Human-specific desmoglein 2 mutations in mice models of arrythmogenic right ventricular cardiomyopathy reproduce patients" phenotype

Funding Acknowledgements Type of funding sources: None. Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease that inevitably leads to terminal heart failure and sudden cardiac death due to ventricular arrhythmias. Excessive exercise accelerates the progress of the disease. At present, there is no curative therapy for ARVC. The disease is mostly caused by mutations in genes encoding desmosomal proteins, and Japanese ARVC patients commonly possess missense mutations in DSG2. Although there are animal models with Dsg2-related cardiomyopathy, they do not reflect the precise human disease caused by missense mutations in DSG2. Purpose We aimed to generate knock-in mouse models of ARVC carrying Dsg2 mutations equivalent to those found in ARVC patients. Methods We introduced Dsg2 mutations (R297C, D499A), which were equivalent to most common mutations in Japanese ARVC patients, to mice by CRSPR/Cas9 genome editing system. Echocardiography and treadmill training were performed to evaluate the cardiac phenotype of the mouse models. Results There is phenotype inequality between the two lines of Dsg2 knock-in mice. In R297C mice, all the homozygotes died suddenly until the age of 20 weeks, and half of the heterozygotes until the age of 25 weeks. Heart examination of the deceased homozygous mice demonstrated enlarged cavities, predominantly in the right ventricles, and scattered pale patches suggesting fibrotic replacement of the myocardium. Echocardiography of four 9-week-old homozygous mice shows acoustically dense zones (white arrows in the left figure) and left ventricular dysfunction (ejection fraction (EF) < 35%). Exploration of the hearts of sacrificed mice confirmed echocardiographical findings (red arrow in the right figure) demonstrating scattered pale regions similar to those in suddenly died mice. In contrast, homozygous and heterozygous D499A mice survived without sudden death. Both mutant mice develop heart failure after the age of 30 weeks. To mimic the effect of exercise, we performed treadmill training for two months started from 11 weeks old in both types of D499A and heterozygous R297C mice. The training significantly accelerated cardiac dysfunction: all D499A mice (homo and hetero) and half of heterozygous R297C developed biventricular dysfunction at the age of 19 weeks though no change in WT. EF for R297C heterozygous mice, D499A heterozygous and D499A homozygous mice decreased from 64.4 ± 4.3%, 71.9 ± 1.7% and 73.9 ± 1.1 to 31.2 ± 5.7%, 16.6 ± 2.1% and 12.4%±1.9, respectively. Treadmill training did not alter significantly EF in WT mice: 71.8 ± 2.3% before and 67.5 ± 1.4 at the end of physical challenge. Conclusion We generated mouse model of ARVC mutations that mimic the exact gene defect found in the humans. Our experimental work confirms significant overlap to the human phenotype. Further investigation will clarify the underlying cellular mechanisms. Abstract Figure. A heart from homozygous R297C mouse