Role Of Voltage-Gated Sodium Channels Mediated Immunomodulation Of Monocytes/Macrophages In Myocardial Ischemia Reperfusion Injury

Objective:Inflammatory response plays an important role in the healing process and ventricular remodeling after myocardial ischemia/reperfusion (I/R) injury. Inhibition of excessive inflammation could limit the infarct area and ameliorate heart function. Monocytes/macrophages are the major inflammatory cells that stay for longest time in the remodeling process after myocardial injury. Monocytes/macrophages are a heterogeneous population:the inflammatory monocytes/macrophages have a strong capacity of phagocytosis and chemotaxis; the anti-inflammatory monocytes/macrophages could promote angiogeness, collage deposition and tissue repairing. Previous studies shown that switching the inflammatory monocytes/macrophages into the anti-inflammatory monocytes/macrophages maybe benefit for the tissue repair and ventricular remodeling. The functional expression of voltage-gated sodium channels (VGSCs/NaV) was demonstrated in the monocytes/macrophages, which participate the phenotypic polariztion of monocytes/macrophages. Our previous work shown that VGSCs antagonist phenytoin (PHT) could regulate the function of macrophage and accelerate the remodeling process after experimental myocardial infarction (MI). All these results suggest that the VGSCs in monocytes/macrophages would be a regulatory target of phenotypic polarization. The expression of VGSCs was documented in many tissues, such as myocardium, skeletal muscle and nervous system. Liposome is a commonly used vehicle in the field of pharmaceutics and molecular biology. Liposome is phagocyted by the monocytes/macrophages via opsonin after intravenous injection and prone to recruit into the sites of inflammation. This effect would accomplish the goal for monocytes/macrophages targeting. The objective of this project is to exploit the effects of VGSCs antagonist and agonist on monocytes/macrophages phenotype polarization, and investigate the role of PHT-loading liposome (PHT-lipo) in the remodeling process after myocardial I/R injury.Methods:This research is composed of two parts:cell experiments and animal experiments. Cell experiments:mice macrophage cell line RAW264.7and peritoneal macrophages of male Wistar rat were investigated. The mRNA expression level of VGSCs and phenotypic markers, the protein expression level of NF-κB singnaling in PHT, tetrodotoxin (TTX) and veratridine treated macrophage were quantified by polymerase chain reaction (PCR) or Western blot. RNA interference (RNAi) plasmid (pGPU6/Neo-NaV1.9-shRNA) was transfected into RAW264.7and G418was used to screen the stable cell line. Experiments were performed in NaV1.9knockdown cell line:knockdown efficiency was qualified by PCR; the proliferation was measured by CCK-8; cell cycle and phagocytic ability were analyzed by flow cytometry; migratory ability was detected by transwell chamber migration assay; the mRNA expression level of phenotypic markers was detected by PCR. Animal experiments:PHT-lipo was made by film dispersion method. The estimation of pharmacokinetic characteristics of PHT-lipo was done by high performance liquid chromatography (HPLC). Rats were randomly divided into sham groups and I/R groups. Each group was divided into3subgroups which were intravenous injected with saline, empty liposome (Emp-lipo) or PHT-lipo respectively on0,2,4days after surgery. I/R model was established by left coronary artery ligation, ischemia for45min and sequentially persistent reperfusion. The left coronary artery was not ligated in sham groups. Peripheral blood was extracted from individual rat through the tail vein at base line and1,3,5,7,14,30d after surgery. The proportions of circulating monocyte CD43+and CD43++subsets were analyzed by flow cytometry. At30days after the induction of I/R, the cardiac function was evaluated by echocardiography and invasive left ventricular hemodynamic analysis hemodynamic parameter. Then rats were sacrificed and hearts were harvested for the histology examination. Index of expansion, collagen volume fraction (CVF) and infaret size were analyzed from Masson staining. Cross-sectional area was investigated by wheat germ agglutinin (WGA). The capillary density was calculated by isolectin B4staining.Results:1) The a subunits of VGSCs expressed in RAW264.7are:NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, NaV1.9and NaVX.2) The mRNA expression level of CCL5, TNF-a, NOS2(M1markers) in uninduced cell and IL-1β, CCL2, CCL5, TNF-a in LPS induced cell were declined after the treatment of PHT. The mRNA expression level of IL-1β，CCL5in uninduced cell and CCL2, CCL5, TNF-a in LPS induced cell were declined after the treatment of TTX. The mRNA expression level of Argl (M2marker) was decreased in IL-4induced cell after the treatment of veratridine.3) VGSCs antagonist inhibited the activation of NF-κB signaling in LPS-induced RAW264.7; while VGSCs agonist facilitated the activation in IL-4induced cell.4) NaV1.9-deficient stable cell line was established and the expression of NaV1.9was reduced80%. Decreased proliferative, phagocytic and migratory abilities were observed in NaV1.9knockdown RAW264.7cell line. The mRNA expression level of CCL5and NOS2were down-regulated, and Argl and mannose were up-regulated in the stable cell line.5) The expression of VGSCs were confirmed in rat peirtoneal macrophages:NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.6, NaV1.7, NaVX, Scnlb, Scn3b and Scn4b. PHT inhibited the enhancement of TNF-a, CCL5in LPS treated cells and promoted the increment of Argl, TGF-β1in IL-4treated cells.2Animal experiments:1) The concentration-time curves of PHT and PHT-lipo fitted two-compartment model in rats. The T1/2a and T1/2β of PHT-lipo were shorter and the AUC was lower than PHT.2) Dynamic changes of monocyte subsets proportions were detected in I/R model. In I/R group, the proportion of CD43+monocyte increased at1d after surgery compared with base line, peaked at3d, and then decreased to the base line at7d. In sham group, the percentage of CD43+monocyte enhanced at1d after surgery compared with base line, declined at3d. The ratio of CD43in I/R group at3d was much higher than sham group at3d. There was no significant difference between the two groups at any other time points. The change of CD43++proportion was contrary to CD43+proportion.3) PHT-lipo significantly decreased circulating CD43+monocytes, which was accompanied by improvement of post-infarction left ventricle (LV) remodeling, as shown by decreased infarct size, enhanced angiogenesis, reduced intersitial fibrosis and cardiomyocyte cross-sectional area, dereased LV expansion index, and improved LV functions.Conclusions:Block the VGSCs in macrophages could switch the transition of M1into M2polarization in macrophages. PHT-lipo modified the dynamic changes of monocyte subsets and improved the post-infarction healing in rat myocardial I/R model. The present study demonstrates a novel role of VGSCs mediated monocyte/macrophage immunomodulation following myocardial ischemic insult, and provides the experimental basis for monocytes/macophages targeted therapy for the treatment of post-infarction LV remodeling and heart failure.