Detrimental Effect Of Fractalkine On Both Myocardial Ischemia And Ischemic Heart Failure In Mice

Background and Objective:Fractalkine (FKN, also called CX3CL1) is a newly identified membrane-bound chemokine that is mainly expressed on endothelial cells. Emerging evidence suggests that FKN is involved in the pathogenesis of various inflammatory diseases, such as atherosclerosis, glomerulonephritis, rheumatoid arthritis, cerebral ischemia, renal failure, coronary artery disease. Inhibition of FKN has been shown to have a favorable impact on inflammatory diseases state. It has been reported that mice deficient in FKN were less susceptible to cerebral ischemic-reperfusion injury. High dose aspirin can suppress the fractalkine expression in murine aorta and improve the atherosclerotic lesion; atherosclerotic lesion formation was decreased in FKN receptor/apolipoprotein E double knockout mice. It has also been demonstrated that some agents can suppress the expression of FKN in vivo or in vitro, such as aspirin, pentoxifylline (PTX), Epigallocatechin-3-O-gallate (EGCG) and resveratrol (RES). However, the role of FKN on myocardial ischemia and ischemic heart failure (HF) was largely unknown. To our knowledge, except a recent study demonstrating that both the myocardium and serum levels of FKN were increased in mice or patients with CHF and FKN itself induced upregulation of hypertrophy and heart failure-related genes, no any else studies were designed to investigate the potential roles of FKN on the pathogenesis of myocardial ischemia and chronic heart failure (CHF).We hypothesized that s-FKN had detrimental effects on cardiac cells and inhibition of s-FKN could be a novel therapeutic approach for myocardial ischemia and chronic heart failure. In this study, we investigated (a):whether the expression of FKN was associated with the development of HF; (b):whether s-FKN had any influences on cardiomyocytes, fibroblasts and ECs under physiological and hypoxemic or ischemic states; (c) whether PTX and RES can inhibit the role of FKN; and (d) whether inhibition of FKN exerts beneficial effects on myocardial ischemia and ischemic HF in mice.Methods:1. cDNA microarray analysisC57BL/6 male mice (aged 8 weeks, weighed 20-25 g) were subjected to either transverse aortic constriction (TAC) or sham operation.8 weeks after surgery, we performed cDNA microarray experiments using the whole hearts of mice showing different degree of HF.2. Cell culture and treatmentThe isolation and culturing of neonatal rat ventricular cardiomyocytes, fibroblasts and cardiac microvascular ECs was performed. In FKN groups, cells were treated with various concentrations (100,150 and 200 ng/ml, respectively) of recombinant mouse s-FKN (chemokine domain, R&D) for 24 hours, while in drug-treatment groups, pentoxifylline (PTX,1 mg/ml dissolved in phosphate buffer solution (PBS)) or resveratrol (RES,25μM dissolved in dimethyl sulfoxide (DMSO)) were added to the cells one hour before stimulation with FKN (100 ng/ml). We designed there control groups including one untreated group and two vehicle groups (treated with PBS and DMSO, respectively). Cells were harvested after 24 h stimulation and analyzed for ANP, MMP-9 and ICAM-1 mRNA expression. 3. Stimulation of cells with anoxia/reoxygenation and oxidative stressThe effects of FKN on cardiomyocytes in the setting of anoxia/reoxygenation (A/R) and H2O2 induced oxidative stress model were also analyzed. The neonatal rat cardiomyocytes were cultured in 96-well plates at 1×105 cells/well and the experimental groups were designed as follows:(1) A/R group:cardiomyocytes were incubated with anaerobic simulated ischemia buffer for 3 h of anoxia followed by reoxygenation for 2 h; (2) A/R+s-FKN groups (100,150 and 200 ng/ml):at the end of A/R, different concentrations of s-FKN were added to the cardiomyocytes and incubated for 12 h; (3) A/R+RES+s-FKN:after A/R, RES (25μM) was added 1 h before FKN (100 ng/ml) addition. Oxidative stress was induced by exposure of cells to H2O2 (100μM) for 6 h. After 6 h, the cardiomyocytes were treated with s-FKN (100,150 and 200 ng/ml) for 12 h with/without pre-treatment using 25μM RES 1 h before FKN (100 ng/ml) addition. Viability of cardiomyocytes was determined by methyl thiazoyltetrazolium (MTT) assay according to the manufacture's instruction.4. Animal model of myocardial infarction and heart failureC57BL/6 male mice (aged 11-12 weeks, weighed 23-30 g) were intraperitoneally anesthetized with pentobarbital (50 mg/kg) and subjected to a left-sided thoracotomy and the left coronary artery (LCA) ligation to induce myocardial infarction with subsequent development of HF. The operated mice that survived for 12 hours were randomized to treatment with resveratrol (RES,20 mg/kg/d intraperitioneally injection) for 42 days or an intraperitoneal injection of propylene glycol (vehicle) alone. Sham operated mice were also given the treatment of vehicle or RES. Cardiac function and remodeling was dynamically evaluated with echocardiography at 7,14, 30,42 days. From M-mode tracing, LV end-diastolic diameter (LVEDd), LV end-systolic diameter (LVESd) and LVFS were measured. LVFS-(LVEDd-LVESd) /LVEDd×100. Hearts for protein analysis were stored at -80℃, while for Immunohistochemistry hearts were fixed in 4% paraformaldehyde and embedded in paraffin 4-6μm sections.5. Preparation of mouse heart Langendorff modelC57BL/6 male mice aged 11-12 weeks were anesthetized with pentobarbital (50 mg/kg, ip), after cannulating the ascending aorta, the hearts were quickly excised and perfused with Krebs-Henseleit (K-H) buffer in a Langendorff apparatus. Hearts were subjected to 20 min perfusion for stabilization, and then exposed to 40 min global ischemia followed by reperfusion with K-H buffer. For for ANP, MMP-9 and ICAM-1 mRNA analysis, four experimental groups were divided as follows:(1) control group (perfusion with K-H buffer for 220 min), (2) IR group (ischemia 40 min, reperfusion with K-H buffer for 180 min), (3) IR+FKN group (ischemia 40 min, reperfusion with K-H buffer and s-FKN 50 ng/ml for 180 min),(4) IR+RES+FKN group (ischemia 40 min, reperfusion with K-H buffer and RES 25μmol/L and s-FKN 50 ng/ml for 180 min).Hearts for RNA analysis were stored in RNA latter liquid. Five experimental groups were divided for measurement of the infarct size with triphenyl tetrazolium chloride (TTC) staining:(1) control group (perfusion with K-H buffer for 100 min), (2) IR group (ischemia 40 min, reperfusion with K-H buffer for 60 min), (3) IR+FKN group (ischemia 40 min, reperfusion with K-H buffer and s-FKN 50 ng/ml for 60 min), (4) IR+RES group (ischemia 40 min, reperfusion with K-H buffer and RES 25μmol/L for 60min), and (5) IR+RES+FKN group (ischemia 40 min, reperfusion with K-H buffer and RES 25μmol/L and s-FKN 50 ng/ml for 60 min).Results:1. Relations between FKN expression and severity of HFMicroarray analysis showed that FKN gene expression was markedly increased in the failing hearts of mice. FKN expression level was positively correlated with lung weight to body weight ratio (LW/BW) and BNP expression level respectively and negatively correlated with LVFS (P< 0.05)2. Effects of s-FKN stimulation on cardiac cellsIn cultured cells, soluble FKN stimulation for 24 h increased atrial natriuretic peptide (ANP) expression in cardiomyocytes, matrix metalloproteinase-9 (MMP-9) in fibroblast, and intercellular adhesion molecule-1 (ICAM-1) expression in microvascular endothelial cells (P<0.05). The up-regulation of those genes was suppressed by co-cultured with pentoxifylline or resveratrol (P<0.05)3. Effects of s-FKN on viability of cardiomyocytesThe MTT assay showed that low concentration of FKN did not significantly reduced the cell viability in the setting of physiological state. However, during pathological stresses, addition of s-FKN further suppressed the viability of cardiomyocytes. A/R and H2O2 stimulations reduced cell viability by 25% and 43%, respectively. Addition of 100-200 ng/ml of s-FKN further reduced the viability (P< 0.05), whereas pretreatment with RES abrogated the damage effect of s-FKN.4. RES improves the survival of HF miceThe treatment with RES increased the accumulated survival rate of mice with MI from about 55% to about 80%(Log-rank test, P=0.022).5. RES attenuates cardiac remodeling and improves HFAt 42 days post-MI, both heart weight to body weight ratio (HW/BW) and LW/BW were significantly lower in RES-treated MI mice than in untreated MI mice (P< 0.001).The fibrosis area of LV in the RES-treated mice was markedly attenuated than in untreated MI mice (P<0.01). Serial echocardiographic examinations in untreated MI mice showed a time-dependently increase of LVEDd and LVESd, and a progressively depression of LVFS over a course of six weeks. In contrast, RES-treated mice had significant higher LVFS in the corresponding time points (P< 0.05) 6. RES suppresses the upregulation of ANP, MMP-9, ICAM-land FKNIn comparison with sham group, the cardiac expression of ANP, MMP-9, ICAM-1 genes, and ICAM-1 immunostaining in the vascular endothelia were significantly increased in MI mice, while treatment with RES abrogated those expressional changes. By immunohistochemistry test, we found stronger FKN immunoreactivity in both myocardium and vascular endothelia in post-MI mice than in RES-treated MI mice. Western blot analysis also demonstrated that cardiac expression levels of FKN were significantly higher than in RES-treated MI group (P<0.01).7. Effects of s-FKN stimulation on acute ischemia in ex vivo heartIn a mouse Langendorff model,3 hours reperfusion with FKN after 40min global ischemia, we found that ANP, MMP-9 and ICAM-1 expression were significantly up-regulated compared to non-ischemic group and FKN-untreated ischemia/ reperfusion group (P<0.01). Interestingly, co-treatment with RES inhibited the upregulation of these genes. Thus we addressed this issue in a mouse Langendorff model. After 40min global ischemia and 60 min reperfusion, we noted that MI size was increased by about 20% in IR+FKN group comparing to the IR group (68.3±1.06% vs.48.7±3.01%, P<0.001). Treatment with RES reduced the infarct size by about 30% and 20% in IR+FKN and IR group, respectively (68.3±1.06% vs.37.5±3.37%; 48.7±3.01% vs.30.6±2.03%).Conclusions:1. Enhanced cardiac FKN expression was associated with the disease severity of HF, FKN may be a biomarker of HF.2. FKN suppressed cardiomyocytes viability, promoted cardiac fibrosis, activated ECs and increased MI size, indicating detrimental effects of FKN on myocardial ischemia and ischemic HF.3. Inhibition of FKN significantly suppressed those detrimental effects and was showed to attenuate cardiac remodeling and increase the survival of CHF mice, and suggesting that FKN inhibition might be a novel therapeutic target for myocardial ischemia and heart failure.