| Background and Objective:Cardiac rupture is a fatal complication of acute myocardial infarction (AMI) and accounts for10-20%of all in-hospital deaths due to AMI. Successful reperfusion therapy may significantly reduce the incidence of cardiac rupture, however thrombolytic therapy appears to increase the risk of early rupture. Therefore, searching the effective preventive approach for cardiac rupture is of important clinical significance. Findings from studies in the last decade have clearly indicated that post-infarct myocardial inflammation, extracellular matrix(ECM) injury and infarct healing play important roles in the development of cardiac rupture. Thus it is plausible to speculate that intervention into the above pathophysiologic process could be a preventive target for cardiac rupture.MI evokes intense inflammatory responses both systemically and within the infarct myocardium. Inflammatory responses are essential for post MI healing and scar formation. However, excessive inflammatory responses and ECM remodeling would contribute to adverse consequences, such as post-infarct cardiac rupture, expansive ventricular remodeling and heart failure. As we discussed (vide supra), regional inflammation is a key feature of the infarcted myocardium characterized by infiltration of monocytes, neutrophils and macrophages. Clinical studies have observed more severe inflammation in hearts of patients who developed rupture than non-rupture patients with acute MI. Some groups have reported that the risk of rupture is correlated with the amplitude of inflammatory cell infiltration, particularly of macrophages. These constitute the major inflammatory cell population in the infarct myocardium at the peak time of rupture occurrence and are found to be significantly higher in rupture-prone hearts. Further, a greater inflammatory infiltration is always associated with upregulated expression and production of pro-inflammatory cytokines and mediators, and enhanced expression and activities of MMPs (including MMP-2,-8,-9and-13subtypes), indicating a close link between regional inflammation and ECM degradation.Fractalkine (FKN, also called CX3CL1) is a chemokine that is reported to involve in the pathogenesis of coronary artery disease and heart failure. FKN-deficient mice are less susceptible to cerebral ischemia-reperfusion injury, while specific mutations of the CX3CR1gene (FKN receptor) are associated with a reduced risk of future coronary events. We have reported that soluble FKN up-regulates intercellular adhesion molecular-1(ICAM-1) and matrix metallopeptidase9(MMP-9)in cardiomyocytes, while resveratrol antagonizes the detrimental effect of FKN and reduces the incidence of cardiac rupture after MI. ICAM-1participates in the inflammatory process, while MMP-9accelerates the degradation of ECM, both of which could have an influence on the occurrence of post-infarct cardiac rupture, but it remains unclear whether direct inhibition of FKN can reduce cardiac rupture after AMI.Another important factor influencing cardiac rupture is the tensile strength. Fibrillar collagen forms a3-dimensional network providing tensile strength to the myocardium and preserving the alignment of adjoining myocytes. Although lacking quantitative measurement, in patients with non-fatal rupture and undergoing emergency cardiac surgery, it was found that the infarct myocardium surrounding the rupture site was found to be fragile and could be easily removed using blunt forceps. Furthermore, closure of the rupture slit by direct stitch was associated with a high incidence of second rupture further. These findings indicate a poor mechanical strength of the infarct myocardium. Earlier studies examined the tensile strength of the infarct myocardium prepared from different species including rabbits, dogs and sheep, and reported no difference versus that of normal heart or non-infarct myocardium. We also observed in the infarct rat heart a well-maintained tensile strength at day4after MI indicated by unchanged tension-to-rupture (TTR). These findings are in keeping with the lack of rupture in all these species. In contrast, a significant reduction in the tensile strength was observed in the infarct myocardium from mice. Importantly, reduced tensile strength in the mouse myocardium is detectable as early as24h post MI, proceeding the time of onset of rupture. At the peak time of rupture (days3-4), the tensile strength of the infarct wall was reduced by about60%. Adherens junction protein aE-catenin is essential to maintain the tensile strength. It was found that a reduced expression and defective localization of aE-catenin occur in the intercalated disc region in patients dying of infarct rupture, indicating an important role of aE-catenin in preventing cardiac rupture. Moreover, inhibition of Rho kinases (ROCK) was reported to increase the levels of E-cadherin and a-catenin and stimulated formation of adherens junctions, while FKN was reported to induce significant cytoskeletal rearrangements, implying that FKN is able to stimulate ROCK. Thus we hypothesized that FKN activation would promote the development of cardiac rupture by down-regulating aE-catenin through stimulating RhoA/ROCK pathway.In this study, we investigated:(i) whether deletion of CX3CR1could improve the survival of mice in response to AMI;(ii) the involvement of aE-catenin, ICAM-1, MMP9· in wild type and CX3CR1-/-mice receiving AMI and in cultured cardiomyocytes subjected to anoxia; and (iii) whether activation of RhoA/ROCK pathway is the underlying mechanism for FKN down-regulating aE-catenin.Methods:1. Animal Model of Myocardial Infarction.CX3CR1+/-male mice were obtained from Jackson laboratory. CX3CR1-/-(knockout mice, KO) and wild type (WT) littermates were bred from CX3CR1+/-couples. KO and WT male mice (aged8-12wks, weighing20-24g) were intraperitoneally anesthetized with xylazine (5mg/kg, intraperitoneal) and ketamine (100mg/kg, intraperitoneal), and the adequacy of anesthesia was monitored from the disappearance of pedal withdrawal reflex. Mice were then subjected to left coronary artery ligation to induce MI with subsequent development of cardiac rupture, as described elsewhere. Ischemia was judged by myocardial blanching and electrocardiogram ST-segment elevation. Sham-operated mice underwent the same procedure without left coronary artery ligation. Four groups were designed:WT sham, WT-MI, KO-sham and KO-MI. Some mice were sacrificed1-3days after MI to measure myocardial infarct size as described elsewhere or to perform molecular biological examinations and histological analysis.2. Cell Culture.The neonatal Sprague-Dawley rats were killed by overdose anesthesia with2%isoflurane. The isolation and culturing of neonatal rat ventricular cardiomyocytes (NRVCs) were performed as previously described. The effects of soluble-FKN(s-FKN) and Y27632on cardiomyocytes in the setting of anoxia model were analyzed. The experimental groups were designed as follows:1) s-FKN group:cardiomyocytes were treated with s-FKN200ng/mL (chemokine domain, R&D, Minneapolis, MN) for24hrs;2) s-FKN+Y27632group:cardiomyocytes were co-treated with s-FKN200ng/mL and Y2763210μmol/L for24hrs;3) Anoxia group:cardiomyocytes were incubated with anaerobic-simulated ischemia buffer for24hrs;4) Anoxia+s-FKN group:in the presence of anoxia, s-FKN was added to the cardiomyocytes and incubated for24hrs; and5) Anoxia+s-FKN+Y27632group:in the presence of anoxia, both s-FKN and Y27632were added to the cardiomyocytes and incubated for24hrs. Cells were harvested after24hrs of incubation and analyzed for aE-catenin expression by RT-PCR, western blot and immunofluorescence.3. Polymerase Chain Reaction.Total RNA was extracted from culture cells and mouse heart tissues (total RNA isolation system, Omega, Norcross, GA). Reverse transcription was carried out in20μL of reaction mixture containing1μg of total RNA. Digital images of aE-catenin and (3-actin bands separated on ethidium bromide-stained agarose gels were obtained. We further quantitatively checked the expression of MMP-9, ICAM-1and β-actin in the whole hearts of mice using Quantitect SYBR Green real-time polymerase chain reaction method, as described elsewhere.4. Western Blotting.Proteins were obtained from whole-heart homogenates or cultured cardiomyocytes. Samples containing equal amounts of protein were loaded onto12%sodium dodecyl sulfate-polyacrylamide gels for electrophoresis and transferred onto polyvinyl difluoride membranes. The membranes were blocked with5%skim milk (0.1%Tween in TBS) at room temperature for2hrs, and then incubated over night at4℃with5%skim milk containing the following antibodies:anti-fractalkine (R&D, MAB571), anti-aE-catenin (Cell Signaling Technology,#3236), and anti-β-actin antibody (Boster, Wuhan, China) for loading control. Immunoreactive bands were visualized with Odyssey(?) InfraRed Imaging System (LI-COR(?) Bioscience, America) and quantified by densitometry using the Image J Analysis software (National Institutes of Health, Bethesda, MD).5. Immunofluorescence assay.Cells were fixed in4%formaldehyde followed by permeabilization in0.1%Triton X-100. After washing, cells were blocked inl%bovine serum albumin (BSA) and then incubated with aE-catenin antibody (1:1000dilution) in phosphate buffered saline (PBS) containing1%BSA. Cells were incubated with tetramethyl-rhodamine-isothiocyanate (fluorogenic secondary antibody to label the aE-catenin) in PBS containing1%BSA. Fluorescence was observed using an Olympus FluoviewFV1000microscope, and images were acquired and analyzed with charge-coupled software.6. Immunohistochemistry.After mice were sacrificed by overdose anesthesia and cervical dislocation, the hearts were rapidly excised, rinsed with PBS, fixed in4%paraformaldehyde, and embedded in paraffin. Then4-6mm sections were cut for immunostaining. Then, after antigen were retrieved in citrate pH6.0, the sections were incubated with rabbit anti-aE-catenin antibody, rabbit anti-MPO (myeloperoxidase) antibody or rabbit anti-CD68antibody overnight at4℃. After secondary antibody incubation, the DakoEVision+System-HRP and3,3’-diaminobenzidine (DAB) were used to visualize the expression of aE-catenin, MPO or CD68. Hematoxylin was used for nuclear dyeing. Semi-quantitative analysis was performed using a score system to reflect the immunoreactivity as we described elsewhere.7. Statistical Analysis.All data were expressed as mean±tandard deviation (x±s), and values of P <.05were considered to be statistically significant. Statistical differences were evaluated by independent-samples t test or factorial design analysis of variance, Satterthwaite t test was used when equal variances not assumed (Levene’s test for homogeneity tests, P<.05). Post Hoc multiple comparison tests were performed using Bonferroni (equal variances assumed) or Dunnett’s T3(equal variances not assumed). The overall survival of MI mice was evaluated using Kaplan-Meier survival analysis. All analyses were performed using SPSS16.0software (SPSS Inc, Chicago, IL).Results:1. CX3CR1Deletion Improves the Survival of Mice subjected to MI.Myocardial FKN mRNA and protein levels in response to3days of MI were both markedly increased. In order to figure out the influence of FKN on AMI, we compared the one week survival rate and the reason for death between FKN receptor knockout mice and the WT littermates. The two groups of animals had their left coronary artery ligation at the same position, and their levels of ST-segment elevation in the electrocardiogram were similar as well. The overall mortality rate of mice during the1st week of MI was significantly lower in KO mice than in their WT littermates. By post mortem examination, we noticed that3KO mice and9WT mice died of cardiac rupture, thus the incidence of cardiac-rupture was significantly lower in KO MI mice.2. CX3CR1Deletion Limits Infarct Size.Since cardiac rupture is associated with the cardiac infarct size, we detected the influence of CX3CR1knocking out on the infarct size. The result shows that the myocardial infarct size in response to ischemia for24hrs in KO mice was markedly smaller than in WT group (53.8%in WT-MI group vs.28.6%in KO-MI group. We then investigated the possible reasons for the reduction of cardiac rupture rate as well as the infarct size in CX3CR1gene knock-out mice. 3. Lacking of FKN Receptor Affects the Expression of MMPs and the Inflammatory Adhesion Molecules.The baseline expression of MMP-9was lower in KO mice than in WT mice.24hrs after MI, the MMP-9mRNA was increased by6folds in WT mice, while only2folds increase in KO mice, suggesting that matrix metal protein degradation is reduced in KO mice. With similar baseline expression in WT and KO mice, the ICAM-1mRNA at24hrs after MI was increased by2.2folds in WT mice, while only by1.6folds in KO mice.To evaluate the extent of infiltrated neutrophils and macrophages, the expression of MPO and CD68was detected by immunohistochemistry. We found that the increased expression of both MPO and CD68in response to1day of ischemia was lower in KO mice than in WT mice.4. CX3CR1Deletion Prevents the Down-regulation and Defective Localization of aE-catenin in the Infarct Region.The semi-quantitative analysisusing Image-J software showed that myocardial expression of aE-catenin mRNA was remarkably lower on day3after MI in WT-MI mice than in WT-sham group, while it was only slightly lower in KO-MI mice than in KO-sham group. We analyzed the expression of aE-catenin mRNA after MI in the infarct area (free wall) and the remote (septum) area respectively, and found it was remarkably decreased in the infarct area but slightly reduced in the remote area of the WT-MI mice, but such distinct difference disappeared in KO-MI mice. The myocardial expression of aE-catenin protein on day3after infarction detected by western blot and immunohistochemistry showed similar change for aE-catenin mRNA.5. The down-regulation of aE-catenin in response to s-FKN stimulation is abrogated by ROCK1Inhibitor. In cultured neonatal rat cardiomyocytes, s-FKN significantly down-regulated aE-catenin mRNA, which was abolished by Y27632, an inhibitor of ROCK1.The expression of aE-catenin mRNA was also reduced in response to anoxia, while treatment with s-FKN further down-regulated it. Furthermore, the down-regulation of aE-catenin mRNA by anoxia in the presence/absence of s-FKN stimulation was inhibited by co-treatment with Y27632. As for the expression of aE-catenin protein, similar results were obtained by western blot and immunofluorescence assay.Conclusions:1. The expression of myocardial FKN significantly increased after myocardial infarction;2. The rupture rate of mice during the1st week of MI was significantly lower in KO mice than in their WT littermates;3. CX3CR1deletion limits infarct size;4. The increasement of inflammatory cell infiltration after myocardial infarction were significantly inhibited in KO mice;5. The increasement of ECM degradation after myocardial infarction were significantly inhibited in KO mice,-6. CX3CR1deletion prevents the down-regulation and defective localization of aE-catenin in the infarct region;7. The down-regulation of aE-catenin in response to s-FKN stimulation is abrogated by ROCK1Inhibitor. |
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