The Role And Mechanism Of Innate Immune Receptor-NOD2in Ventricular Remodeling

ObjectiveVentricular remodeling is the changes of structure and function in the incidence of heart damage, such as myocardial necrosis and apoptosis, cardiac hypertrophy, interstitial fibrosis, change in cardiac ion channels and sympathetic innervations. Many cardiovascular diseases such as hypertension, myocardial infarction, heart failure, dilated cardiomyopathy, valvular heart disease have the phenomenon of ventricular remodeling. In addition, hyperhomocysteinemia, atherosclerosis and diabetic complications also have the phenomenon of ventricular remodeling. Ventricular remodeling is the combined effects produced by a variety of morphological, molecular biology, pathophysiology changes, electrophysiological environment, its mechanisms including inflammation, hemodynamic load, neurohormonal activation, cytokine production and protease-induced extracellular matrix degradation.NOD-like receptors (NOD-like receptor, NLRs) are innate immune pattern recognition receptors which recognise intracellular pathogen-associated molecular patterns or endogenous danger signals related molecules, thereby initiating an immune response.NLRs plays an important role in the regulation of immune response. So far,22NLR family members are currently discovered, the most representative NLRs are NOD1and NOD2. It has been reported that activation of NOD1with the specific agonist iEDAP induces a time-and dose-dependent cardiac dysfunction that occurs concomitantly with cardiac fibrosis and apoptosis. The administration of iEDAP promotes the activation of the NF-κB and TGF-β pathways and induces apoptosis in the heart. But so far, the expression of NOD2and the role of NOD2involved in ventricular remodeling in the heart keeps unknown. Therefore, in the present study we selected two different exent ventricular remodeling model--yperhomocysteinemia and myocardial infarction to investigate whether NOD2is involved in ventricular remodeling and to explore the possible mechanisms by which NOD2regulates the ventricular remodeling.MethodsSection one:Hyperhomocysteinemia model was established by WT and CBS+/" mouse non-folate diet, left ventricular internal diameter and cardiac function was acquired by echocardiography. Western Blot, RT-PCR and immunohistochemistry was done to evaluate the expression of NOD2in the myocardium. To investigate the mechanisms of cardiac dysfunction and ventricular remodeling caused by hHcys, we used NOD2gene knockout mouse to establish hyperhomocysteinemia model. Left ventricular internal diameter and cardiac function acquired by echocardiography was to observe whether there was a little amelioration in ventricuar remodeling in NOD2knockout mouse; Masson staining was to observe the degreement of fibrosis in mice; the protein level of matrix metalloproteinase MMP2, MMP9and matrix metalloproteinase inhibitor TIMP-1was detected by Western Blot.Section two:Mouse MI model was induced by permanent left anterior descending coronary artery ligation. Sham operated ones underwent the same procedure but ligation. Both infarct and remote myocardium from sham and post-MI3,7,14and28days were homogenized to evaluate mRNA expression of NOD2by quantitative real-time polymerase chain reaction(qPCR), and detect its protein level by Western Blot and immunohistochemistry post-MI28days. To further investigate the role of NOD2in post-MI LV remodeling process, NOD2gene knockout mouse were used to establish myocardial infarction model. Left ventricular internal diameter and cardiac function post-MI28days were acquired by echocardiography; Masson staining and Tunel staining were used to observe the degreement of fibrosis and apoptosis; the enzyme-linked immunosorbent assay (ELISA) measured tissue proinflammatory factor levels; the protein level and activity changes of matrix metalloproteinase MMP2, MMP9of cardiac tissue lysates were detected by Western Blot and gelatin zymography. The inflammatory cells infiltration after myocardial infarction and cardiac fibroblasts (cardiac fibroblast) trans-differentiation into cardiomyocytes fibroblasts were observed by immunohistochemical. In vitro, use mouse fibroblasts to investigate the molecular mechanisms of ventricular remodeling which NOD2was involved in.The phosphorylation levels of ERK1/2and p38in fibroblasts stimulated with MDP were detected by Western Blot which was also used to acquire NOD2and MMP9protein levels in cardiac fibroblasts under hypoxia/reoxygenation conditon; RT-PCR detected the transfection efficiency of NOD2; ELISA determined relative levels of proinflammtory mediators in cardiac fibroblasts with shRNA-NOD2transfection. ResultsSection one:First of all, NOD2expression was improved with increasing concentrations of Hcy. The level of NOD2in CBS+-/-model group was highest, and then WT model group. Immunohistochemistry and PCR results also confirmed that NOD2expression in CBS+-/-model group was the strongest.②NOD2expression was significantly increased in H9C2stimulated with different concentrations of L-Hcy. NOD2expression was the strongest when L-Hcy concentration was25μmol/L. The same result was found in SMC.③The expression of MMP9which was a ventricular remodeling marker was significantly increased in WT model group, while reduced in NOD2knockout model group; the expression of COL1was significantly decreased in WT model group, while increased in NOD2knockout model group. Although NOD2can affect the expression of MMP9, COL1, but failed to change hHcys-induced ventricular remodeling. Compared to WT controls group, LVIDd LVIDs increased in WT model group indicating left ventricular hypertrophy; LVPWd and LVPWs decreased indicating left ventricular wall thinning. NOD2gene knockout failed to alleviate the left ventricular hypertrophy and ventricular wall thinning.Section two:①NOD2expression was significantly increased in the cardiac infarcted area in post-MI mice. NOD2mRNA levels were significantly increased in the tissue from the cardiac infracted area in post-MI mice at different time points. The enhanced NOD2protein levels were further confirmed by Western blot and immunochemical.②NOD2deficiency exhibited improvements in cardiac dysfunction and remodeling after MI. Cardiac functions were examined by echocardiography at28days after MI.MI significantly decreased cardiac function in WT mice as evidenced by decreases in EF%, FS%, when compared with WT basal controls, which can be recovered in NOD2-/-mice. In NOD2-/-mice, the MI-induced LV enlargement and wall thinning were significantly improved. Furthermore, TUNEL staining and Masson trichrome staining indicated that NOD2deficiency protected against ischemia-induced cell death and cardiac fibrosis.③NOD2deficiency reduced the levels of proinflammatory mediators, inflammatory cell infiltration and MMP-9activity after MI. NOD2deficiency significantly reduced the levels of proinflammatory cytokines and chemokines including IL-1β, TGF-P, TNF-a and MCP-1in the cardiac infarcted area in mice after MI. Moreover, elevated MMP-9levels and activity in the cardiac tissues were blocked by NOD2deficiency after MI.The infiltration of inflammation cells and the expression of a-SMA was significantly reduced in NOD2-/-mice.④MDP induced activation of MAPK signaling pathways, production of proinflammatory mediators in primary cultured cardiac fibroblasts. MDP induced the activation of MAPKs as assessed by measuring the levels of phospho-specific ERK-1/2, p38MAPK. Also MDP enhanced the production of proinflammatory mediators in primary cultured cardiac fibroblasts.⑤Gene silencing of NOD2attenuated hypoxia-induced MMP-9expression.In primary cultured cardiac fibroblasts, hypoxia/reoxygenation significantly enhanced NOD2and MMP9enpression while hypoxia-induced MMP-9expression was markedly blocked by shRNA-NOD2. Additionly, hypoxia-induced the production of proinflammatory mediators was attenuated by gene silencing of NOD2.Conclusion1. We investigated for the first time the role of NOD2in ventricular remodeling caused by hHcys. In this study, we found that the mRNA and protein levels of NOD2significantly increasedin myocardial tissue of CBS+-/-hyperhomocysteinemia mouse. Further study observed that although NOD2deficiency reduced the levels of MMP expression which is a ventricular remodeling marker, but it had no improvement for hyperhomocysteinemia-induced ventricular remodeling.The possible reasons were that hyperhomocysteinemia induced less damage to heart, only had the effects on the left ventricular diastolic inner diameter and left ventricular posterior wall thickness and has no effect on systolic and diastolic function, thus the improving role of NOD2failed to be reflected. Secondly some other compensatory mechanisms may be involved in the development of hyperhomocysteinemia.2. We further explored whether NOD2participated in ventricular remodeling caused by mycardial infarction with the model of left anterior descending coronary artery ligation. We confirmed for the first time that mRNA levels of NOD2were significantly higher in the infarcted area and non-infarcted area, expecially in the infarcted area. Further study found that NOD2deficiency attenuated cardiac dysfunction and remodeling after myocardial infarction and protectd against ischemia-induced cell death, also cardiac fibrosis was significantly reduced in NOD2-/-mice. Studies had shown that NOD2mediated left ventricular remodeling after myocardial infarction by regulating the levels of MMP9protein and activity, inflammatory mediators production and inflammatory cell infiltration. In vitro studies had shown that NOD2agonist MDP induced the activation of MAPKs in cardiac fibroblasts as assessed by measuring the levels of phospho-specific ERK1/2p38MAPK. We also observed that MDP enhanced the production of proinflammatory mediators and hypoxia/reoxygenation significantly enhanced NOD2and MMP-9expression in primary cultured cardiac fibroblasts. After silencing NOD2gene, hypoxia-induced MMP9expression was markedly blocked and hypoxia-induced the production of proinflammatory mediators was attenuatd.