Deficiency Of SM22α Promotes Myocardial Fibrosis In Mice

Objective:SM22αis a cytoskeleton-associated protein,which is mainly expressed in mature vascular smooth muscle cells and involves in maintaining phenotypic homeostasis of smooth muscle cells.The expression of SM22αcan be detected in adult mice myocardium,but its relationship with cardiac function and myocardial remodeling is unclear.The model of aortic arch coarctation（transverse aortic constriction,TAC）was established in wild-type（WT）and SM22αknockout(sm22α^-/-)mice,which is used to investigate the role of SM22αin myocardial fibrosis.Methods:1 Genotype identification of mice.SM22αheterozygous(sm22α^+/-)female and male mice aged 6-8 weeks were randomly selected and mated at 2:1 to generate the offsprings.The genomic DNA was extracted from the ears of 4-week-old mice.The genotypes of mice were identified through polymerase chain reaction（PCR）.2 Expression of SM22αin heart tissues and cardiomyocytesThe expression of SM22αin heart tissues was detected by Western blot and immunohistochemical staining（IHC）.0.05%clostridiopeptidase was used to isolate the cardiomyocytes from heart tissues at 37℃and we analyzed the expression of SM22αin the cardiomyocytes by Western blot.3 Establishment of model of transverse aortic constriction（TAC）in miceTwenty of adult male wild-type and 20 sm22α^-/-mice aged 8 weeks were randomly selected to establish TAC model by ligating the aortic arch region between the left common carotid artery and the arteriae anonyma,in which the sham operation groups were used as the control groups,with 10mice in each group.The left ventricular ejection fraction of mice was recorded on the day of operation and the day after 2 weeks and 4 weeks,respectively.4 Fibrosis staining of cardiac tissues of miceMasson’s trichrome stain was used to investigate the content of cardiac fibrosis.The staining results of fibrosis were counted by Image J software.5 Detection of collagen in mouse heart tissuesThe expressions of type I collagen and type III collagen in the heart tissues were measured via Western blot,quantitative real time polymerase chain reaction（qRT-PCR）and IHC,respectively.6 Statistical analysisAll the above experiments were repeated for three times.The measurement datas were expressed as mean±standard deviation.Student’s t-test was used to compare the mean in each group.There were statistical difference between the two groups when P<0.05.Results:1 Genotype identification of mice offspringThe PCR results showed that there was only one clear band near 500 bp in wild-type mice,only one clear band near 750 bp in sm22α^-/-mice,and two clear bands in sm22αheterozygous mice(sm22α^+/-)near 500 bp and 750 bp,respectively.2 SM22αexpressed in heart tissues and cardiomyocytesThe expression of SM22αwas detected not only in the heart tissues but also in cardiomyocytes of WT male mice by Western blot and IHC.3 Effect of sm22αgene deficiency on cardiac functionThere were no significant difference in LVEF of wild mice at 0 weeks,2weeks and 4 weeks after TAC（P>0.05）,but the significant difference in LVEF of sm22α^-/-mice were observed after TAC and declined more obviously with the prolongation of time.Compared with WT TAC mice,the LVEF of KO TAC mice descended significantly（P<0.05）,and declined more obviously as time went on.4 Effect of SM22αon fibrosis in TAC miceThe integral optical density（IOD）of the blue areas,stained by masson staining and analysed by Image J software,were observed in the heart tissues of WT sham,KO sham,WT TAC and KO TAC mice.The IOD in WT shamgroup and KO sham group were 10.18±1.13 and 12.19±1.22,respectively（P>0.05）.The IOD in WT TAC group and KO TAC group were 98.28±3.66and 220.37±1.55,respectively（P<0.05）.The results indicated that the deletion of sm22αgene did not significantly affect the ejection fraction of mice in sham group,but obviously promoted myocardial fibrosis in TAC mice.5 Expression of type I collagen and type III collagen in TAC model miceType I collagen and type III collagen,expressed in cardiac tissue of WT sham group,KO sham sham group,WT TAC group and KO TAC group mice,were detected by Western blot,qRT-PCR and IHC,respectively.Compared with WT sham group,there were no significant difference of type I collagen and type III collagen in KO sham group（P>0.05）.Compared with WT TAC group,the expression of type I collagen and type III collagen in KO TAC group were up-regulated significantly（P<0.05）.Conclusion:1 The deletion of SM22αdid not affect the cardiac function in physiological conditions.2 The deletion of SM22αpromotes pressure overload-induced cardiac fibrosis and cardiac dysfunction.