Zurich ARVC Program

Establishing an Academic ARVC Program in Zurich

We have initiated an interdisciplinary clinical and scientific Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) Program at the University Hospital Zurich in order to give greater consideration to this underestimated disease. This program’s clinical and research goals include the optimization of the interdisciplinary team-work for complex cases, state-of-the-art imaging, optimal therapy for progressive disease, ICD-therapy, catheter ablations, genetic testing and genetic counseling. We have started assembling a large clinical registry of ARVC patients in order to investigate clinical and genetic factors conferring an increased risk for disease manifestation, progression and a worse outcome. Furthermore, we have installed a multicenter ARVC biobank to study the molecular basis of ARVC and to define novel molecular mechanisms implicated in this complex disease.

Our multidisciplinary Zurich ARVC Program is kindly supported by the “Georg und Bertha Schwyzer-Winiker Stiftung” and the University of Zurich.

Research projects

Several research projects are ongoing at our institution. Our goal is to improve our understanding of ARVC at different stages of the disease and to improve treatment and risk stratification for potentially lethal arrhythmias and heart failure.

All the data that is going to be obtained via these research projects will be kept confidential and stored in an anonymized manner. The electronic database is coordinated by the Clinical Trials Center (CTC) Zurich. Ongoing studies were approved by the Zurich local ethical committee (KEK Zurich). We have been or are currently conducting the following studies:

- Genotype-Phenotype Correlation (details below)
- Immunohistochemistry of the intercalated disc (details below)
- Search for tissue and serum biomarkers for early diagnosis and disease prognosis by using Next Generation RNA and Protein Sequencing
- 2/3-D Echo studies and strain imaging
- Electrocardiographical predictors of arrhythmias
- Electrophysiological properties of ARVC patients
- Electroanatomical voltage mapping (details below)
- Development of a novel in-vitro silicone right heart model

Development of a novel silicone right heart model

Since 2015 there is an ongoing collaboration with the Environmental Fluid Mechanics (EFM) group at the Institute of Environmental Engineering (IFU) at ETH Zurich.. The group has already studied different cardiovascular flow conditions, e.g. aneurysm, valve dysfunction, and aortic stenosis, as well as physiological flows and orientation of heart valves in an aortic phantom under clinically realistic conditions using three-dimensional particle tracking velocimetry (3D-PTV). There is considerable joint interest to develop an in vitro right heart model to study structural RV diseases as well as the impact of sports on RV physiology.
Recent data suggests that regional structural alterations in the RV may not only be found in patients with ARVC, but also occur in apparently healthy endurance athletes in the absence of desmosomal mutations. The extent of these alterations seems to be associated with the type, intensity and duration of exercise. Abnormal blood flow and mechanical stresses may contribute to structural alterations in the athlete’s heart albeit to a different extent compared to patients with ARVC. Therefore, it seems reasonable to create a right heart model to study the effects of abnormal blood flow and mechanical stresses on the development and progression of structural remodeling in ARVC as well as in the athlete’s heart using similar experimental setup.
In the clinical setting non-invasive cardiac imaging tools such as cardiac magnetic resonance imaging (MRI) are also capable of showing RV structural alterations and blood flow patterns. Nevertheless, blood flow patterns in the RV assessed by MRI have not been validated by in vitro models. The creation of a right heart model to study flow dynamics and mechanical stresses may increase our understanding of the processes that initiate structural alterations in RV diseases as well as adaptation of the healthy heart to excessive exercise. Furthermore, such a model may also be used to select optimal MRI parameters for visualization and quantification of abnormal blood flow, which may have clinical implications with respect to choice of type and degree of exercise for affected subjects. This project is supported by a grant from the Swiss National Science Foundation (SNF).

Electroanatomical voltage mapping (EAM)

A gold standard for diagnosis of ARVC is still lacking and currently, diagnosis is made according to the 2010 revised Task Force Criteria. However, the authors of these revised criteria emphasize that current criteria are “not perfect”, still lead to underrecognition of the disease and that future improvements in diagnostic tools are urgently needed, especially for depicting early stages of the disease. In recent years, EAM has been used to define pathological myocardial substrate. It is an evolving elegant minimal-invasive technique increasingly performed in patients with suspected ARVC in order to detect low voltage areas corresponding to fibrofatty tissue in subtle ARVC. Several studies have addressed the ability of unipolar and bipolar electroanatomic mapping to identify dysplastic areas identified by echocardiography, CMR and endomyocardial biopsy in ARVC. In symptomatic ARVC patients, success of ventricular tachycardia ablation can be increased with EAM.
Currently, two electroanatomic systems are available: the commonly used CARTO system (Biosense Webster Inc., Diamond Bar, California) and the EnSite NavX system (St. Jude Medical).

EAM may importantly contribute to early diagnosis of ARVC, but did not find its place in current task force criteria yet. This can be explained by the fact that the method, as currently performed, has certain limitations:
- varying cut-off values (for bipolar RV signals between 1.2 and 1.5mV)
- Previous studies were performed in a relatively small number of patients (n < 35)
- There are important statistical flaws associated with previous studies that defined reference values
- The way EAM was performed in these studies did not add much information for diagnosis of ARVC. With conventional catheters it is very difficult to distinguish between low voltage areas resulting from poor tissue contact and low voltage resulting from scar and fibrosis. This problem is particularly important when mapping the perivalvular region of the pulmonary and tricuspid valve, as catheter contact with the myocardium is difficult in these regions. Novel catheters that we use at our institution may overcome these problems.

Genotype-Phenotype Correlation

Understanding the Genetic Basis of Sudden Cardiac Death: Genetic Predictors of Risk and Novel Mechanisms Implicated in Arrhythmogenic Right Ventricular Cardiomyopathy
Sudden cardiac death (SCD) is a major public health issue worldwide. Near 80% of SCD are attributed to coronary artery disease. Nevertheless, in children and adolescents between the ages of 1 and 22 years, SCD account for 11% of all deaths. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart-muscle disease that is a cause of sudden death in young people and athletes. The disease is characterized by either massive or partial progressive replacement of myocardium by fatty or fibro-fatty tissue. This infiltration provides a substrate for electrical instability and leads to life-threatening ventricular arrhythmias. In the last two decades the extraordinary advances in molecular biology of ARVC have provided significant insights into our understanding of the disease etiology. Interactions between mechanical disruption of cell-cell adhesion and defects of desmosomal-mediated intracellular signalling are likely to be involved in the pathogenesis of the ARVC phenotype. To date, 10 genes associated with ARVC have been described, of which 5 contain 90% of the reported mutations. Overall, ~50% of the patients still remain genotype elusive. The discovery of the causative genes for ARVC offers the possibility of identifying genetically-affected individuals before potentially malignant clinical phenotype occurs. Early detection of ARVC and preventive therapy of young individuals at highest risk of experiencing sudden cardiac death may be improved by molecular genetic screening within affected families and may alter the clinical management of patients.

Methods
This project aims to perform a genotype-phenotype correlation and to elucidate novel genetic mechanisms in ARVC that may help to understand the pathophysiological basis of the disease to aid in clinical stratification and potentially improve the treatment. For our aim 1, we plan to perform a genotype-phenotype correlation in a large cohort of ARVC patients with the hypothesis that patients with ARVC and different genetic background have different clinical outcome. For this aim we will perform a comprehensive genotype analysis of a large ARVC using polymerase chain reaction (PCR), and direct DNA sequencing of the 5 major ARVC-associated genes: Plakoglobin (JUP), Desmoplakin (DSP), Plakophilin-2 (PKP2), Desmoglein-2 (DSG2) and Desmocollin-2 (DSC2). Our aim 2 focuses on the identification of novel ARVC genes through next generation sequencing. We hypothesize that other mechanisms may be implicated in the ARVC pathophysiology, since 60% of the cases with clear phenotype remain genetically elusive. Families with ARVC, who remain genotype negative, are excellent candidates for this novel approach. Lastly, in our aim 3, these results will be validated in the remaining genotype negative cohort, we will perform comprehensive genetic analysis of the coding region of the novel putative genes detected in aim 2.

Novel Biomarkers for diagnosis and prognosis

ARVC patients have several mutations in genes encoding desmosomal proteins. The most frequent mutations are found in the plakophilin-2 (PKP2), desmoplakin (DSP) and desmoglein-2 (DSG2) gene. We performed gene expression profiling of proteins that are part of the desmosomes, adherens junctions, gap junctions and tight junctions. Cards with 384 reaction chambers with specific primers for 64 different mRNAs are utilized. The RNA analyses are performed in a two-step RT-PCR process: 1. Reverse trancription of cDNA from total RNA sample. 2. Real-Time PCR products are synthesized using TaqMan® Universal PCR Master Mix. For immunohistochemistry studies, specific monoclonal antibodies binding to proteins using FFPE (Formalin-Fixed, Paraffin-Embedded) samples are utilized using both direct and indirect methods with antibodies labelled with a fluorescent dye. Our first results indicate that non desmosomal tissue markers may exist in ARVC patients (Figure 1).
In a next step our aim is to measure all mRNAs and proteins in ARVC patients´ heart tissue and compare it to patients with dilated cardiomyopathy and healthy controls in order to search for molecules that are differently regulated, and may interact in similar disease pathways. This will be done in order to have novel insights into disease mechanisms and to find novel biomarkers for early diagnosis and disease progression.