Endogenous interleukin-22 prevents cardiac rupture after myocardial infarction in mice.

Myocardial infarction (MI) can result in fatal myocardial rupture or heart failure due to adverse remodeling and dysfunction of the left ventricle. Although recent studies have shown that exogenous interleukin (IL)-22 shows cardioprotective effect after MI, the pathophysiological significance of endogenous IL-22 is unknown. In this study, we investigated the role of endogenous IL-22 in a mouse model of MI. We produced MI model by permanent ligation of the left coronary artery in wild-type (WT) and IL-22 knock-out (KO) mice. The post-MI survival rate was significantly worse in IL-22KO mice than in WT mice due to a higher rate of cardiac rupture. Although IL-22KO mice exhibited a significantly greater infarct size than WT mice, there was no significant difference in left ventricular geometry or function between WT and IL-22KO mice. IL-22KO mice showed increase in infiltrating macrophages and myofibroblasts, and altered expression pattern of inflammation- and extracellular matrix (ECM)-related genes after MI. While IL-22KO mice showed no obvious changes in cardiac morphology or function before MI, expressions of matrix metalloproteinase (MMP)-2 and MMP-9 were increased, whereas that of tissue inhibitor of MMPs (TIMP)-3 was decreased in cardiac tissue. Protein expression of IL-22 receptor complex, IL-22 receptor alpha 1 (IL-22R1) and IL-10 receptor beta (IL-10RB), were increased in cardiac tissue 3 days after MI, regardless of the genotype. We propose that endogenous IL-22 plays an important role in preventing cardiac rupture after MI, possibly by regulating inflammation and ECM metabolism

Early and sustained expression of SOCS3 protein during myocardial IRI.

<p>(<b>A</b>) Total cell lysates (TCL) were prepared from the left ventricle of wild-type (WT) and SOCS3-CKO mice at the indicated times after reperfusion and were immunoprecipitated with anti-SOCS3 antibodies. The immunoprecipitates and total cell lysates were subjected to western blotting and probed with antibodies against SOCS3 and GAPDH, respectively. The graphs represent the time course expression of SOCS3 protein in WT mice after reperfusion (left) and the quantitative difference between WT and SOCS3-CKO mice 3 h after reperfusion (right; n = 5 per group). *<i>P</i> < 0.05 vs. WT pre-ischemia. §<i>P</i> < 0.05 vs. WT 3 h after reperfusion. AU indicates arbitrary units; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IRI, ischemia reperfusion injury. (<b>B</b>) Real-time PCR analysis for SOCS3 mRNA expression in mouse hearts pre-ischemia or 3 h after reperfusion (n = 5 for each group). *<i>P</i> < 0.05 vs. pre-ischemia.</p

Confirmation of myocardial-specific SOCS3 deletion in SOCS3-CKO mice.

<p>mRNA was prepared from the hearts and livers of wild-type (WT) mice 3 h after lipopolysaccharide (LPS) injection, and real-time PCR analyses for SOCS3 were performed. Values normalized to GAPDH are expressed (n = 5 per group). *<i>P</i> < 0.05 vs. WT pre-injection, §<i>P</i> < 0.05 vs. WT LPS injection.</p

Activation of cardioprotective signaling molecules in wild-type (WT) mice during myocardial IRI.

<p>Total cell lysates were prepared from the left ventricle of WT mice at the indicated times after reperfusion. Blots were probed using antibodies against tyrosine-phosphorylated STAT3 (pY-STAT3), STAT3, phosphorylated AKT (p-AKT), AKT, phosphorylated ERK1/2 (p-ERK1/2), and GAPDH. The graphs represent quantitative differences in expression between the ratios of pY-STAT3 to STAT, p-AKT to AKT, and p-ERK1/2 to ERK1/2 (n = 6 per group). *<i>P</i> < 0.05 vs. pre-ischemia, §<i>P</i> < 0.05 vs. 1 h (pY-STAT3 and p-AKT) or 0.3 h (p-ERK1/2) after reperfusion. AU, arbitrary units; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IRI, ischemia reperfusion injury.</p

(A) Sustained activation of cardioprotective signaling molecules in SOCS3-CKO mice during IRI.

<p>Western blot analysis of total cell lysates prepared from the left ventricle of WT or SOCS3-CKO mice at the indicated times after reperfusion. The blots were probed using antibodies against tyrosine-phosphorylated STAT3 (pY-STAT3), STAT3, phosphorylated AKT (p-AKT), AKT, phosphorylated ERK1/2 (p-ERK1/2), and GAPDH. The graphs represent quantitative differences in the ratios of the expression of pY-STAT3 to STAT3, p-AKT to AKT, and p-ERK1/2 to ERK1/2 (n = 6 per group). *<i>P</i> < 0.05 vs. WT mice. AU, arbitrary units; IRI, ischemia reperfusion injury. (<b>B</b>) Immunostaining of tyrosine-phosphorylated STAT3 (pY-STAT3) in hearts pre-ischemia or 3 h after reperfusion (n = 5 for each group). Representative photographs of the hearts from each group are shown. Values are expressed as the percentage of pY-STAT3–positive cells in the hearts pre-ischemia or 6 h after reperfusion. *<i>P</i> < 0.05 vs. pre-ischemia. Scale bar = 100 μm. AU = arbitrary units.</p

Echocardiography measurements in wild-type (WT) and cardiac-specific SOCS3-CKO mice during the chronic phase.

<p>Echocardiography was performed at pre-ischemia and from day 1 to day 28 after reperfusion (n = 8 per group). Pooled data for echocardiographic measurements in WT and SOCS3-CKO mice. *<i>P</i> < 0.05 vs. WT mice. FS indicates fractional shortening; LVESD, left ventricular end systolic dimension; LVEDD, left ventricular end diastolic dimension; IVST, interventricular septum thickness; PWT, posterior left ventricular wall thickness.</p

Expression of genes related to oxidative stress during myocardial IRI.

<p>mRNA was prepared from the left ventricles of WT or SOCS3-CKO mice after reperfusion and subjected to real-time PCR analysis regarding the indicated genes. Values normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) are expressed as fold change from the values of sham mice (n = 5 for each group). *<i>P</i><0.05 versus WT sham. Gpx = glutathione peroxidase; Prdx = peroxiredoxin; HO = heme oxygenase; Sod = superoxide dismutase.</p

Reduction of apoptosis and mitochondrial cytochrome c release in SOCS3-CKO mice during IRI.

<p>(<b>A</b>) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays of hearts from wild-type (WT) or SOCS3-CKO mice 6 h after reperfusion (n = 5 per group). Representative images of the ischemic areas of hearts from each group. The graph shows the percentage of TUNEL-positive cells. *<i>P</i> < 0.05 vs. WT sham, §<i>P</i> < 0.05 vs. WT IRI. (<b>B</b>) The cytosolic (Cyto) and mitochondrial (Mito) fractions were prepared from the left ventricles of WT or SOCS3-CKO mice 6 h after reperfusion and subjected to western blotting using an antibody against cytochrome c. The cytosolic marker α-tubulin and the mitochondrial marker VDAC served as internal controls for WT mice (n = 6 per group). The graph represents quantitative differences in cytochrome c expression. *<i>P</i> < 0.05 vs. WT sham, §<i>P</i> < 0.05 vs. WT IRI. AU, arbitrary units; IRI, ischemia reperfusion injury. (<b>C</b>) Total cell lysates were prepared from the left ventricles of wild-type (WT) or SOCS3-CKO mice pre-ischemia or 6 h after reperfusion, and western blots were probed with antibodies raised against cleaved caspase 8, cleaved caspase 3, and serine-phosphorylated STAT3 (pS-STAT3). The graphs represent quantitative differences in expression among cleaved caspase 8, cleaved caspase 3, and PS-STAT3 (n = 5 per group). §<i>P</i> < 0.05 vs. WT pre-ischemia, *<i>P</i> < 0.05 vs. WT 6 h after IRI. AU, arbitrary units; IRI, ischemia reperfusion injury.</p

Reduced infarct size and preserved left ventricular systolic function after IRI in SOCS3-CKO mice.

<p>(<b>A</b>) Representative images of Evans blue and triphenyltetrazolium chloride (TTC) staining in wild-type (WT) and SOCS3-CKO mice 24 h after reperfusion (top) (n = 8 per group). The infarct size of the left ventricle (LV) was expressed as a percentage of the area at risk (AAR) of each group (left). The graphs show quantification of AAR/LV and infarct area/AAR. *<i>P</i> < 0.05 vs. WT mice. (<b>B</b>) Echocardiography was performed pre-ischemia and 24 h after reperfusion (n = 8 per group). Pooled data for echocardiographic measurements in WT and SOCS3-CKO mice. *<i>P</i> < 0.05 vs. WT mice. FS indicates fractional shortening; LVESD, left ventricular end systolic dimension; IRI, ischemia reperfusion injury.</p