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Background: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by high propensity to life-threatening arrhythmias and progressive loss of heart muscle. More than 40% of reported genetic variants linked to ARVC reside in the PKP2 gene, which encodes the PKP2 protein (plakophilin-2)., Methods: We describe a comprehensive characterization of the ARVC molecular landscape as determined by high-resolution mass spectrometry, RNA sequencing, and transmission electron microscopy of right ventricular biopsy samples obtained from patients with ARVC with PKP2 mutations and left ventricular ejection fraction >45%. Samples from healthy relatives served as controls. The observations led to experimental work using multiple imaging and biochemical techniques in mice with a cardiac-specific deletion of Pkp2 studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes., Results: Samples from patients with ARVC present a loss of nuclear envelope integrity, molecular signatures indicative of increased DNA damage, and a deficit in transcripts coding for proteins in the electron transport chain. Mice with a cardiac-specific deletion of Pkp2 also present a loss of nuclear envelope integrity, which leads to DNA damage and subsequent excess oxidant production (O 2 .- and H 2 O 2 ), the latter increased further under mechanical stress (isoproterenol or exercise). Increased oxidant production and DNA damage is recapitulated in human induced pluripotent stem cell-derived PKP2-deficient myocytes. Furthermore, PKP2-deficient cells release H 2 O 2 into the extracellular environment, causing DNA damage and increased oxidant production in neighboring myocytes in a paracrine manner. Treatment with honokiol increases SIRT3 (mitochondrial nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-3) activity, reduces oxidant levels and DNA damage in vitro and in vivo, reduces collagen abundance in the right ventricular free wall, and has a protective effect on right ventricular function., Conclusions: Loss of nuclear envelope integrity and subsequent DNA damage is a key substrate in the molecular pathology of ARVC. We show transcriptional downregulation of proteins of the electron transcript chain as an early event in the molecular pathophysiology of the disease (before loss of left ventricular ejection fraction <45%), which associates with increased oxidant production (O 2 .- and H 2 O 2 ). We propose therapies that limit oxidant formation as a possible intervention to restrict DNA damage in ARVC.

Background: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca 2+ (Ca 2+ i ) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca 2+ i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts., Methods: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo., Results: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca 2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca 2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca 2+ i transient amplitude showed a differential response to β-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca 2+ i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels., Conclusions: Exercise disproportionately affects Ca 2+ i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular β-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable β-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

Aims: Exercise increases arrhythmia risk and cardiomyopathy progression in arrhythmogenic right ventricular cardiomyopathy (ARVC) patients, but the mechanisms remain unknown. We investigated transcriptomic changes caused by endurance training in mice deficient in plakophilin-2 (PKP2cKO), a desmosomal protein important for intercalated disc formation, commonly mutated in ARVC and controls., Methods and Results: Exercise alone caused transcriptional downregulation of genes coding intercalated disk proteins. The changes converged with those in sedentary and in exercised PKP2cKO mice. PKP2 loss caused cardiac contractile deficit, decreased muscle mass and increased functional/transcriptomic signatures of apoptosis, despite increased fractional shortening and calcium transient amplitude in single myocytes. Exercise accelerated cardiac dysfunction, an effect dampened by pre-training animals prior to PKP2-KO. Consistent with PKP2-dependent muscle mass deficit, cardiac dimensions in human athletes carrying PKP2 mutations were reduced, compared to matched controls., Conclusions: We speculate that exercise challenges a cardiomyocyte "desmosomal reserve" which, if impaired genetically (e.g., PKP2 loss), accelerates progression of cardiomyopathy., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2021. For permissions, please email: journals.permissions@oup.com.)

Purpose: The genetic architecture of Plakophilin 2 (PKP2) cardiomyopathy can inform our understanding of its variant pathogenicity and protein function., Methods: We assess the gene-wide and regional association of truncating and missense variants in PKP2 with arrhythmogenic cardiomyopathy (ACM), and arrhythmogenic right ventricular cardiomyopathy (ARVC) specifically. A discovery data set compares genetic testing requisitions to gnomAD. Validation is performed in a rigorously phenotyped definite ARVC cohort and non-ACM individuals in the Geisinger MyCode cohort., Results: The etiologic fraction (EF) of ACM-related diagnoses from truncating variants in PKP2 is significant (0.85 [0.80,0.88], p < 2 × 10 -16 ), increases for ARVC specifically (EF = 0.96 [0.94,0.97], p < 2 × 10 -16 ), and is highest in definite ARVC versus non-ACM individuals (EF = 1.00 [1.00,1.00], p < 2 × 10 -16 ). Regions of missense variation enriched for ACM probands include known functional domains and the C-terminus, which was not previously known to contain a functional domain. No regional enrichment was identified for truncating variants., Conclusion: This multicohort evaluation of the genetic architecture of PKP2 demonstrates the specificity of PKP2 truncating variants for ARVC within the ACM disease spectrum. We identify the PKP2 C-terminus as a potential functional domain and find that truncating variants likely cause disease irrespective of transcript position., (© 2021. The Author(s).)

Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates., Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately., Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca 2+ transients, increased Ca 2+ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca 2+ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca 2+ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca 2+ sensitivity and preferential phosphorylation in a domain known to modulate Ca 2+ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca 2+ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca 2+ , increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca 2+ ] i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels., Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca 2+ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca 2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca 2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Objectives: This study determined if radical plakophilin-2 (PKP2) variants might underlie some cases of clinically diagnosed catecholaminergic polymorphic ventricular tachycardia (CPVT) and exercise-associated, autopsy-negative sudden unexplained death in the young (SUDY)., Background: Pathogenic variants in PKP2 cause arrhythmogenic right ventricular cardiomyopathy (ARVC). Recently, a cardiomyocyte-specific PKP2 knockout mouse model revealed that loss of PKP2 markedly reduced expression of genes critical in intracellular calcium handling. The mice with structurally normal hearts exhibited isoproterenol-triggered polymorphic ventricular arrhythmias that mimicked CPVT., Methods: A PKP2 gene mutational analysis was performed on DNA from 18 unrelated patients (9 males; average age at diagnosis: 19.6 ± 12.8 years) clinically diagnosed with CPVT but who were RYR2-, CASQ2-, KCNJ2-, and TRDN-negative, and 19 decedents with SUDY during exercise (13 males; average age at death: 14 ± 3 years). Only radical (i.e., frame-shift, canonical splice site, or nonsense) variants with a minor allele frequency of ≤0.00005 in the genome aggregation database (gnomAD) were considered pathogenic., Results: Radical PKP2 variants were identified in 5 of 18 (27.7%) CPVT patients and 1 of 19 (5.3%) exercise-related SUDY cases compared with 96 of 138,632 (0.069%) individuals in gnomAD (p = 3.1 × 10 -13 ). Cardiac imaging or autopsy demonstrated a structurally normal heart in all patients at the time of their CPVT diagnosis or sudden death., Conclusions: Our data suggested that the progression of the PKP2-dependent electropathy can be independent of structural perturbations and can precipitate exercise-associated sudden cardiac arrest or sudden cardiac death before the presence of overt cardiomyopathy, which clinically mimics CPVT, similar to the PKP2 knockout mouse model. Thus, CPVT and SUDY genetic test panels should now include PKP2., (Copyright © 2019 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for Ca V 1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Mutations in proteins of the desmosome are associated with arrhythmogenic cardiomyopathy (AC; also referred to as "ARVC" or "ARVD"). Life-threatening ventricular arrhythmias often occur in the concealed phase of the disease before the onset of structural changes. Among the various potential mechanisms for arrhythmogenesis in AC, in this article, we concentrate on the relation between desmosomes and sodium channel function. We review evidence indicating that (1) loss of desmosomal integrity (including mutations or loss of expression of plakophilin-2; PKP2) leads to reduced sodium current (INa), (2) the PKP2-INa relation could be partly consequent to the fact that PKP2 facilitates proper trafficking of proteins to the intercalated disc, and (3) PKP2 mutations can be present in patients diagnosed with Brugada syndrome (BrS), thus supporting the previously proposed notion that AC and BrS are not two completely separate entities, but "bookends" in a continuum of variable sodium current deficiency and structural disease., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Brugada syndrome (BrS) primarily associates with the loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (or arrhythmogenic right ventricular cardiomyopathy). Experimental models showed correlation between the loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced INa. We hypothesized that PKP2 variants that reduce INa could yield a BrS phenotype, even without overt structural features characteristic of arrhythmogenic right ventricular cardiomyopathy., Methods and Results: We searched for PKP2 variants in the genomic DNA of 200 patients with a BrS diagnosis, no signs of arrhythmogenic cardiomyopathy, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L, and MOG1. We identified 5 cases of single amino acid substitutions. Mutations were tested in HL-1-derived cells endogenously expressing NaV1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased INa and NaV1.5 at the site of cell contact. These deficits were restored by the transfection of wild-type PKP2, but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes from a patient with a PKP2 deficit showed drastically reduced INa. The deficit was restored by transfection of wild type, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related INa deficiency to the reduced number of channels at the intercalated disc and increased separation of microtubules from the cell end., Conclusions: This is the first systematic retrospective analysis of a patient group to define the coexistence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.

Aims: The shRNA-mediated loss of expression of the desmosomal protein plakophilin-2 leads to sodium current (I(Na)) dysfunction. Whether pkp2 gene haploinsufficiency leads to I(Na) deficit in vivo remains undefined. Mutations in pkp2 are detected in arrhythmogenic right ventricular cardiomyopathy (ARVC). Ventricular fibrillation and sudden death often occur in the 'concealed phase' of the disease, prior to overt structural damage. The mechanisms responsible for these arrhythmias remain poorly understood. We sought to characterize the morphology, histology, and ultrastructural features of PKP2-heterozygous-null (PKP2-Hz) murine hearts and explore the relation between PKP2 abundance, I(Na) function, and cardiac electrical synchrony., Methods and Results: Hearts of PKP2-Hz mice were characterized by multiple methods. We observed ultrastructural but not histological or gross anatomical differences in PKP2-Hz hearts compared with wild-type (WT) littermates. Yet, in myocytes, decreased amplitude and a shift in gating and kinetics of I(Na) were observed. To further unmask I(Na) deficiency, we exposed myocytes, Langendorff-perfused hearts, and anaesthetized animals to a pharmacological challenge (flecainide). In PKP2-Hz hearts, the extent of flecainide-induced I(Na) block, impaired ventricular conduction, and altered electrocardiographic parameters were larger than controls. Flecainide provoked ventricular arrhythmias and death in PKP2-Hz animals, but not in the WT., Conclusions: PKP2 haploinsufficiency leads to I(Na) deficit in murine hearts. Our data support the notion of a cross-talk between desmosome and sodium channel complex. They also suggest that I(Na) dysfunction may contribute to generation and/or maintenance of arrhythmias in PKP2-deficient hearts. Whether pharmacological challenges could help unveil arrhythmia risk in patients with mutations or variants in PKP2 remains undefined.

Inheritable cardiac disorders, which may be associated with cardiomyopathic changes, are often associated with increased risk of sudden death in the young. Early linkage analysis studies in Mendelian forms of these diseases, such as hypertrophic cardiomyopathy and long-QT syndrome, uncovered large-effect genetic variants that contribute to the phenotype. In more recent years, through genotype-phenotype studies and methodological advances in genetics, it has become evident that most inheritable cardiac disorders are not monogenic but, rather, have a complex genetic basis wherein multiple genetic variants contribute (oligogenic or polygenic inheritance). Conversely, studies on genes underlying these disorders uncovered pleiotropic effects, with a single gene affecting multiple and apparently unrelated phenotypes. In this review, we explore these 2 phenomena: on the one hand, the evidence that variants in multiple genes converge to generate one clinical phenotype, and, on the other, the evidence that variants in one gene can lead to apparently unrelated phenotypes. Although multiple conditions are addressed to illustrate these concepts, the experience obtained in the study of long-QT syndrome, Brugada syndrome, and arrhythmogenic cardiomyopathy, and in the study of functions related to SCN5A (the gene coding for the α-subunit of the most abundant sodium channel in the heart) and PKP2 (the gene coding for the desmosomal protein plakophilin-2), as well, is discussed in more detail. [ABSTRACT FROM AUTHOR]