Involvement of glycogen synthase kinase-3β in liver ischemic conditioning induced cardioprotection against myocardial ischemia and reperfusion injury in rats

Abstract

Remote ischemic conditioning has been convincingly shown to render the myocardium resistant to a subsequent more severe sustained episode of ischemia. Compared with other organs, little is known regarding the effect of transient liver ischemic conditioning. We proposed the existence of cardioprotection induced by remote liver conditioning. Male Sprague-Dawley rats were divided into sham-operated control (no further hepatic intervention) and remote liver ischemic conditioning groups. For liver ischemic conditioning, three cycles of 5 min of liver ischemia-reperfusion stimuli were conducted before-(liver preconditioning), post-myocardial ischemia (liver postconditioning), or in combination of both (liver preconditioning + liver postconditioning). Rats were exposed to 45 min of left anterior descending coronary artery occlusion, followed by 3 h of reperfusion thereafter. ECG and hemodynamics were measured throughout the experiment. The coronary artery was reoccluded at the end of reperfusion for infarct size determination. Blood samples were taken for serum lactate dehydrogenase and creatine kinase-MB test. Heart tissues were taken for apoptosis measurements and Western blotting. Our data demonstrate that liver ischemic preconditioning, postconditioning, or a combination of both, offered strong cardioprotection, as evidenced by reduction in infarct size and cardiac tissue damage, recovery of cardiac function, and inhibition of apoptosis after ischemia-reperfusion. Moreover, liver ischemic conditioning increased cardiac (not hepatic) glycogen synthase kinase-3β (GSK-3β) phosphorylation. Accordingly, inhibition of GSK-3β mimicked the cardioprotective action of liver conditioning. These results demonstrate that remote liver ischemic conditioning protected the heart against ischemia and reperfusion injury via GSK-3β-dependent cell-survival signaling pathway.NEW & NOTEWORTHY Remote ischemic conditioning protects hearts against ischemia and reperfusion (I/R) injury. However, it is unclear whether ischemic conditioning of visceral organs such as the liver, the largest metabolic organ in the body, can produce cardioprotection. This is the first study to show the cardioprotective effect of remote liver ischemic conditioning in a rat model of myocardial I/R injury. We also, for the first time, demonstrated these protective properties are associated with glycogen synthase kinase-3β-dependent cell-survival signaling pathway.

Keywords: GSK-3β; cardioprotection; liver ischemic conditioning; myocardial ischemia and reperfusion injury.

Fig. 1.

Experimental protocols. In experiment 1, all hearts were subjected to 45-min ligation of the left anterior descending coronary artery (LAD), followed by 3 h of reperfusion (R), except for the sham-operated ones. For remote liver ischemic preconditioning (RPre), three cycles of 5 min of liver ischemia with 5-min intermittent reperfusions were conducted before myocardial ischemia. For remote liver ischemic postconditioning (RPost), the above three cycles of liver I/R stimulus were induced after myocardial ischemia (at the onset of myocardial reperfusion). For the combination of remote liver ischemic preconditioning and postconditioning (RPre+RPost), the liver conditioning stimulus was induced before myocardial ischemia and at the onset of myocardial reperfusion. The GSK-3β inhibitor SB-216763 was applied 5 min before reperfusion. Thirty minutes of acute memory phase were allowed before being followed by a 45-min LAD occlusion and a subsequent 180 min of reperfusion (I/R). Arrows indicate the time points at which tissue samples were harvested. In experiment 2, hepatic stimuli were conducted without myocardial I/R intervention. Liver was taken at the same time points as in experiment 1.

Fig. 2.

Liver conditioning ameliorated myocardial damage post reperfusion. A: hepatic ischemia and reperfusion cycles did not cause liver damage. Representative (of n = 5 rats/group) hematoxylin-and-eosin-stained micrographs of liver section are shown. Scale bars, 100 μm. B: representative images of terminal transferase dUTP nick-end labeling (TUNEL)-stained liver sections. Nuclei were counterstained with 3,3-diaminobenzene (n = 4–5). RPre, remote liver ischemic preconditioning; RPost, remote liver ischemic postconditioning; RPre+RPost, the combination of remote liver ischemic preconditioning and postconditioning. Scale bars, 10 μm. C: representative sections of triphenyltetrazolium chloride (TTC)-stained heart subjected to 45-min myocardial ischemia followed by 3-h reperfusion. CON, control; RPre, remote liver ischemia preconditioning; RPost, remote liver ischemia postconditioning; RPre+RPost, the combination of remote liver ischemia preconditioning with remote liver ischemic postconditioning. D: quantification of myocardial infarct size expressed as a percentage of left ventricular (LV) area at risk (AAR) (top) and AAR expressed as a percentage of LV area (bottom). Values are means ± SE; n = 5 each group. *P < 0.05 and ***P < 0.001 compared with CON; ^##P < 0.01 compared with RPre; and †P < 0.05 compared with RPost (by one-way ANOVA). E: representative images of TUNEL-stained heart sections. Myocardial apoptosis was determined by measurement of TUNEL-positive cardiomyocyte nuclei in the AAR of myocardium obtained from liver-conditioned and CON rats after coronary artery reperfusion injury. TUNEL-positive (red) cardiomyocytes were identified as apoptotic cells. Positive cells were not detected in the nonischemic zone. Arrows denote TUNEL-positive nucleus. Scale bars = 10 μm. F: bar graph showing the TUNEL-positive nuclei expressed as a percentage of total nuclei in the heart AAR sections. Values are means ± SE; each group, n = 4–6. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with CON; ^#P < 0.05 compared with RPre (by one-way ANOVA). G: serum levels of lactate dehydrogenase (LDH) in rats subjected to 45 min of left anterior descending artery occlusion followed by 3 h of reperfusion. Values are means ± SE; n = 11–13 per group. **P < 0.01 and ***P < 0.001 vs. sham; ^#P < 0.05 vs. CON. H: post-reperfusion injury mean serum levels of creatine kinase MB (CK-MB) for CON and liver ischemic conditioned rats. Values are means ± SE; n = 7–12 per group. ***P < 0.001 vs. sham; ^#P < 0.05 and ^##P < 0.01 vs. CON.

Fig. 3.

Myocardial apoptotic protein expression. A: representative immunostainings of Bcl-2 and Bax protein in the cytoplasm of the left ventricular myocytes isolated after 3 h of reperfusion. CON, control; RPre, remote liver ischemia preconditioning; RPost, remote liver ischemia postconditioning; RPre+RPost, the combination of remote liver ischemia preconditioning with remote liver ischemic postconditioning. Densitometric analysis of Bcl-2 (B), Bax (C), and Bcl-2/Bax (D) protein expression in rat left ventricles post-I/R is shown. Values are means ± SE; n = 4–6 per group. PEI, positive expressive index. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham; ^#P < 0.05 and ^##P < 0.01 vs. CON; †P < 0.05 vs. RPre. Representative Western blots (top) and quantification (bottom) of cleaved caspase-3 (E) and caspase-3 (F) protein band density (normalized to GAPDH) in sham, CON, and liver-conditioned rat left ventricles are shown. Values are means ± SE; n = 4–5 per group. *P < 0.05, **P < 0.01, and ***P < 0.001 vs. sham; ^##P < 0.01 and ^###P < 0.001 vs. CON.

Fig. 4.

Liver ischemic conditioning stimulates ventricular GSK-3β phosphorylation. Representative immunoblots (top) and densitometric analysis (bottom) of phosphorylated GSK-3β (Ser9) (p-GSK-3β) and total GSK-3β in rat livers (A) and ventricles (B) are shown. Sham animals did not undergo liver stimulus. Sham, sham-operated group; RPre, remote liver ischemic preconditioning; RPost, remote liver ischemic postconditioning; RPre+RPost, the combination of remote liver ischemic preconditioning and postconditioning. All band densities were normalized to sham group. Values are means ± SE; n = 5 in each group. NS, no significant difference. *P < 0.05 and **P < 0.01 vs. sham; ^##P < 0.01 vs. CON (by one-way ANOVA).

Fig. 5.

The protective effect of pharmacological inhibitors on I/R injury. A: representative sections of TTC-stained heart subjected to 45-min myocardial ischemia followed by 3-h reperfusion. CON, control; RPre+RPost, the combination of remote liver ischemia preconditioning with remote liver ischemic postconditioning. B: quantification of myocardial infarct size expressed as a percentage of left ventricular area at risk. Values are means ± SE; n = 4–5 each group. Values for CON and RPre+RPost rats are repeated from Fig. 2C for comparison. **P < 0.01 and ***P < 0.001 compared with CON (by one-way ANOVA). C: mean serum levels of LDH of CON and liver ischemic conditioned rats after I/R injury with or without SB-216763 or U-0126. Values are means ± SE; n = 7–13. Values for CON and RPre+RPost rats are repeated from Fig. 2G for comparison. **P < 0.01 and ***P < 0.001 compared with CON (by one-way ANOVA). D: mean serum levels of CK-MB of CON and liver ischemic conditioned rats after I/R injury with or without SB-216763 or U-0126. Values are means ± SE; n = 7–12. Values for CON and RPre+RPost rats are repeated from Fig. 2H for comparison. *P < 0.05 and **P < 0.01 compared with CON (by one-way ANOVA). E, left: apoptotic nuclei detected by TUNEL technique in the area of myocardium at risk. Arrow indicates TUNEL-positive nuclei (red). Scale bars, 10 µm. C, control; R, the combination of remote liver ischemia preconditioning with remote liver ischemic postconditioning; S, SB-216763. Right: graph showing the averaged percentage of TUNEL-positive cells in the ischemic regions of LVs. Values are means ± SE; n = 4–6, each group. Values for CON and RPre+RPost rats are repeated from Fig. 2, E and F for comparison. **P < 0.01 and ***P < 0.001 compared with CON (by one-way ANOVA). F: representative Western blots (top) and densitometric analysis (bottom) of phosphorylated GSK-3β (Ser9) (p-GSK-3β) and total GSK-3β in rat ventricles with (+) or without (−) SB-216763 after 180 min of cardiac I/R injury. Sham, sham-operated group; RPre, remote liver ischemic preconditioning; RPost, remote liver ischemic postconditioning; RPre+RPost, the combination of remote liver ischemic preconditioning and postconditioning. All band densities were normalized to sham group. Values are means ± SE; n = 5 in each group. ***P < 0.001 vs. sham; ^###P < 0.001 vs. CON (by one-way ANOVA).