| Backgroud: Ventricular remodeling is the general course of heart failure.Not only the structural remodeling of the ventricle,but also electrical remodeling at the molecular level of the ion channel may be associated with ventricular fibrillation after heart failure.The structural reconstruction of the ventricle is mainly reflected in cardiac hypertrophy and myocardial fibrosis.NLRP3 inflammasome is the key regulator of inflammatory response.The NLRP3 inflammasome is a large multiprotein complex with Caspase 1 and ASC to promote the activation of the classical inflammatory molecule interleukin-1?(IL-1?),and also to participate in the regulation of the typical pro-fibrotic factor transforming growth factor beta(TGF-?).NLRP3 mediates inflammatory cascade response of cardiomyocytes,induces cardiomyocyte death and fibrosis.However,NLRP3 affects the expression of Nav1.5 and its mechanism remains to be studied.Objective: After establishing the heart failure model,and then ventricular fibrillation was induced to study the expression changes of NLRP3 and Nav1.5.Based on this,investigating the mechanism of the NLRP3 inflammasome was mediated by IL-1? and TGF-? to regulate the Nav1.5 expression of cardiomyocytes.Methods: 1.Rats were randomly divided into surgery group(abdominal aortic cconstraction)and sham operation group.Rat echocardiograph was used to evaluate cardiac function in rats.On the basis of establishing a heart failure model,ventricular fibrillation was induced by transesophageal electrical stimulation,and the same evoked condition stimulated the sham operation group.The left ventricular myocardium of the two groups was examined by Western Blotting to detect the expression of Nav1.5 and NLRP3 core proteins.2.Vehicle,NLRP3-shRNA,IL-1?-shRNA,TGF-?-shRNA adenovirus were stablely transfected,and two kinds of viruses were both transfected to H9C2 rat cardiomyocyte cell line.The cells consist of 6 groups: 1)LV-shRNA Group(empty virus transfected with vehicle,control group);2)NLRP3-shRNA group;3)IL-1?-shRNA group;4)TGF-?-shRNA group.These four groups are single virus transfection groups.5)NLRP3-IL-1?-shRNA group;6)NLRP3-TGF-?-shRNA group,which continued to transfect IL-1? or TGF-? virus on the basis of successful transfection of NLRP3 virus.RT-PCR and fluorescently labeled protein expression were used to identify transfections efficiency.3.The expression levels of Nav1.5,NLRP3,IL-1?,TGF-?,ASC and Caspase 1 in 6 groups of cells were detected.The distribution and expression of Nav1.5 in LV-shRNA group and NLRP3-shRNA group were observed by immunofluorescence.To explore the relationship between NLRP3 and TGF-?,IL-1? and the effect of this factors on the expression of Nav1.5.Data statistics were processed by SPSS 21.0,Western Blotting grayscale values were selected for Adobe Photoshop CS6 calculation,the violin and histograms were produced using R 3.5 and GraphPad Prism 5,respectively.Each index of echocardiography was expressed as mean ± standard deviation.The comparison between the continuous variables of multiple groups was analyzed by one-way analysis of variance,and the continuous variables between the two groups were analyzed by two independent samples t-test.P < 0.05 was considered statistically significant.Results: 1.After 8 weeks of abdominal aortic coarctation surgery,colour Doppler echocardiography showed that left ventricular ejection fraction,left ventricular short axis shortening rate and cardiac output were significantly lower than that of sham-operated group,suggesting that heart failure rats was built successfully.The voltage of 12 V was stimulated for 16 seconds,and the heart failure rats stably induced ventricular fibrillation.Under the same conditions,no ventricular fibrillation occurred in the sham operation group.The expression of NLRP3 core protein in left ventricular myocardium of ventricular fibrillation rats was higher than that of the control group.Conversely,Nav1.5 was lower than the control group.2.Transfection efficiency in one transfection group: Fluorescence of LV-shRNA group,NLRP3-shRNA group,IL-1?-shRNA group and TGF-?-shRNA group was successful transfected.Transfection efficiency of two viruses co-transformed: RT-PCR was used to detect the expression of TGF-? in the IL-1? and NLRP3-TGF-?-shRNA groups in the NLRP3-IL-1?-shRNA group.The expression levels of IL-1? and TGF-? mRNA were inhibited,respectively,and the expression of fluorescent protein under the microscope was satisfied,indicating that each group was successfully transfected.Compared with the LVshRNA group,the expression levels of Nav1.5 in the NLRP3-shRNA group,NLRP3-TGF-?-shRNA group and IL-1?-shRNA group were up-regulated,and the NLRP3-TGF-?-shRNA group showed upward expression too.The upregulation was more pronounced than in the NLRP3-shRNA group,while the NLRP3-IL-1?-shRNA group was not up-regulated.The IL-1?-shRNA group was the most highly expressed in all silencing groups.After NLRP3 silencing,the protein expression of Nav1.5 was increased,accompanied by inflammatory factors such as NLRP3,ASC,Caspase 1,and TGF-? and IL-1?,they reduced during the same period.After transfection of IL-1? and TGF-? viruses,the protein content of NLRP3 increased,while the NLRP3-TGF-?-shRNA group was downregulated.The expression of IL-1? and TGF-? in each experimental group was lower than that in the empty virus group,and mature TGF-? could not be detected in each group.Compared with the NLRP3-IL-1?-shRNA group,the expression of NLRP3,ASC,Caspase 1,IL-1?,pro-IL-1? and pro-TGF-? was inhibited in the IL-1?-shRNA group,while Nav1.5 was observed.Was promoted.In addition,the expression of ASC and Caspase 1 in each group was high,low,and not completely synchronized.Their expression and relationship were without regularity to conform to,but ASC and Caspase 1 are most promoted in the NLRP3-TGF-?-shRNA group and NLRP3-shRNA,respectively.The expression was most positive in the shRNA group,and both were most significantly down-regulated in the IL-1?-silencing group.4.Immunofluorescence showed a significant increase in the abundance of Nav1.5 expression in the NLRP3 silenced group.Conclusion: 1.After 8 weeks of abdominal aortic coarctation surgery,the heart failure model can be successfully constructed.The voltage of 12 V with 16 seconds can induce ventricular fibrillation in heart failure rats by esophageal stimulation.2.NLRP3,IL-1? and TGF-? are involved in the regulation of Nav1.5 protein expression,and NLRP3 can directly down-regulate the expression of Nav1.5.Moreover,NLRP3 inflammatory bodies inhibit the expression of Nav1.5 in cardiomyocytes by TGF-?,but have less possibility of IL-1?,but the direct repression of IL-1? to Nav1.5 is very strong.3.The protein expression levels of ASC and Caspase 1 in each group were inconsistent with the trend of NLRP3.What's more,NLRP3,IL-1?,TGF-?,Caspase 1,and ASC interact with each other. |
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