, polymorphic VT; SVT, sustained ventricular tachycardia (over 10 sec); *P < 0.05, **P < 0.01, ***P < 0.001 versus Kcne2 ‐/‐ mice without RIPC treatment. (B) Mean VT durations for Kcne2 +/+ and Kcne2 ‐/‐ mice with or without RIPC (Liver or Limb) treatment (n = 8–15). Mice without VT were indicated as 0 sec duration. ***P < 0.001, versus Kcne2 ‐/‐ mice without RIPC treatment (by one‐way ANOVA). (C) Latency to first run of VT after the onset of reperfusion in Kcne2 +/+ and Kcne2 ‐/‐ mice with or without RIPC (Liver or Limb) treatment (n = 9–15). ***P < 0.001, versus Kcne2 ‐/‐ mice without RIPC treatment (by one‐way ANOVA).

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  Figure 4. Liver remote ischemic preconditioning (RIPC) stimulates ventricular ERK1/2 and AKT phosphorylation in Kcne2‐/‐ mice post cardiac IR injury. (A‐D) Left, representative western blots of phospho‐(p) ERK1/2 and total (t)ERK1/2 (A), phospho‐(p) AKT and total (t) AKT (B), phospho‐(p) GSK3β and total (t) GSK3β (C), phospho‐(p) STAT‐3 and total (t) STAT‐3 (D) from Kcne2 +/+ and Kcne2 ‐/‐ mice with or without RIPC(Liver) treatment; one mouse per lane. Right, mean ratio of band densities of pERK/tERK (A, **P < 0.01, ***P < 0.001, vs. baseline Kcne2 +/+ mice, ## P < 0.01, ### P < 0.001 vs. baseline Kcne2 ‐/‐ mice, † P < 0.05 vs. Kcne2 +/+ mice after RIPC(Liver) treatment), pAKT/tAKT (B, *P < 0.05, vs. baseline Kcne2 +/+ mice, # P < 0.05 vs. baseline Kcne2 ‐/‐ mice, † P < 0.05 vs. Kcne2 +/+ mice after RIPC(Liver) treatment), pGSK3β/tGSK3β (C), pSTAT‐3/tSTAT‐3 (D, **P < 0.01, vs. baseline Kcne2 +/+ mice, ## P < 0.01, vs. baseline Kcne2 ‐/‐ mice). n = 4–5 each group.

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  Figure 5. Limb remote ischemic preconditioning (RIPC) stimulates ventricular AKT phosphorylation in Kcne2‐/‐ mice post cardiac IR injury. (A–D) Left, typical blots of phospho‐(p) ERK1/2 and total (t)ERK1/2 (A), phospho‐(p) AKT and total (t) AKT (B), phospho‐(p) GSK3β and total (t) GSK3β (C), phospho‐(p) STAT‐3 and total (t) STAT‐3 (D) from Kcne2 +/+ and Kcne2 ‐/‐ mice with or without RIPC(Limb) treatment; one mouse per lane. Right, mean ratio of band densities of pERK/tERK (A, *P < 0.05, ***P < 0.001, vs. baseline Kcne2 +/+ mice, # P < 0.05, ### P < 0.001 vs. baseline Kcne2 ‐/‐ mice, † P < 0.05 vs. Kcne2 +/+ mice after RIPC(Limb) treatment), pAKT/tAKT (B, **P < 0.01, vs. baseline Kcne2 +/+ mice, ## P < 0.01, vs. baseline Kcne2 ‐/‐ mice, † P < 0.05 vs. Kcne2 +/+ mice after RIPC(Limb) treatment), pGSK3β/tGSK3β (C), pSTAT‐3/tSTAT‐3 (D, ***P < 0.01 vs. baseline Kcne2 +/+ mice, ### P < 0.01 vs. baseline Kcne2 ‐/‐ mice). n = 4–5 each group.

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  Figure 6. AKT and ERK1/2 activation is essential for prevention of SCD by remote ischemic preconditioning (RIPC)‐liver in Kcne2‐/‐ mice postIR. (A) The change of cardiac arrhythmia incidence in the absence (−) or presence (+) of liver I/R preconditioning stimulus when applying ERK inhibitor U0126 or AKT inhibitor Wortmannin (Wort) in Kcne2 ‐/‐ mice (n = 8–15 per group). Numbers of mice per category are indicated in parentheses. Values for Kcne2 mice without inhibitors are repeated from Fig. 2 for comparison. AVB, AV block; SCD, sudden cardiac death; VT, ventricular tachycardia; PVT, polymorphic VT; SVT, sustained ventricular tachycardia over 10 sec. *P < 0.05,**P < 0.01, versus RIPC‐treated Kcne2 ‐/‐ mice post I/R. (B) Durations of VT for RIPC (Liver)‐treated or nontreated Kcne2 ‐/‐ mice in the presence (+) or absence (−) of ERK inhibitor U0126 or AKT inhibitor Wortmannin (Wort) application. Mice without VT were indicated as 0 sec duration. Values for Kcne2 ‐/‐ mice without inhibitors are repeated from Figre 2 for comparison. *P < 0.05, versus RIPC‐treated Kcne2 ‐/‐ mice post I/R. (C) Left, typical blot of phospho‐(p) ERK1/2 and total (t)ERK1/2 from Kcne2 ‐/‐ mice with or without…

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  Figure 7. AKT phosphorylation is essential for remote ischemic preconditioning (RIPC)‐limb‐induced antiarrhythmic activity in Kcne2‐/‐ mice post‐IR. (A) The effect of inhibiting AKT (Wortmannin, Wort) on postreperfusion arrhythmia characteristics in Kcne2 ‐/‐ mice in the absence (−) or presence (+) of limb I/R preconditioning stimulus. Values in parentheses indicate the numbers of mice per category. n = 8–15 each group. Values for Kcne2 mice without inhibitor are repeated from Figure 2 for comparison. AVB, AV block; SCD, sudden cardiac death; VT, ventricular tachycardia; PVT, polymorphic VT; SVT, sustained ventricular tachycardia over 10 sec. (B) Durations of VT for RIPC‐Limb‐treated or nontreated Kcne2 ‐/‐ mice in the presence (+) or absence (−) of AKT inhibitor Wortmannin (Wort) application. Mice without VT were indicated as 0 sec duration. Values for Kcne2 ‐/‐ mice without inhibitors are repeated from Figure 2 for comparison. *P < 0.05 versus Kcne2 ‐/‐ mice after RIPC‐limb treatment without inhibitor. (C) Left, typical blot of phospho‐(p) AKT and total (t) AKT from Kcne2 ‐/‐ mice with or without RIPC‐limb treatment in the presence (+) or absence (−) of AKT inhibitor Wortmannin (Wort)…