IL-6 plays an obligatory role in late preconditioning via JAK-STAT signaling and upregulation of iNOS and COX-2

Abstract

Objective: We sought to determine whether interleukin (IL)-6 modulates myocardial infarction or the late phase of preconditioning (PC).

Methods: Wild-type and IL-6(-/-) mice underwent a 30-min coronary occlusion followed by 24 h of reperfusion with or without six cycles of coronary occlusion/reperfusion 24 h earlier. Myocardial IL-6 protein expression, activation of Janus kinase (JAK) 1 and JAK2, and signal transducers and activators of transcription (STAT) 1 and STAT3 after ischemic PC protocol were examined. The expression of the inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 was determined 24 h after the PC ischemia.

Results: In preconditioned wild-type mice, infarct size was reduced from 60.5+/-2.6% of the risk region to 33.5+/-3.6%, indicating a late PC effect. In nonpreconditioned IL-6(-/-) mice, infarct size was similar to that observed in wild-type mice (59.9+/-3.8%), indicating that the deletion of IL-6 has no effect on infarct size. However, in preconditioned IL-6(-/-) mice, infarct size was not reduced (65.1+/-3.1%), indicating that the infarct-sparing effect was completely abrogated. Ischemic PC increased the expression of IL-6 in the cytoplasm of cardiomyocytes in the ischemic/reperfused zone. In IL-6(-/-) mice, the ischemic PC-induced activation of JAK1 and JAK2 and STAT1 and STAT3 was significantly reduced, and the increase in iNOS and COX-2 protein expression 24 h after the PC ischemia was markedly attenuated.

Conclusion: IL-6 does not modulate myocardial infarct size in naïve myocardium. However, following a PC stimulus, IL-6 is obligatorily required for the activation of the JAK-STAT pathway, the ensuing upregulation of iNOS and COX-2 (co-mediators of late PC), and the development of a cardioprotective phenotype.

Fig. 1

Experimental protocol. Twenty groups of mice were used. On day 2, mice in groups I–IV underwent a 30-min coronary occlusion followed by 24 h of reperfusion. On day 1, mice in group II (n = 9, late PC group) and group IV (n = 9, IL-6^−/− late PC group) underwent a sequence of six 4-min coronary occlusion (O)/4-min reperfusion (R) cycles, while mice in group I (n = 17, control group) and group III (n = 9, IL-6^−/− control group) did not undergo any intervention. Mice in group V (n = 6) underwent 1 h of open-chest state (n = 3) or six cycles of 4-min coronary occlusion/4-min reperfusion (n = 3), and the hearts were harvested 30 min later for IL-6 immunolocalization. In groups VI–VIII (n = 6 in each group), wild-type mice underwent no intervention (group VI), 1 h of open-chest state (group VII), or six cycles of 4-min coronary occlusion/4-min reperfusion (group VIII), and myocardial tissue samples were harvested 2 h later for the measurement of myocardial IL-6 levels by ELISA. In groups IX–XX (n = 6 in each group), wild-type (groups IX, X, XIII, XIV, XVII, and XVIII) and IL-6^−/− (groups XII, XV, XVI, XIX, and XX) mice underwent six cycles of 4-min coronary occlusion/4-min reperfusion (groups X, XII, XIV, XVI, XVIII, and XX) or 1-h open-chest state (groups IX, XIII, XV, XVII, and XIX), and myocardial tissue samples were harvested 5 min [groups IX–XII (JAK activity assays)], 30 min [groups XIII–XVI (STAT activation assays)] and 24 h [groups XVII–XX (iNOS and COX-2 levels)] later for biochemical assays.

Fig. 2

Myocardial infarct size in groups I (control group), II (PC group), III (IL-6^−/− control group), and IV (IL-6^−/− PC group). Infarct size is expressed as a percentage of the region at risk of infarction. (○) Individual mice. (●) Mean ± S.E.M. (n, number of mice).

Fig. 3

Relationship between size of the region at risk and size of myocardial infarction. Illustrated are both individual values and regression lines obtained by linear regression analysis for groups I (control group), II (PC group), III (IL-6^−/− control group), and IV (IL-6^−/− PC group). In groups I, III, and IV, infarct size was positively and linearly related to the risk region size. Linear regression equations were as follows: group I, y = 0.572x + 1.37 (r = 0.83); group III, y = 0.784x − 6.234 (r = 0.92); and group IV, y = 0.910x − 11.42 (r = 0.99). For any risk region size, infarct size was smaller in group II compared with groups I and IV. In contrast, for any risk region size, infarct size was similar in groups IV and III. These data demonstrate that late PC reduced infarct size independent of risk region size and that this effect was abrogated in mice lacking IL-6.

Fig. 4

Immunolocalization of myocardial IL-6. Representative lower magnification images (100×, panels A and B) demonstrate increased expression of IL-6 in the ischemic/reperfused myocardium. Minimal IL-6 immunoreactivity (green fluorescence) was noted in the sham-operated hearts (panel B). In contrast, extensive IL-6 expression was noted in the ischemic/reperfused zone in the hearts of preconditioned mice 30 min after the preconditioning ischemia (panel A). Higher magnification images (630 ×, panels C–E) demonstrate the cytoplasmic distribution of IL-6 (panel C, green fluorescence) in cardiomyocytes. Cardiomyocytes are identified by the red fluorescence of troponin T (panel D). Colocalization of IL-6 and troponin T in yellow fluorescence (panel E) confirms the cytoplasmic distribution of intracellular IL-6 in cardiomyocytes. Nuclei are identified by DAPI (panel E, blue fluorescence).

Fig. 5

Tyrosine phosphorylation of JAK1 and JAK2 by ischemic PC in wild-type and IL-6^−/− mice. Myocardial samples were obtained from mice that underwent a sham operation (control) and from the ischemic/reperfused region of preconditioned (PC) wild-type and IL-6^−/− mice. All mice were euthanized 5 min after the sixth reperfusion. Upper panels: Western blots showing that the immunoreactivity of the tyrosine-phosphorylated forms of JAK1 (panel A) and JAK2 (panel B) increased markedly 5 min after ischemic PC in wild-type mice, and that this increase was ablated in preconditioned IL-6^−/− mice. Lower panels: Densitometric analysis of tyrosine-phosphorylated JAK1 and JAK2 signals. Data are mean ± S.E.M.

Fig. 6

Tyrosine phosphorylation of STAT1 and STAT3 by ischemic PC in wild-type and IL-6^−/− mice. Nuclear extracts were prepared from mice that underwent a sham operation (control), and from the ischemic/reperfused region of preconditioned (PC) wild-type and IL-6^−/− mice that were euthanized 30 min after the sixth reperfusion. Upper panels: Western blots showing that the immunoreactivity of tyrosine-phosphorylated STAT1 (panel A) and STAT3 (panel B) in nuclear extracts increased markedly 30 min after ischemic PC in wild-type mice, and that this increase was significantly attenuated in preconditioned IL-6^−/− mice. Lower panels: Densitometric analysis of tyrosine-phosphorylated STAT1 and STAT3 in nuclear extracts at 30 min after PC. Data are mean ± S.E.M.

Fig. 7

Myocardial iNOS expression 24 h after ischemic PC in wild-type and IL-6^−/− mice. Myocardial samples were obtained from mice that underwent a sham operation (control) and from the ischemic/reperfused region of preconditioned (PC) wild-type and IL-6^−/− mice. Upper panels: Western blot showing that the immunoreactivity of iNOS in the cytosolic fraction increased 24 h after ischemic PC, and that this increase was ablated in the absence of IL-6. Lower panels: Densitometric analysis of immunoreactive iNOS. Data are mean ± S.E.M.

Fig. 8

Myocardial COX-2 expression 24 h after ischemic PC in wild-type and IL-6^−/− mice. Myocardial samples were obtained from mice that underwent a sham operation (control) and from the ischemic/reperfused region of preconditioned (PC) wild-type and IL-6^−/− mice. Upper panel: Western blots showing a marked increase in COX-2 immunoreactivity in the membranous fraction in the ischemic/reperfused region 24 h after ischemic PC in wild-type mice. Lower panel: Densitometric analysis of COX-2 signals in the membranous fraction. In all samples, the densitometric measurements of COX-2 immunoreactivity were expressed as a percentage of the average value measured in the anterior left ventricular wall of sham-operated wild-type mice. Data are mean ± S.E.M.