Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC) Cell Models for Research

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare inherited cardiomyopathy with an estimated prevalence of 1 in 2,000 to 1 in 5,000 in the general population, according to Orphanet and NCBI GeneReviews. It accounts for approximately 10-20% of sudden cardiac deaths in young athletes and up to 5% of all sudden cardiac deaths in individuals under 35 years. The disease is characterized by progressive fibrofatty replacement of the right ventricular myocardium, leading to ventricular arrhythmias and heart failure. Major risk factors include pathogenic variants in desmosomal genes, male sex, intense endurance exercise, and a family history of ARVC. According to the National Center for Biotechnology Information (NCBI), the median age at diagnosis is 30-40 years, and the lifetime risk of ventricular arrhythmia is high. Five-year survival rates vary; with early diagnosis and management, survival exceeds 90%, but undiagnosed cases may present with sudden cardiac death as the first manifestation. The NCI does not provide stage-based survival for ARVC as it is not a cancer, but the clinical course is marked by arrhythmic risk and progressive heart failure.

ARVC is an ideal disease for mechanistic studies due to its well-defined genetic basis, primarily mutations in desmosomal genes. Subtypes include classic ARVC, left-dominant arrhythmogenic cardiomyopathy (ALVC), and biventricular forms. Public datasets such as ClinVar, gnomAD, and the ARVC registry provide rich genetic and phenotypic data. Open questions include the molecular mechanisms linking desmosomal dysfunction to fibrofatty replacement, the role of exercise in disease penetrance, and the development of targeted therapies. The disease's monogenic nature facilitates the creation of gene-edited cell models to dissect pathways and test therapeutics.

ARVC is not a cancer, but the term carcinogenic pathways is not applicable. Instead, the pathogenesis involves desmosomal and non-desmosomal pathways. Key pathways include:

1. Desmosomal dysfunction: Mutations in desmosomal genes (PKP2, DSG2, DSP, DSC2, JUP) lead to impaired cell-cell adhesion, increased susceptibility to mechanical stress, and activation of apoptotic and necrotic cell death.

2. Wnt/β-catenin signaling: Desmosomal proteins interact with the Wnt pathway; their loss leads to decreased β-catenin signaling, promoting adipogenesis and fibrosis.

3. TGF-β signaling: Upregulation of TGF-β1 and TGF-β3 contributes to fibrosis and inflammation.

4. Calcium handling and ion channel remodeling: Dysregulation of calcium homeostasis and ion channels (e.g., RyR2, SERCA2a) promotes arrhythmogenesis.

These pathways are interconnected, leading to the classic ARVC phenotype of fibrofatty replacement and arrhythmias.

The table below summarizes high-frequency genetic alterations in ARVC, based on ClinVar and published cohort studies. Frequencies are approximate and vary by population.

Data compiled from ClinVar, COSMIC (for somatic mutations in related pathways), and published ARVC cohorts.

Key deregulated signaling networks in ARVC include:

• • Wnt/β-catenin pathway:
• • Reduced β-catenin nuclear translocation due to desmosomal protein loss.
• • Increased adipogenic differentiation of cardiac progenitor cells.
• • Downregulation of Wnt target genes (e.g., c-Myc, cyclin D1).
• • TGF-β pathway:
• • Upregulation of TGF-β1, TGF-β2, and TGF-β3.
• • Activation of SMAD2/3, leading to fibrosis.
• • Cross-talk with Wnt pathway.
• • Hippo/YAP pathway:
• • Dysregulation of YAP/TAZ contributes to cardiomyocyte proliferation and fibrosis.
• • Mechanical stress activates Hippo kinases, altering YAP localization.
• • Calcium signaling:
• • Abnormal RyR2 function leads to diastolic calcium leak.
• • Reduced SERCA2a expression impairs calcium reuptake.
• • Calmodulin-dependent kinase II (CaMKII) hyperactivation.
• • MAPK/ERK pathway:
• • Activated in response to stress, promoting apoptosis and fibrosis.
• • Potential target for therapeutic intervention.

Several cell lines and organoid models are used in ARVC research. The table below lists commonly used cell lines with their origin and key mutations.

Organoid advantages: 3D structure mimics native tissue, allows study of cell-cell interactions, fibrosis, and arrhythmias. Patient-derived iPSC organoids retain genetic background, enabling personalized drug testing.

Animal models are crucial for understanding ARVC pathogenesis and testing therapies. Examples include:

• • Genetically engineered mouse models (GEMMs):
• • PKP2 conditional knockout mice: Develop right ventricular dysfunction and arrhythmias.
• • DSG2 knockout mice: Show cardiomyocyte necrosis and fibrosis.
• • DSP knockout mice: Exhibit severe cardiomyopathy.
• • TMEM43 S358L knock-in mice: Recapitulate human ARVC with high penetrance.
• • Zebrafish models:
• • pkp2a and pkp2b mutants: Display desmosome defects and arrhythmias.
• • dsp mutants: Show cardiac dysfunction.
• • Rat models:
• • Transgenic rats overexpressing mutant PKP2: Develop ARVC-like phenotype.
• • Induced models:
• • Exercise-induced ARVC in mice: Endurance exercise accelerates disease.
• • Pharmacological models: Use of drugs to induce arrhythmias.

These models help study disease mechanisms and evaluate potential therapies.

CRISPR/Cas9 gene editing enables the creation of isogenic cell lines with precise mutations, offering powerful tools for ARVC research. Examples include:

• • PKP2 knockout human iPSC-derived cardiomyocytes: Generated by CRISPR, these cells show loss of plakophilin-2, disrupted desmosomes, and increased adipogenic gene expression. They are used to study the role of PKP2 in ARVC and to test drugs.
• • DSG2 knock-in with patient-specific missense mutation: Isogenic lines carrying the DSG2 c.1481A>G (p.Asn493Ser) mutation exhibit impaired cell adhesion and increased susceptibility to mechanical stress.
• • DSP knockout HEK293T cells: Used for biochemical studies of desmoplakin interactions.
• • Reporter lines: CRISPR knock-in of fluorescent tags (e.g., GFP) into desmosomal genes allows live-cell imaging of desmosome dynamics.

Commercially available, sequence-verified gene-edited cell models accelerate research by providing ready-to-use, validated tools. These models are engineered using CRISPR and other gene-editing technologies, ensuring reproducibility and reducing the time required for model generation. They are available from commercial sources without naming specific companies.

Gene-edited cell models are essential for functional genomics in ARVC. Knockout and knock-in lines allow researchers to:

• • Validate the pathogenicity of genetic variants identified in patients.
• • Dissect molecular pathways affected by desmosomal mutations.
• • Study gene-gene interactions and compensatory mechanisms.
• • Examples:
• • A PKP2 knockout iPSC line can be used to test whether reintroduction of wild-type PKP2 rescues the phenotype.
• • A DSG2 mutant knock-in line helps determine the specific effects of the mutation on cell adhesion and signaling.
• • CRISPR screens in ARVC patient-derived cells can identify modifiers of disease severity.

Isogenic pairs of ARVC cell lines (wild-type vs. mutant) are ideal for drug screening. They enable:

• • High-throughput screening of compound libraries to identify drugs that rescue disease phenotypes.
• • Assessment of drug efficacy and toxicity in a genetically defined background.
• • Modeling of drug resistance by introducing secondary mutations.

For example, isogenic PKP2 knockout and wild-type cardiomyocytes can be used to screen for small molecules that restore desmosome function or reduce arrhythmias. Resistance modeling can be achieved by chronic exposure to a drug and subsequent identification of resistance mutations via CRISPR screens.

CRISPR synthetic lethality screens in ARVC cell models can uncover novel biomarkers and therapeutic targets. By systematically knocking out genes in ARVC mutant cells, researchers can identify genes that are essential for survival only in the mutant context. These synthetic lethal interactions can reveal:

• • Potential biomarkers for early diagnosis.
• • New drug targets that selectively kill or rescue ARVC cells.
• • Pathways that contribute to disease progression.

For instance, a genome-wide CRISPR screen in PKP2 knockout cells might identify genes in the Wnt pathway as essential for the mutant phenotype, suggesting they could serve as biomarkers or therapeutic targets.

The following public databases provide valuable data for ARVC research.