Desmoglein 2 mutant mice reproduce arrhythmogenic right ventricular cardiomyopathy patients' phenotype

Abstract

Abstract Background Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is inherited cardiac disease with unresolved treatment. ARVC is progressive and leads to lethal arrhythmias and terminal heart failure. Most often encountered ARVC mutations are in desmosomal genes [1]. Purpose We aimed to generate transgenic mice with exact copy of genetic defects found in Japanese cohort of ARVC patients, describe the phenotype, and identify target for curative therapy. Methods CRISPR/Cas9 genome editing was used to generate knock-in mice with the two most common point mutations in Japanese ARVC patients: DSG2 292R>C and 494D>A [2] (mouse equivalent positions are 297R and 499D, respectively). To analyze the phenotype of mice we used imaging techniques - cardiac echography and MRI as well as telemetry, treadmill, immunohistochemistry, confocal microscopy, histology, Western blot. Results In vivo observations of transgenic mice demonstrated inequalities in phenotypical presentation between the two mutations. Some of 297C mice (both hetero- and homozygous) died suddenly, starting from the age of 9 weeks, in contrast to surviving all 499A carriers. Dissection of suddenly died mice reveals enlarged cardiac cavities, mainly in the right heart. In addition, 297C homozygous hearts present with pale zones scattered all over the heart. Paraffin sections from this hearts stained with hematoxilin/eosin and Masson's trichrome show myocardial areas with absent myocytes, collagen accumulation and calcifications. With exception of homozygous 297C mice, spontaneous development of cardiac phenotype in Dsg2 knock-in mice starts after 25th weeks of age. Both mutant mice gradually developed cardiac dysfunction (echography and MRI, Fig. 1) and echographically visible left ventricular wall infiltrations. Heterozygous 297C mutation produces more severe phenotype that develops earlier. Similar to human ARVC, the degree of cardiac damage vary significantly. It is known from the human ARVC that physical activity aggravates the ARVC phenotype and may cause sudden cardiac death. To evaluate the effect of physical stress on the phenotype of Dsg2 knock-in mice we subjected 11 weeks old animals on treadmill exercise. Training for 8 weeks provoked development of heart failure in both 297C and 499A mutant mice, significantly earlier than natural progress of the phenotype. No mouse died suddenly. Telemetry experiment demonstrated electrical instability in 297C homozygous hearts showing conduction and rhythm abnormalities (Fig. 2). Apoptosis was detected in both mutant mice by TUNEL staining and confocal microscopy. Dsg2 protein expression was not affected by the mutations. Conclusion We generated mouse model of ARVC that reproduces exact genetic defect of the human disease. There are significant similarities between our model and ARVC patients' phenotype. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Japanese KAKENHI funding