A Preliminary Study Of The Pathological Phenotype And Pathogenic Mechanism Of The DSG2 F536C Knock-In Mouse Model

Part I:Construction and pathological phenotype analysis of DSG2^F536C Knock-In miceBackground:Arrhythmogenic right ventricular cardiomyopathy（ARVC）,characterized by progressive ventricular fibrofatty replacement and various ventricular arrhythmias,is a common reason for cardiac sudden death.In recent years,molecular genetics researchs had revealed that more than 50%of ARVC patients carry desmosomes gene mutation.In our previous study,we screened the patients with ARVC in DS family of Chinese Han.We found that the Desmoglein（DSG2）gene mutated c.1592T>G,leading to the substitution of cysteine at position 531 by phenylalanine（DSG2F531C）.But whether the mutation is pathogenic,there still lack direct evidence.Objective:To establish the DSG2^F536C Knock-In mouse model,and to observepathological phenotype characteristics of the mouse heart.Methods:DSG2^F536C Knock-In mice were generated through homologous recombination of target gene Knock-In strategy.And the genotypes of DSG2^F536C Knock-In mice were identified by PCR amplification of mouse tail DNA.The pathological characteristics of DSG2^F536CKnock-In mice were detected by Masson’s trichrome staining,H&E staining and Oil Red O staining.The ultrastructures of mouse heart were observed by transmission electron microscopy（TEM）.The expression and subcellular localization of DSG2 protein in mouse heart were detected by Immunohistochemistry（IHC）and Western blot respectively.Results:DSG2^F536C Krnock-In mice with three genotypesWere generated,including homozygous mice（DSG2m/m）,heterozygous mice（DSG2m/+）and wild-type mice（DSG2+/+）.The results are as follows:（1）Routine pathology:At 2 weeks of age,no significant abnormality were observed in DSG2+/+ mice,DSG2m/+ mice and DSG2m/m mice.At 10 weeks of age,DSG2m/mmice were observed to have disordered cardiac muscle,fibrosis and inflammatory cellinfiltrates of cardiac myocytes in the right ventricle,and left ventricle was affected in 1/3 of them,accumulation of fat droplets could be visible in fibrous area,but DSG2m/+mice were noted to have disordered cardiac muscle only.（2）collogen volume fraction（CVF）:Compared with DSG2+/+ and DSG2m/+ mice（p<0.001）,CVF in right ventricular of DSG2m/m mice were significantly increased（DSG2+/+ vs.DSG2m/m,3.46%± 1.94%vs.24.72%±2.23%,p<0.001;DSG2m/+ vs.DSG2m/m,3.14%± 1.46%vs.24.72%± 2.23%,p<0.001）,but there was no significant difference between DSG2+/+ and DSG2m/m mice（p= 0.84）.Moreover,there was no statistically significant difference among three groups for CVF in left ventricular（p = 0.31）.（3）TEM:DSG2+/+ and DSG2m/+micecould observed typical desmosome structure,while DSG2m/m mice could not detected the similar structure,and could discovered significantly widened of intercalated disk and reduction of mitochondria.（4）IHC and Western blot:IHC results displayed that expression of DSG2 were not found in DSG2m/m mice intercalated disks area,but normalized in DSG2+/+ and DSG2m/+ mice.Western blot results showed that compared with DSG2+/+ mice（0.482±0.188）and DSG2m/+ mice（0.388±0.090）,expression of DSG2 protein in the heart of DSG2m/m mice（0.004±0.003）were almost not expressed,but there are no significant difference between DSG2+/+ mice and DSG2m/+ mice（p>0.05）.Conclusions:At 10 weeks,DSG2m/m mice were detected pathological phenotype similar to those of humans ARVC,including right ventricular dominated inflammatory response,calcification,ventricular fibrosis and lipid droplet infiltration;and abnormal desmosome structure,widened of intercalated disk and reduction of mitochondria;DSG2 protein expression abnormalities,it indicated that the mutation are pathogenic.It indicates that DSG2^F536C might be a pathogenic mutation of ARVC.In addition,DSG2^F536C Knock-In mice could be a useful model for the study of the pathogenesis of ARVC.Part Ⅱ:Preliminary study on the pathogenesis of DSG2^F536Cmutation induced ARVCBackground:It is known that the pathogenesis of arrhythmia right ventricular cardiomyopathy（ARVC）involves the regulation of multiple signaling pathways,including inhibition of Wnt signaling pathway,activation of Hippo signaling pathway,down-regulation of iASPP signaling pathway and activation of TGF-β1 signaling pathway.We successfully constructed the DSG2^F536C Knock-In mice with inflammatory response,calcification,ventricular fibrosis and lipid droplet infiltration,which are similar to the phenotype of human ARVC,suggesting that the mutation is pathogenic mutation.However,the molecular mechanism of DSG2^F536C to ARVC is not yet clear.Objective:To explore whether the pathogenesis of ARVC induced by DSG2F636C mutation involves Wnt signaling pathway,Hippo signaling pathway,iASPP signaling pathway and TGF-β1 signaling pathway.Methods:The DSG2^F536C Knock-In mice were used as experimental animal model,including:homozygous mice group（DSG2m/m）,heterozygous mice group（DSG2m/+）and wild-type micegroup（DSG2+/+）.To observe the expression of the main protein for each signaling pathway:（1）Wnt signaling pathway:The subcellular localization of plakoglobin（PG）were measured by IHC.The expression of PG in mouse heart were detected by Western blot.（2）Hippo signaling pathway:The expression of Yes associated protein（YAP）and its phosphorylated products（P-YAP）were observed by Western blot（3）iASPP signaling pathway:The expression of inhibitor of apoptosis-stimulating protein of p53（iASPP）were evaluated by Western blot（4）TGF-β1 signaling pathway:The expression of transforming growth factor β1（TGF-β1）were studied by Western blot.Results:（1）Wnt signaling pathway:The results of IHC showed that PG were located ordinarily in the heart intercalated discsarea,and the results of Western blot were observed no significant difference in the expression of PG protein among the three groups（p>0.05）;（2）Hippo signaling pathway:Western blot results showed that the expression of P-YAP,YAP and P-YAP/YAP were no significantly different among three groups（p>0.05）（3）iASPP signaling pathway:Western blot results suggested that the expression of iASPP were no statistically different among three groups（p>0.05）.（4）TGF-β1 signaling pathway:Western blot results showed that compared with DSG2+/+ mice,the expression of TGF-β1 in DSG2m/+ and DSG2m/m mice were significantly increased（DSG2+/+ vs.DSG2m/+,0.202±0.010 vs.1.048±0.444,p<0.05;DSG2+/+ vs.DSG2m/m,0.202±0.010 vs.0.814±0.347,P<0.05）,but there was no statistically different between DSG2m/+ and DSG2m/m,mice（p>0.05）.Conclusion:The activation of TGF-β1 signaling pathway may be involved on the pathogenesis of DSG2^F536C induced ARVC of DSG2^F536C Knock-In mice.This study provides a basis for further study on the pathogenesis of DSG2^F536C induced ARVC.