Remote Liver Ischemic Preconditioning Protects against Sudden Cardiac Death via an ERK/GSK-3β-Dependent Mechanism

Fig 1. Experimental protocols. All groups were subjected to 5-min left main coronary artery occlusion followed by 20-min reperfusion except for the sham-operated group. Remote liver ischemia preconditioning (RLIPC) was induced by four cycles of 5 min of liver ischemia with 5 min intermittent reperfusions. Pharmacological inhibitors (SB216763 and U0126) were administered as a bolus 30 min prior to myocardial ischemia. Two baseline values were recorded: Baseline 1, ten minutes stabilization after instrumentation, and Baseline 2, twenty-five minutes post liver I/R stimulus.

Fig 2. RLIPC elevates ST segment prior to induction of cardiac ischemia in rat model of arrhythmia. A) Left, typical ECGs from CON and RLIPC rats during baseline periods showing elevated ST segment in RLIPC-treated rats. Right, quantification of parameters from ECGs showing elevated ST segment in RLIPC rats post liver I/R stimulus (n = 9–10, each group). *P<0.05, **P<0.01, vs. RLIPC in the presence of liver I/R cycles. B) Variables recorded during the experimental protocol investigating the effect of liver I/R stimulus on hemodynamics prior to coronary artery ligation. LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; ±dP/dtmax, maximum rate of increase/decrease in left ventricular pressure. Data are the mean±SEM (n = 6–7 in each group). *P<0.05 compared with values at Baseline 1. Baseline 1 indicates the first baseline value after 10 minutes stabilization; Baseline 2 indicates the second baseline value obtained 5 minutes prior to coronary ligation. Hearts in RLIPC group experienced four cycles of I/R stimulus, indicated as 1, 2, 3, 4.

Fig 3. RLIPC improves cardiac function post-ischemic arrhythmia. A) Representative hemodynamic traces from CON and RLIPC rats prior to- or post-myocardial ischemia showing preservation of cardiac function with liver ischemic preconditioning. B) Left ventricular ±dP/dtmax values measured during and after myocardial ischemia in control and RLIPC-treated rats; n = 6–8 per group. *P<0.05 and ***P<0.001 vs. CON.

Fig 4. RLIPC confers cardioprotection against post-ischemic arrhythmia and SCD. A) Quantification of the incidence of different cardiac arrhythmia characteristics and mortality during post ischemia reperfusion in CON (n = 16) and RLIPC (n = 14) rats. Numbers of animals per category are indicated in parentheses. Sinus rhythm, remained in sinus rhythm; AVB, AV block; VT, ventricular tachycardia; SVT, sustained VT (>1min VT); PVT, polymorphic VT; VF, ventricular fibrillation; SCD: sudden cardiac death. *P<0.05 and **P<0.01 vs. CON. B) Representative surface ECGs from CON and RLIPC rats during reperfusion showing greater severity of arrhythmia in the former. C) Mean VT parameters including the onset time of first VT episode after reperfusion (left) and durations of VT (middle) and the longest episode of VT duration (LVT) (right) for control (n = 16) and RLIPC (n = 14) rats. Rats without VT were indicated as 0 duration. **P<0.01 vs CON, NS: P>0.05 between two groups. D) Left, VF incidence during the first 5 minutes of reperfusion for CON (n = 16) and RLIPC (n = 14) rats. Numbers in parentheses indicate the numbers of rats exhibiting VF; right, the starting time of the first run of VF after…

Fig 5. RLIPC phosphorylates ERK1/2 and GSK-3β post cardiac IR injury. A) Left, Western blots of ventricular phosphorylated ERK1/2 and total ERK1/2 isolated from sham, control and RLIPC rats after 20 minutes of reperfusion; right, band density of p-ERK/ERK (n = 6–8 per group). ***P<0.001 vs. RIPC. B) Left, Western blots of ventricular phosphorylated GSK-3β(Ser9) and total GSK-3β isolated from sham, control and RLIPC rats after 20 minutes of reperfusion; right, band density of p-GSK-3β(Ser9)/GSK-3β (n = 6–8 per group). ***P<0.001 vs. RIPC.

Fig 6. The influence of pharmacological inhibitors on ST elevation in rats. A) The change of ST height in the absence (-) or presence (+) of liver I/R preconditioning stimulus when applying GSK-3β inhibitor SB216763 (n = 6–9 per group). NS: P>0.05 among groups. ST height for control group is repeated from Fig 2 for comparison. C, control; R, RLIPC; SB, SB216763. B) The change of ST height in the absence (-) or presence (+) of liver I/R preconditioning stimulus when applying ERK inhibitor U0126 (n = 6–9 per group). NS: P>0.05 among groups. ST height for control group is repeated from Fig 2 for comparison. C, control; R, RLIPC; U, U0126.

Fig 7. Pharmacological inhibition of ERK or GSK-3β abolishes RLIPC-induced anti-arrhythmic cardioprotection. A) The effect of inhibiting GSK-3β on post-reperfusion arrhythmia characteristics and mortality in CON (n = 10) and RLIPC (n = 12) rats upon administration of GSK-3β inhibitor SB216763 prior to coronary ligation. Numbers of animals per category are indicated in parentheses. CON, control; RLIPC, remote liver ischemia preconditioning. AVB, AV block; VT, ventricular tachycardia; SVT, sustained VT (>1min VT); PVT, polymorphic VT; VF, ventricular fibrillation; SCD: sudden cardiac death. B) Mean values for onset time of first VT (upper left)/VF (upper right) episode after reperfusion and durations of VT (lower left), the longest episode of VT duration (LVT, lower right) for control (n = 10) and RLIPC (n = 12) rats upon GSK-3β inhibitor SB216763 application. Values for control and RLIPC rats are repeated from Fig 3 for comparison. NS, no significant difference, **P<0.01, ***P<0.001 compared withRLIPC (by One-way ANOVA). C) The effect of administering ERK inhibitor U0126 to CON (n = 8) and RLIPC (n = 8) rats on post-reperfusion arrhythmia characteristics and mortality. Values in…

Fig 8. The influence of pharmacological inhibitors on cardiac ERK and GSK-3β phosphorylation status in rats. A) Upper panel, Representative western blots of phosphorylated ERK1/2 and ERK1/2 isolated from CON and RLIPC rats in the presence or absence of pharmacological inhibitors. CON, control; RLIPC, remote liver ischemia preconditioning. Lower panel, Quantification of p-ERK/ERK protein band density; n = 6–12, each group. S, sham-operated group, C, control; R, RLIPC; SB, SB216763; U, U0126. ***P<0.0001 compared with RLIPC (by One-way ANOVA). All other group comparisons showed P>0.05. B) Upper panel, Representative western blots of phosphorylated GSK-3β(Ser9) and total GSK-3β isolated from control and RLIPC rats in the presence or absence of pharmacological inhibitors. Lower panel, Quantification of p-GSK-3β(Ser9)/GSK-3β protein band density; n = 5–10, each group. S, sham-operated group, C, control; R, RLIPC; SB, SB216763; U, U0126. ***P<0.0001 compared with RLIPC (by One-way ANOVA). All other group comparisons showed P>0.05. C) Upper panel, Representative western blots of phosphorylated GSK-3β(Tyr216) and total GSK-3β isolated from control and RLIPC rats in the presence or absence of…