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Early Cardiac Inflammation As a Driver of Murine Model of Arrhythmogenic Cardiomyopathy

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Early Cardiac Inflammation As a Driver of Murine Model of Arrhythmogenic Cardiomyopathy bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.169664; this version posted June 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Early cardiac inflammation as a driver of murine model of Arrhythmogenic Cardiomyopathy K.E. Ng1,2, P.J. Delaney2, D. Thenet2, S. Murtough2, C.M.Webb2, E.Tsisanova1, S.L.M Walker1, J.D. Westaby4, D.J Pennington2, R.Pink3, D.P. Kelsell2* & A. Tinker1* 1William Harvey Research Institute, Barts & The London School of Medicine & Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. 2Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, London, E1 2AT, UK. 3Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Headington Campus, Oxford, OX3 0BP. 4CRY Dept. of Cardiovascular Pathology, Cardiology Clinical Academic Group, Molecular and Clinical Sciences Research Institute, Jenner Wing, St. George’s University of London, Cranmer Terrace, London,SW17 0RE. *Joint senior and corresponding authors: Professor Andrew Tinker E-mail: [email protected] and Professor David Kelsell E-mail: [email protected] Running title: Desmoglein 2 and cardiac inflammation Keywords:, Desmoglein 2 (Dsg2), arrhythmogenic cardiomyopathy (AC), desmosome, cardiac inflammation, macrophages, fibrosis, rhomboid family member 2 (iRHOM2) , ADAM metallopeptidase domain 17 (Adam17/TACE), tumor necrosis factor alpha (TNFα), interleukin-6 (Il-6), RNA-Sequencing (RNA-Seq). 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.169664; this version posted June 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Abstract: Both may present with prominent extracardiac features involving the skin (Palmoplantar The study of a desmoglein 2 murine model of keratoderma) and/or woolly hair. The genetic arrhythmogenic cardiomyopathy revealed cardiac basis for the disease was first revealed by the inflammation as a key early event leading to study of autosomal recessive forms of the fibrosis. Arrhythmogenic Cardiomyopathy (AC) cardiocutaneous form of AC and demonstrated is an inherited heart muscle disorder leading to loss of function mutations in desmoplakin ventricular arrhythmias and heart failure due to (Carvajal syndrome) and plakoglobin (Naxos abnormalities in the cardiac desmosome. We disease) (3, 4). Both of these genes encode examined how loss of desmoglein 2 (Dsg2) in the proteins that are part of the desmosome. This young murine heart leads to development of AC. prompted studies in autosomal dominant AC Cardiomyocyte apoptosis was an early cellular including the examination of other desmosomal phenotype and RNA-Seq analysis revealed early genes and revealed mutations in other activation of inflammatory-associated pathways components of the cardiac desmosome including in Dsg2 null (Dsg2-/-) hearts at postnatal day 14 desmoglein (DSG2), desmocollin2 (DSC2) and (Two weeks) that were absent in the fibrotic heart plakophilin2 (PKP2) (5-7). Desmosomes tether of adult mice (Ten weeks). This included cardiomyocytes together by linking cells at the upregulation of iRhom2/ADAM17 and its intercalated disc with the intermediate filaments, associated pro-inflammatory cytokines and specifically desmin, in the cytoskeleton (8). receptors such as TNFα, IL6R and IL-6. Together with gap junctions and adherens Furthermore, genes linked to specific junctions in the area composita of the intercalated macrophage populations were upregulated. This disc; they provide mechanical and electrical suggests cardiomyocyte stress triggers an early connection between cells (9). immune response to clear apoptotic cells AC can be a largely asymptomatic allowing tissue remodelling later on in the fibrotic dormant condition until the initial cardiac episode heart. Our analysis at different disease stages occurs in later life. These disease triggers implicate inflammation related to loss of underlying AC are still largely unknown though desmoglein 2 as a major mechanism for disease exercise and/or infection are proposed external progression. factors with desmosomal dysregulation and cell adhesion as likely consequences. Early Introduction: observations from myocarditis patient biopsies previously suggested the involvement of a Arrhythmogenic cardiomyopathy (AC), also systemic viral trigger however; no research has referred to as arrhythmogenic right ventricular been able to confirm this as the cause (10-13). cardiomyopathy (ARVC), is an inherited heart Interestingly, subsequent studies described the muscle disease characterised by ventricular presence of inflammatory infiltrates in post arrhythmias notably ventricular tachycardia and mortem biopsies from patients with AC (13). fibrillation and later in the disease process heart Interestingly, clinical reports of myocarditis in failure (1, 2). It is often a cause of cardiac arrest recent paediatric cases highlight the connection in young athletes. The condition appears to have between inflammation and AC (14). The acute arrhythmogenic phases before a decline in examination of the early cardiac development in ventricular function that occurs later in the murine models of AC may provide further insight disease. Pathologically it is characterised by to the mechanisms that link the loss of cardiac chamber dilatation and fibrofatty desmoglein 2 and the inflammatory response. replacement of myocytes. Historically, it was There are a number of murine models for thought to be a predominantly a right ventricular AC, including those involving genetic disease but biventricular and left ventricular manipulation of Dsg2 (15-20), that have been patterns are now widely recognised (1, 2). The developed and studied. Global genetic deletion of disease is hereditary in origin occurring in about Dsg2 is embryonically lethal whilst 1 in 5,000 births and is generally autosomal heterozygotes with haplo-insufficiency appear dominant though recessive forms are recognised. normal (17). In contrast, transgenic mice 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.169664; this version posted June 26, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. overexpressing a missense dsg2 mutation or a response has not been examined in detail in these knock-in mouse lacking a domain of Dsg2 transgenic mice. We hypothesized the immune develop cardiac dysfunction similar to that seen response and stress pathways in the weeks in AC (15, 18). AC can be characterised by acute following birth may play a larger role in the episodes of worsening pathology punctuating a immediate stages of the disease than initially more stable disease course (1). In this study, we reported (19). We studied the identity of this examine mice with the cardiac specific deletion inflammatory infiltrate at two weeks of age and of Dsg2 (19). We describe experimental evidence how inflammation may be associated with the and identify an early inflammatory response to progressive changes that lead to cardiac cardiomyocyte apoptosis that leads to cardiac dysfunction in the adult mouse (Ten weeks). fibrosis over time. Adult Dsg2-/- hearts exhibit fibrosis whereas Results: adult Dsg2+/+ and Dsg2+/- hearts appear morphologically similar Mice harbouring the tm1c allele for the Dsg2 gene (Supporting Figure S1A) were crossed with We first examined this murine model of AC by the alpha MHC Cre mice (21) and then bred to a directly comparing three groups: Dsg2 WT second generation to give the study cohort: Cre+, (Dsg2+/+), heterozygous (Dsg2+/-) and knockout Dsg2flx\flx (Dsg2-/-) or Dsg2flx\flx (Dsg2+/+) mice. (Dsg2-/-) mice at ten weeks (Figure 1). The results from morphometric analysis showed Histological analysis with i) Haematoxylin and no differences between the adult Dsg2+/+ and Eosin and ii) Trichrome stain revealed prominent Dsg2-/- groups (Supporting Figure S1B). We also structural differences between Dsg2+/+ and Dsg2- generated a small cohort of heterozygous mice /- hearts (Figure 1A). The changes in the Dsg2-/- (Dsg2+/-) to examine cardiac function and the heart were pronounced at adulthood with effect of gene dosage in AC disease development evidence of myocyte death and interstitial fibrosis (20). Most studies to date have characterised this i), collagen deposits ii) and in some severe cases, murine model therefore, we initially focused on calcification which appear as dark purple deposits recapitulating this information. DAB staining, within the fibrotic area. No abnormal RNA-Seq and qPCR confirmed substantial morphological changes or signs of fibrosis were decreases in transcripts translated into protein for observed in the Dsg2+/+ and Dsg2+/- hearts. Dsg2 (Supporting Figure 1C). The short axis The echocardiographic assessments from view of Dsg2 +/+ and Dsg2-/- hearts shows two the three genotypes are shown in Figure 1B. We morphologically different hearts at ten weeks of measured cardiac function in younger juvenile age. At post-mortem, the majority of Dsg2-/- mice (Six weeks) and adult mice (Ten weeks). hearts showed chamber dilatation in the left The heterozygous group were also included in the ventricle and sometimes right ventricle. Some analysis to reveal if the decline
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