Study On The Regulation Of SCAD/IRF7/CCL2/STAT3 Signal Pathway To Myocardial Hypertrophy

Objective:Short-chain acyl-Co A dehydrogenase（SCAD）is a rate-limiting enzyme forβoxidation of mitochondrial fatty acids.Previous studies have shown that SCAD has a negative regulatory effect on pathological myocardial hypertrophy caused by hypertension.This paper aims to study the regulation of SCAD/IRF7/CCL2/STAT3 signal pathway on myocardial hypertrophy and clarify the new mechanism of SCAD regulating myocardial hypertrophy.Method:1.Subjected to transverse aortic constriction（TAC）,SCAD knockout mice(SCAD^-/-)were regarded as the animal model of cardiac hypertrophy.The genotype of SCAD^-/-mice were confirmed by mouse tail identification.Western blot was used to detect the change of SCAD protein expression in the left ventricle of mice.Evaluation of cardiac function in mice by systolic blood pressure and echocardiography.Ratios of heart weight to body weight or tibia length,hematoxylin-eosin（HE）staining,wheat germ agglutinin（WGA）staining and picrosirius red staining（PSR）were used to observe and analyze the change of cardiac physiological structure.The m RNA expression changes of left ventricular hypertrophy genes ANF,BNP andβ-MHC were detected by real-time quantitative polymerase chain reaction（q PCR）to observe the changes of embryonic genes caused by myocardial hypertrophy.Simultaneously,free fatty acid content（FFA）and ATP content were also detected to analyze the change of energy metabolism in mice.To investigate the relationship between SCAD deficiency and cardiac hypertrophy.2.Phenylephrine（PE）was used to stimulate H9C2（rat-like H9C2）cardiomyocytes to construct an in vitro model of myocardial hypertrophy,and SCAD recombinant adenovirus was used for pretreatment.The changes of SCAD expression,m RNA levels and enzyme activity were also observed in H9C2 cardiomyocytes.The cell surface areas of cardiomyocytes were identified byα-actinin staining.At the same time,ANF,BNP andβ-MHC m RNA levels,ATP content and FFA content were also detected.This part intends to explore the effect of adenovirus-mediated SCAD overexpression on cardiomyocytes hypertrophy induced by PE.3.Twelve-week-old spontaneously hypertensive rats（SHR）were used as pathological myocardial hypertrophy model,and SCAD recombinant adenovirus(5×10¹⁰VP/m L/d)was intravenously injected for8 weeks.Detection of left ventricular SCAD content by SCAD immunofluorescence staining.Examining SCAD protein expression,m RNA levels and enzyme activity in left ventricular.Evaluation of cardiac function in rats by systolic blood pressure and echocardiography.Left ventricular mass index,HE staining,WGA staining and PSR staining were used to observe and analyze the changes of cardiac physiological structure.ANF,BNP andβ-MHC m RNA expression levels,FFA and ATP content were detected in left ventricule.To explore the effect of tail vein injection of SCAD recombinant adenovirus on myocardial hypertrophy in rats.4.Western blot was used to detect the protein expression of interferon regulatory transcription factor 7（IRF7）,CC motif chemokine ligand 2（CCL2）,signal transducer and activator of transcription 3（STAT3）and p-STAT3 in SCAD^-/-mice,hypertrophic cardiomyocytes of SCAD-overexpressing and SHR myocardium of SCAD-overexpressing SHR via tail vein injection.To explore whether SCAD negatively regulates myocardial hypertrophy by activating IRF7/CCL2/STAT3signal pathway.5.H9C2 cardiomyocytes were respectively pretreated with si RNA to knock down IRF7,and then infected with SCAD recombinant adenovirus.Western blot and q PCR were used to detect the changes of the protein content and m RNA levels of IRF7 after si RNA interference sequence treatment to screen the optimal si IRF7 sequence.α-actinin immunofluorescence staining was used to observe and analyze the surface area changes of H9C2 rat cardiomyocytes infected with SCAD recombinant adenovirus under the interference of optimal si IRF7sequence si-1452.ANF,BNP andβ-MHC m RNA expression levels,SCAD enzyme activity were detected.The protein expression levels of IRF7,CCL2,p-STAT3 and STAT3 were also detected.To study whether SCAD regulates myocardial hypertrophy by mediating CCL2/STAT3signal pathway through IRF7.Result:1.10-14 weeks old wild-type mice and SCAD knockout mice did not show significant cardiac pathological changes.Compared with the corresponding genotypes,TAC caused significant myocardial hypertrophy and fibrosis in mice.Compared with WT+TAC group,the cardiac dysfunction,myocardial hypertrophy and fibrosis in KO+TAC group were significantly enhanced,the FFA content was significantly increased,and the ATP content was significantly reduced.2.In the H9C2 cardiomyocytes hypertrophy model induced by PE,H9C2 cardiomyocytes pretreated with SCAD recombinant adenovirus can resist PE-induced cell hypertrophy.Compared with the Ad-GFP+PE group,the protein content,m RNA level and enzyme activity of SCAD in the Ad-SCAD+PE group were significantly increased,the surface area of H9C2 cardiomyocytes induced by PE was significantly reduced,the m RNA expression levels of hypertrophic genes such as ANF,BNP andβ-MHC were significantly downregulated,the oxidation ability of mitochondrial fatty acidβwas improved,and the level of cell energy metabolism was significantly increased.3.Compared with Wistar+Ad-GFP group,SHR+Ad-GFP group showed obvious pathological myocardial hypertrophy.Compared with the SHR+Ad-GFP group,the SCAD content and enzyme activity of the left ventricle in the SHR+Ad-SCAD group were significantly increased,the cardiac function was significantly improved,the m RNA expression levels of ANF,BNP andβ-MHC were significantly down-regulated,myocardial hypertrophy and fibrosis were significantly reduced,in myocardial tissue,the ATP content was significantly increased,the FFA content was significantly reduced.4.SCAD negatively regulates myocardial hypertrophy through IRF7/CCL2/STAT3 signal pathway.Compared with wild type mice,the expression levels of IRF7,CCL2 and p-STAT3 in left ventricle of SCAD^-/-mice were significantly decreased.Compared with the Ad-GFP group,the expression levels of IRF7,CCL2 and p-STAT3 in H9C2cardiomyocytes in the Ad-GFP+PE group were significantly down-regulated.Compared with the Ad-GFP+PE group,the expression levels of IRF7,CCL2 and p-STAT3 in H9C2 cardiomyocytes of the Ad-SCAD+PE group were significantly up-regulated after pretreatment with SCAD recombinant adenovirus for 48 h.Compared with the Wistar+Ad-GFP group,the expression levels of IRF7,CCL2 and p-STAT3 in the left ventricle of SHR+Ad-GFP group were significantly down-regulated.Compared with SHR+Ad-GFP group,the protein expression levels of IRF7,CCL2 and p-STAT3 were significantly up-regulated in SHR+Ad-SCAD group after tail vein injection of SCAD recombinant adenovirus for 8 weeks.5.WB and q PCR results showed that si IRF7 with different sequences could effectively reduce the protein content and m RNA level of IRF7 compared with the control group,and the knock-down effect of si-1452 sequence was the most significant.Therefore,in the subsequent experiment,si-1452 was selected as the IRF7 knock-down sequence of H9C2 rat cardiomyocytes.Compared with the Ad-GFP+NC group,the H9C2 rat cardiomyocytes of the Ad-GFP+si-1452 group showed significant hypertrophy,the m RNA expression levels of ANF,BNP andβ-MHC were significantly up-regulated the protein expression levels of IRF7,CCL2 and p-STAT3 were significantly down-regulated,but the expression of SCAD did not change significantly.Compared with Ad-GFP+si-1452 group,the expression of SCAD protein and m RNA in Ad-SCAD+si-1452 group was significantly up-regulated after 48 h of SCAD recombinant adenovirus pretreatment,and the activity of SCAD enzyme was significantly increased.However,the protein levels of IRF7,CCL2 and p-STAT3 were not significantly up-regulated,and the hypertrophy of H9C2 rat cardiomyocytes was not significantly improved,suggesting that SCAD regulates CCL2/STAT3 signaling pathway through IRF7 to affect the occurrence and development of myocardial hypertrophy.Conclusion:SCAD has a negative regulatory effect on cardiac remodeling probably by regulating CCL2 and STAT3 signaling molecules by mediating IRF7,thereby regulating the occurrence and development of myocardial hypertrophy.