Intracellular Function of Interleukin-1 Receptor Antagonist in Ischemic Cardiomyocytes

Background: Loss of cardiac myocytes due to apoptosis is a relevant feature of ischemic heart disease. It has been described in infarct and peri-infarct regions of the myocardium in coronary syndromes and in ischemia-linked heart remodeling. Previous studies have provided protection against ischemia-induced cardiomyocyte apoptosis by the anti-inflammatory cytokine interleukin-1 receptor-antagonist (IL-1Ra). Mitochondria triggering of caspases plays a central role in ischemia-induced apoptosis. We examined the production of IL-1Ra in the ischemic heart and, based on dual intra/extracellular function of some other interleukins, we hypothesized that IL-1Ra may also directly inhibit mitochondria-activated caspases and cardiomyocyte apoptosis. Methodology/Principal Findings: Synthesis of IL-1Ra was evidenced in the hearts explanted from patients with ischemic heart disease. In the mouse ischemic heart and in a mouse cardiomyocyte cell line exposed to long-lasting hypoxia, IL-1Ra bound and inhibited mitochondria-activated caspases, whereas inhibition of caspase activation was not observed in the heart of mice lacking IL-1Ra (Il-1ra−/−) or in siRNA to IL-1Ra-interfered cells. An impressive 6-fold increase of hypoxia-induced apoptosis was observed in cells lacking IL-1Ra. IL-1Ra down-regulated cells were not protected against caspase activation and apoptosis by knocking down of the IL-1 receptor, confirming the intracellular, receptor-independent, anti-apoptotic function of IL-1Ra. Notably, the inhibitory effect of IL-1Ra was not influenced by enduring ischemic conditions in which previously described physiologic inhibitors of apoptosis are neutralized. Conclusions/Significance: These observations point to intracellular IL-1Ra as a critical mechanism of the cell self-protection against ischemia-induced apoptosis and suggest that this cytokine plays an important role in the remodeling of heart by promoting survival of cardiomyocytes in the ischemic regions

IL-1Ra protects cardiomyocytes from ischemia-induced apoptosis.

<p>(a) Hystochemistry of IL-1Ra expression (purple) in the heart following coronary artery ligation in mice: ventricle cross section, and (b) specific, diffuse IL-1Ra staining of cardiomyocytes in the ischemic heart area. (c) Time course of secreted (s) and intracellular (ic) IL-1Ra mRNA expression in the hypoxic heart of WT (Il-1ra+/+) mice. The graphs represent the fold change after normalization with the expression of β-actin. (d) Histology of TUNEL staining (red stain) of Il-1ra+/+ and (e) Il-1ra−/− mouse hearts after 6 hr hypoxia, and of (f) Il-1ra+/+ and (g) Il-1ra−/− mouse hearts not exposed to hypoxia. (h) Rate of TUNEL staining in d-g conditions. Results are means ± SE, n = 3, **p<0.001 for Il-1ra−/− vs control Il-1ra+/+ mouse hearts after 6 hr hypoxia, *p<0.001 for Il-1ra+/+ mouse hearts after 6 hr hypoxia vs hearts not exposed to hypoxia. Bars, a 2 mm, b 20 um; d, e, g, h 40 um.</p

Expression of IL-1Ra in hearts explanted from patients with end stage ischemic heart disease.

<p>(a) Immunofluorescence co-staining for IL-1Ra and PECAM-1. Several cardiomyocytes show positive staining for IL-1Ra (green), whereas PECAM-1 positive (red) endothelial cells of myocardial microvessels do not co-stain with IL-1Ra. Cell nuclei are evidenced by DAPI (blue) stain. (b) Co-staining of IL-1Ra (brown) and fibroblast specific vimentin (red), and (c) of IL-1Ra (brown) and leukocyte/macrophage specific CD14 (red). Nuclei are lightly counterstained by Mayer’s Hematoxylin. (d) In situ hybridization for IL-1Ra mRNA. Several cardiomyocytes stained positive for the in situ hybridization in a large area of peri-infarct scar viable myocardium. The inset shows how in situ hybridization is localized mainly in perinuclear areas within cardiomyocytes. (e) Co-staining for IL-1Ra and active caspase3 in a peri-infarct scar area. Besides IL-1Ra positive cardiomyocytes (brown) there are several caspase3-positive cells (red). Bars: a–c, e 20 um, d 40 um, insets 10 um. (f) qRT-PCR analyses of sIL-1Ra and icIL-1Ra (type-1, and type-3) mRNA in ischemic cardiomyopathy, corrected for mRNA expression of β-actin. The graph compares heart regions with macroscopic features of normal blood supply and trophism (remote) to heart areas close to post infarct scars (peri infarct-scar) and regions 1 cm away from the scars (intermediate). The bars show mean ± SE of five experiments.</p

Coimmunoprecipitation of IL-1Ra with mitochondria-activated caspases.

<p>(a) Coimmunoprecipitation of IL-1Ra with caspase-9 and (b) with caspase-3, -6, and -7 in cultured HL-1 cardiomyocytes after 6 hr hypoxia. Detection by western blot with monoclonal Abs to caspases or to IL-1Ra, or to control proteins IL-1beta, IL-1 type I receptor (IL-1R1) and IL-1R Ancillary Protein (IL-1R AcP). Proteins immunoprecipitated (IP) by Abs to caspases or to IL-1Ra, or to IL-1beta (control) are compared to unbound (free) supernatant proteins. The data are compiled from different gels in three separate experiments; [ ] not detected.</p

IL-1R1-independent anti-apoptotic function of IL-1Ra.

<p>(a) Immunofluorescence of IL-1Ra (green) and nuclear DAPI (blue) staining of cultured mouse cardiomyocytes (HL-1 cells) incubated for 6 hr in normoxia (panel i), or hypoxia (95%N₂-5%C0₂ panel ii) conditions, and (b) rate of IL-1Ra positive cells (%) in fig. a conditions. (c) Double-immunofluorescence for IL-1Ra and IL-1R1 (both green, panel i), or (d) for IL-1Ra (green, panel i), or (e) for IL-1R1 (green, panel i), or (f) for IL-1Ra and IL-1R1 (both green, panel i), respectively, together with TUNEL co-staining (red, panel ii) in the same field (merge, panel iii) of cultured cardiomyocytes treated with siRNA to both IL-1Ra and IL-1R1, or to IL-1Ra alone, or to IL-1R1 alone, or with control siRNA, respectively, and exposed to 6 hr hypoxia. Bars, 20 um. (g) Rate of TUNEL positive cells (%) in fig. c-f conditions. Results are means ± SE, and were obtained using three siRNA probes to IL-1Ra. n = 8. *p<0.001 vs controls. (h) Western blot detection of IL-1Ra and IL-1R1 protein expression in fig. c-f conditions. (i) RTqPCR analysis of IL-6 mRNA expression in HL-1 cardiomyocytes treated with siRNA to both IL-1Ra and IL-1R1, or control siRNA, and cultured for 5 hr in the presence or absence of IL-1 beta ((40 pg/ml) or TNF alpha (10 ng/ml), corrected for mRNA expression of beta-actin. The results confirm down regulation of the IL-1 receptor (IL-R1) in siRNA-treated HL-1 cells. The bars show mean ± SE of four experiments; *p<0.001 vs activity of TNF alpha-treated controls.</p

In vitro activity of terminal caspases in cardiomyocytes lacking IL-1Ra.

<p>Activity of mitochondria-activated terminal caspases in cytosols of cultured HL-1 cardiomyocytes untreated or treated with siRNA to IL-1Ra RNA, or both IL-1Ra and IL-1R1 RNAs, and then incubated for 6 hr in normoxia or hypoxia conditions. Ac-DEVD-AMC assays compare enzyme activity in the absence (controls) or presence of anti-IL-1Ra Abs. Bars show means ± SE of 3 exp.; *p<0.01 vs activity of controls.</p

Hypothetical model of IL-1Ra in the inhibition of apoptosis.

<p>Smac released from ischemia-induced mitochondria which inhibits the neutralizing effect of IAPs on caspases. Ischemia-induced IL1ra interacts with caspase-9 and blocks cell death.</p