Physiological Function Of Plackoglobin Revealed By Jup Mutation Mice

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is one of the most common inherited cardiomyopathies and a prevalent cause of sudden cardiac death (SCD) in young adults, reportedly contributing to up to10%of SCD in patients less than65years old. Almost half of the ARVC patients have a mutation in one or more of the genes encoding desmosomal proteins expressed in the heart, containing JUP, PKP2, DSP, DSC2and DSG2. On the basis of these finding ARVC is considered a disease of the desmosome. Patients suffering from ARVC have diminished PKG protein localization at ICD, regardless of their desmosomal gene mutation, suggesting an important role in disease progression. Thus ARVC has been proposed that PKG protein loss from the ICD may serve as a diagnostic marker of the disease.The first genetic mutation discovered leading to ARVC was a homozygous2base pair deletion in the JUP gene causing a frame-shift and premature truncation of the PKG protein, named Naxos disease, which characterized by distinct pathology, resulting in dramatic cardiomyocyte cell death and subsequent fibro-fatty replacement of the muscle. In addition to heart changes, Naxos patients exhibit palmoplantar keratoderma and woolly hair. Disorder.in desmosome structure affects tissue systems that undergo significant mechanical stress, implicating the heart and skin.Objective:To generate a mouse model carrying TG deletion mutation in Jup gene by the gene knock-in technique; to determine the role of PKG in cell adhesion and signaling pathways in Jup mutant mice; to give new insight into the molecular mechanisms of ARVC.Methods:Analyze mutations in Naxos patients; construct vectors with the mutation; electroporate the vector into mouse embronic stem cells; inject positive clones into mouse blastocysts; selection and identification chimera; obtain mutant mice carrying TG deletion mutation in Jup gene. Taking advantage of Western-blot, qRT-PCR, Masson trichrome staining and immuno staining, detect expression and location of intercalated disc-related proteins in mutant mice cardiomyocytes. Generate a new mutant mice carrying TG deletion and exon combined in Jup gene.Results:TG null mutation in Jup gene died within24hours after birth, with acantholysis. However, no apparent phenotype was observed by histology analysis, neither was ventricular arrhythmia by ECG analysis. During embryonic development, no apparent phenotype in heart was observed. The expression of truncated PKG in mutant cardiomyocytes was obviously down-regulated. No significant expression changes were observed for Colla, TGF-β1, NFκB, LPL, β-catenin, adiponectin, cyclinDl genes. However, expression of c-Myc was upregulated apparently. Also, in the mutant, expression of plakoglobin, desmoplakin, desmocollin2, desmoglein2and plakophilin2was down-regulated, with up-regulated β-catenin in protein expression. No significant expression changes were observed of aE-catenin pan-cadherin、Connexin43、ZO-1、desmin、ILK、PINCH-1、β1D-integrin. In addition,β-catenin accumulated in intercalated disc, with no PKG and CX43accumulation observed.The mutants, carrying TG deletion and exon combined in Jup gene, survive more than two months after birth and express truncated PKG protein, with similar quantity of PKG in the control. Conclusion:1. Homo-mutant mice carrying TG deletion in Jup gene died at PD1with acantholysis, but no obvious cardiac phenotype.2. Protein expression of truncated PKG in mutant heart was distinctly reduced, with that of desmosomal components containing DSC2, DSG2and DSP.3. Protein expression of β-catenin in mutant heart increased, with upregulating of canonical Wnt signaling target gene c-Myc.4. Homo-mutant mice carrying TG deletion and exon combined in Jup gene survived more than two months and expressed truncated PKG protein, with similar quantity of PKG in the control.