Arrhythmogenic cardiomyopathy, historically known as arrhythmogenic right ventricular cardiomyopathy, is an autosomal dominant genetic disorder affecting an estimated 1 in 1,000 to 1 in 5,000 people. The disease is characterized by frequent ventricular arrhythmias, progressive ventricular dysfunction, and the gradual replacement of heart muscle with fibrofatty tissue, a combination that carries a high risk of sudden cardiac death. Most patients develop symptoms during adolescence or early adulthood, although genetically predisposed individuals may remain asymptomatic until later in life. Epidemiological studies have shown that young athletes face a significantly higher risk of sudden cardiac death than non-athletes, largely because structural abnormalities of the heart predispose them to unstable arrhythmias under physical stress.

The plakophilin-2 gene, abbreviated PKP2, encodes a protein belonging to the arm-repeat family that contains a highly conserved Armadillo domain. It is abundantly expressed in cardiac tissue and serves as a critical molecular linker between desmosomal cadherins, desmoplakin, and the intermediate filament system. Together with desmosomes, fascial adhesion structures, and gap junctions, these proteins form the intercalated disc, the specialized junction that maintains both the mechanical integrity and the electrical coupling of cardiac tissue. Truncating mutations in PKP2, which often lead to loss of one functional copy of the gene, are considered the primary genetic cause of the disease, accounting for approximately 19 to 46 percent of cases in adult cohorts.

Mouse models have dominated mechanistic research on PKP2-associated disease, but they come with intrinsic limitations. Mice have an average heart rate of roughly 600 beats per minute, far faster than the human range of 60 to 90 beats per minute, and their hearts lack epicardial fat, making it difficult to reproduce the characteristic fibrofatty infiltration seen in patients. Zebrafish, by contrast, have an average heart rate of about 100 beats per minute and possess cardiac adipose tissue that is evolutionarily conserved with that of humans. They also share key electrophysiological characteristics with the human heart, breed prolifically, and are well suited to large-scale genetic and pharmacological screening. While a transient morpholino-based knockdown of pkp2 had been reported in zebrafish embryos, no genetically stable pkp2 mutation model existed until now.

Before building the model, the research team analyzed publicly available single-cell RNA sequencing datasets from human, mouse, and zebrafish hearts to compare PKP2 expression across species. The comparison revealed marked interspecies differences. Human PKP2 was highly expressed in most cardiomyocytes, whereas in mice the gene was active only in a small subset of these cells. Zebrafish pkp2 expression was similarly restricted to a limited number of cardiomyocytes, with additional expression in endocardial cells. Amino acid sequence comparisons showed that zebrafish Pkp2 shares 39.1 percent homology with the human protein, while the mouse ortholog shares 81.3 percent identity, underscoring the evolutionary divergence in this gene’s function and expression patterns.

To generate the model, the researchers used CRISPR/Cas9 gene editing to introduce a truncating mutation in exon 3 of the zebrafish pkp2 gene. A single-guide RNA targeting that exon produced a four-base-pair deletion, causing a frameshift at amino acid 250 and premature termination of the protein at amino acid 267, a change designated p.Glu250Glyfs*17. Quantitative RT-PCR showed that pkp2 mRNA levels were not significantly altered in mutant hearts, suggesting that nonsense-mediated mRNA decay was not efficiently triggered. Homozygous mutant fish, designated pkp2e3/e3, displayed gross morphology comparable to their wild-type siblings, maintained normal reproductive capability up to at least one year of age, and showed no significant difference in baseline survival up to 12 months.

Despite their outwardly normal appearance, the mutant hearts told a different story. Prominent lipid accumulation appeared on the ventricular surface, particularly in homozygous mutants, and Oil Red O staining confirmed lipid deposits within the ventricular myocardium of both homozygous and heterozygous fish. Transmission electron microscopy revealed marked ultrastructural abnormalities of the intercalated discs, including abnormal extension and twisting, and the desmosomal gap width was significantly increased compared with controls. Quantitative RT-PCR analyses showed significant upregulation of the inflammation-related genes myd88 and caspa, along with significant upregulation of the Wnt signaling genes gsk3bb and axin2. The authors caution that these molecular changes should be interpreted as supportive evidence accompanying the observed phenotypes rather than conclusive mechanistic drivers of the disease.

Functional testing deepened the picture. High-frequency echocardiography at 6 months of age showed that ejection fraction, fractional shortening, and fractional area change were all significantly reduced in both homozygous and heterozygous mutants, with stroke volume also decreased. Cardiac output and ventricular ejection time were significantly increased in homozygotes but not in heterozygotes. Electrocardiogram recordings from homozygous mutants revealed abnormal features including sinus arrest and T-wave inversion: 48 percent of homozygotes displayed ECG abnormalities, with 36 percent showing sinus arrest and 12 percent showing T-wave inversion, compared with only 6 percent of wild-type siblings showing sinus arrest. The mutants also had significantly reduced heart rates along with prolonged QRS duration and PP intervals.

The exercise experiments produced some of the most clinically resonant findings. In a swimming endurance test, 6-month-old homozygous mutants showed significantly reduced swimming capacity compared with wild-type siblings, and 45 percent of mutants died suddenly immediately after the test, versus 10 percent of controls. Electrocardiogram monitoring 30 minutes after swimming revealed that only 33 percent of mutants displayed normal ECG patterns, while the rest showed T-wave height tip, T-wave inversion, sinus arrest, or tachycardia. The authors note that although these post-exercise abnormalities indicate electrical instability, they do not establish a direct causal relationship between arrhythmias and sudden death; hemodynamic instability or acute physiological stress responses may also contribute. To rule out allele-specific effects, the team generated a second independent truncating allele carrying a five-base-pair deletion in exon 1, and this line recapitulated the key cardiac phenotypes, strengthening the genetic robustness of the model.

Comparative transcriptomic analysis linked the fish model to human disease. Examining single-cell RNA sequencing data from human hearts carrying pathogenic PKP2 variants, the researchers found significant downregulation of pathways associated with muscle system processes and muscle contraction, while genes involved in the fatty acid response pathway were upregulated. Consistently, homozygous mutant zebrafish hearts showed downregulated expression of myocardial development genes including myh7bb, cmlc2, and ryr2b, and upregulated expression of adipogenesis-related genes such as apoea and apoa1a. The study also revealed dose-dependent phenotypes, with homozygotes more severely affected than heterozygotes, aligning with recent gene dose-dependent findings in desmoplakin cardiomyopathy models and patients. The authors acknowledge limitations, including the zebrafish two-chambered heart, their robust cardiac regenerative capacity, and the lack of validated antibodies for assessing Pkp2 protein directly. Even so, the model offers a tractable in vivo platform for investigating disease mechanisms, gene-environment interactions, and potential genetic modifiers, complementing ongoing efforts such as adeno-associated virus-mediated PKP2 gene replacement therapy that has shown promise in murine preclinical studies.

Subject of Research: A stable CRISPR-generated zebrafish model of plakophilin-2 truncating mutations that recapitulates arrhythmogenic cardiomyopathy phenotypes including lipid accumulation, conduction abnormalities, and exercise-induced sudden death.

Article Title: Stable truncating mutations in plakophilin-2 induce arrhythmogenic cardiomyopathy-like phenotypes in adult zebrafish

Article References: Stable truncating mutations in plakophilin-2 induce arrhythmogenic cardiomyopathy-like phenotypes in adult zebrafish. (n.d.). https://doi.org/10.1016/j.isci.2026.117755

Image Credits: AI Generated

DOI: Not provided

Keywords: arrhythmogenic cardiomyopathy, plakophilin-2, PKP2, zebrafish model, CRISPR/Cas9, desmosome, intercalated disc, sudden cardiac death, cardiac arrhythmia, fibrofatty replacement, echocardiography, gene dosage