The effect of interleukin 6 deficiency on myocardial signal transduction pathways activation induced by bacterial lipopolysaccharide in young and old mice.

PURPOSE: Exaggerated release of proinflammatory mediators during sepsis contributes to inadequate vasodilatation and depressed myocardial contractility, which lead to development of shock and circulatory collapse. The aim of the study was to evaluate the effect of IL-6 and aging on activation of intracellular signaling pathways in the myocardium induced by bacterial lipopolysaccharide (LPS) administration. MATERIAL/METHODS: LPS was injected intraperitoneally to male 3- and 24-month old mice with systemic IL-6 gene knock-out (IL-6KO) and the reference strain (WT). LPS was given intraperitoneally in single low (0.1 mg/kg) or high (10 mg/kg) dose, or in two doses (0.1 + 10 mg/kg) with 24-h delay. The expression and phosphorylation of STAT3, ERK1/2, Akt1/2/3 proteins in the left ventricular myocardium was evaluated after 24 h using Western blotting. RESULTS: Low LPS dose induced higher STAT3 phosphorylation only in old IL-6KO mice, not affecting ERK1/2 and Akt1/2/3 phosphorylation in any group. High LPS dose upregulated STAT3 phosphorylation similarly in all groups, reduced ERK1/2 expression in young WT mice and upregulated Akt1/2/3 expression and phosphorylation in young IL-6KO mice. Pretreatment with low LPS dose attenuated phosphorylation of STAT3 in both old groups and phosphorylation of Akt1/2/3 in young IL-6KO group. Two-dose approach also significantly potentiated ERK1/2 phosphorylation in both old groups. CONCLUSIONS: Obtained results show that IL-6 deficiency alters the activity of intracellular signaling pathways: JAK/STAT in old and Akt in young LPS-treated mice. This may indicate that lack of IL-6 attenuates Akt-related cytoprotective effect of pretreatment with low LPS dose in young but not in aged animals.

The role of interleukin-6 in intracellular signal transduction after chronic ß-adrenergic stimulation in mouse myocardium.

INTRODUCTION: Inflammatory mediators play an important role in development and progression of cardiovascular disease. Both adrenergic stimulation and high levels of interleukin-6 (IL-6) indicate an unfavorable outcome in patients with myocardial infarction or heart failure. Understanding the interaction between ß-adrenergic stimulation and IL-6 in the myocardium may contribute to developing more effective treatments. The aim of this study was to verify the role of IL-6 in the effects of ß-adrenergic stimulation in activating selected intracellular signaling pathways in mouse myocardium. MATERIAL AND METHODS: Experiments were performed on 12-week-old male mice: 16 C57BL/6JIL6­/­TMKopf (IL-6 KO) and 17 C57BL/6J (WT). Animals received intraperitoneal injections of isoproterenol (ISO, 50 mg/kg) or placebo (0.9% NaCl) once a day for 16 days. The phosphorylation of STAT3 (signal transducer and activator of transcription 3), ERK1/2 (extracellular-regulated kinases 1/2), Akt1/2/3, p-38, c-Raf and expression of SOCS3 (suppressor of cytokine signaling 3), PIAS1/3 (protein inhibitors of activated STAT) was assessed by western blotting in the myocardium 24 h after the last injection. Evaluation of gene expression downstream of these pathways was performed by real-time PCR. RESULTS: Chronic ISO treatment leads to increased fibrosis of the myocardium in mice lacking IL-6, which is accompanied by increased activity of ERK1/2, p38 and reduced expression of SOCS3. Administration of ISO in IL-6 KO animals intensified gene expression of proteins activated by MAPK/ERK (myc; CEBPB; BMP4; Fasn; Tank), while it reduced expression of genes repressed by ERK 1/2 (Wisp1, Wnt1). CONCLUSIONS: IL-6 plays an important role in regulating the activation of MAPK pathways in the mouse myocardium in response to chronic ß-adrenergic stimulation.

Interleukin 6 Knockout Inhibits Aging-Related Accumulation of p53 in the Mouse Myocardium.

Interleukin 6 (IL6) and p53 are linked by mutual regulatory mechanisms and are both upregulated in aging. The aim of this study was to evaluate the effects of aging and IL6 on expression of p53 in the mouse heart. Male C57BL6/J wild-type and IL6 knockout mice at the age of 4-5 months (young adult) and 24-30 months (old) were used. Myocardial expression of proteins such as p53, p21, Mdm2, and phospho-Akt/Akt was estimated using Western blotting and expression of p53 and p21 mRNA using real-time polymerase chain reaction. Expression of p53 protein was lower in IL6 knockout hearts than in wild-type hearts. Aging caused significant upregulation of p53 protein level; however, it was significantly higher in old wild-type hearts than in old IL6 knockout hearts (p < .05). Similar p53 mRNA levels in all groups implied IL6 influence on age-related proteasomal degradation of p53. Localization of p53 mainly in the extranuclear compartment and lack of p21 upregulation in aged hearts may suggest quenched transcriptional activity of p53 despite increased abundance of p53. We conclude that lack of IL6 attenuates expression of p53 protein in the hearts of young mice and diminishes its accumulation with aging by post-transcriptional mechanisms; however, this is not related to altered phenotype of aging heart.

Interleukin-6 Affects Aging-Related Changes of the PPARα-PGC-1α Axis in the Myocardium.

Aging is related to gradual increase of interleukin 6 (IL-6) plasma level that affects peroxisome proliferator-activated receptor (PPAR) expression. We evaluated age-related changes in cardiac expression of PPARα, its coactivator PGC-1α, and selected downstream proteins in mice with systemic IL-6 knockout (IL6KO). Male C57BL6/J wild-type (WT) and IL6KO mice were used at the age of 16-20 weeks (young) and 24-30 months (senescent). Echocardiography and electron microscopy were applied to assess the function and ultrastructure of the heart. Western blotting and quantitative real-time PCR were used to estimate protein and mRNA levels of selected genes. PPARα expression in the myocardium of young IL6KO animals is lower and remains unchanged with aging, whereas in WT mice it declines with age and in both senescent groups it is equal. We observed aging-related upregulation of PGC-1α and less pronounced decline of Sirt3 in IL6KO animals; the level of cytochrome C was significantly decreased in IL6KO group only, suggesting disturbed mitochondrial function, which was not sufficient to evoke obvious changes in cardiac performance and function assessed by echocardiography. IL-6 and aging are involved in regulation of PPARα and PGC-1α expression and may influence the mitochondrial function.

Interleukin 6 modulates PPARα and PGC-1α and is involved in high-fat diet induced cardiac lipotoxicity in mouse.

BACKGROUND: Interleukin 6 (IL-6) may be involved in regulation of cardiac lipid metabolism and mitochondrial function through its influence on peroxisome proliferator-activated receptors (PPARs). In this study we evaluated the impact of the physiological level of IL-6 on the expression of PPARα and PGC-1α in the heart and the effect of lack of this cytokine on high-fat diet (HFD) induced lipotoxicity. METHODS: Male C57BL6/J wild type (WT) and IL-6 knock-out (IL-6KO) mice were used. 20 animals of each genotype were fed with HFD for 15-18weeks. Cardiac function was assessed using echocardiography and cardiomyocyte ultrastructure was examined using electron microscopy. QT-PCR and Western blotting were applied to estimate the expression of PPARα and PGC-1α at the transcriptional and protein levels. RESULTS: At baseline WT and IL-6KO mice had similar size and function of the left ventricle. HFD induced similar left ventricular hypertrophic response in both groups without causing heart failure, but only WT animals had increased resting ejection fraction of the LV. Ultrastructure of HFD groups showed markers of lipotoxicity, that were more pronounced in IL-6KO group. In basal conditions IL-6KO animals had lower PPARα and similar PGC-1α expression as compared to WT. HFD induced downregulation of both PPARα and PGC-1α in WT animals, while in IL-6KO mice this effect was constrained. CONCLUSION: IL-6 is involved in basal regulation of PPARα and PGC-1α expression in cardiomyocytes. The lack of this cytokine promotes high-fat diet induced lipotoxicity but without overt manifestations of cardiac failure.

Remodeling of the intercalated disc related to aging in the mouse heart.

BACKGROUND: Aging is related to declined cardiac hemodynamic function. As pumping performance may be significantly related to slowed ventricular depolarization and non-synchronous contraction, we hypothesized that aging may cause dysfunction of intercalated disc (ID), which is the structure responsible for intercellular electrical communication between cardiomyocytes. METHODS: Male C57BL/6J mice were used for the study at two ages: 4 and 24 months. Electrocardiographic recording was made to analyze the time of ventricular depolarization. Then mice were killed, and the hearts were harvested for examination in transmission electron microscopy (TEM) and immunofluorescence imaging. The expression of connexin 43 (Cx43), N-cadherin, and ß-catenin in the myocardium of the left ventricle was evaluated using Western blotting. RESULTS: In senescent mice, analysis of averaged QRS complex showed its significant prolongation. At the ultrastructural level, we found frequent disruptions of the ID (affecting 29±5% of them), mainly at the site of adherens junction, with relatively preserved desmosomal intercellular connections and diminished number of gap junctions. Western blotting revealed significantly decreased abundance of Cx43 protein in aged animals, which may cause slowed impulse propagation through the gap junctions and contribute to the observed electrocardiographic alterations. The level of RNA for Cx43 is similar between young and old animals, which suggests a post-transcriptional mechanism of Cx43 protein downregulation. CONCLUSIONS: Our study shows age-related disorganization of ID, which may be responsible for slowed conduction of the depolarization wave within the heart, and supports the hypothesis of cardiac dysfunction in senescence.

CCN1 expression in interleukin-6 deficient mouse kidney in experimental model of heart failure.

Chronic heart failure often leads to worsening of the renal function. Mediators of this process include inflammatory and neuroendocrine factors. CCN1 (Cyr 61), a member of growth factor-inducible immediate early genes, which modulates inflammation and fibrogenesis, is excreted with urine in the early phase of acute renal injury and may be involved in the pathogenesis of the cardiorenal syndrome. The aim of the study was to evaluate CCN1 protein abundance and localization in the kidney of IL-6-deficient C57BL/6J (IL-6 KO) mice and respective wild-type (WT) animals in basal conditions and in animals with chronic heart failure twelve weeks after myocardial infarction. Age- and sex-matched mice from both strains subjected to sham operation served as controls. One group of WT animals subjected to myocardial infarction was treated with antagonist of AT1 receptor telmisartan over 12 weeks. Abundance and localization of CCN1 protein in kidney were assessed with Western blotting and immunohistochemistry, respectively. In all groups the strongest immunohistochemical reaction for CCN1 was observed in distal convoluted tubules and in smaller arteries, however, the total expression of CCN1 protein was lower in IL-6 KO mice in comparison to WT animals. The main difference in CCN1 distribution between the examined genotypes was lack of reaction in internal renal medulla and very weak reaction in proximal convoluted tubules in IL-6 KO mice. Experimental heart failure only slightly attenuated the expression of CCN1 protein in the kidney of WT mice and had no effect in IL-6 KO mice. Although, blockade of AT1 receptor did not alter CCN1 protein expression in kidneys of WT mice after myocardial infarction, it significantly changed its CCN1 distribution in the renal tubular system.

Atrial expression of the CCN1 and CCN2 proteins in chronic heart failure.

Previous studies have reported the upregulation of CCN proteins early after acute heart injury. The aim of the present work was to evaluate the expression of the CCN1 and CCN2 proteins and their regulation by angiotensin II in the atrial myocardium of a chronically failing heart. Male adult mice were subjected to ligation of the left coronary artery to produce myocardial infarction (the MI group), and 16 of them were treated for 12 weeks with the AT1 receptor antagonist telmisartan (the MI-Tel group). Sham-operated mice served as controls. The expression of proteins was evaluated by immunohistochemistry 12 weeks after the operation. In shamoperated mice, stainings for CCN1 and CCN2 proteins were positive within atrial cardiomyocytes. CCN1-positive reaction revealed diffused cytoplasmic localization, while CCN2 was present mainly within the perinuclear cytoplasm. CCN1 was upregulated in the MI group, while CCN2 remained at basal level. Telmisartan prevented the upregulation of CCN1 and decreased CCN2 level. We compared the experimental data with the expression of CCN1 and CCN2 proteins in human right atrial appendages. We found an inverse, but not significant, relation between the level of either protein and the left ventricular ejection fraction. This suggests a similar atrial regulation of CCN1 and CCN2 expression also in humans. We conclude that in the murine atria, CCN1 and CCN2 proteins are expressed constitutively. In chronic heart failure, CCN proteins tend to be upregulated, which may be related to the action of angiotensin II.