Faculty Bibliography

OBJECTIVES/OBJECTIVE: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:). 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.

Plakophilin-2 (PKP2) is a component of the desmosome complex and known for its role in cell-cell adhesion. Recently, alterations in the Pkp2 gene have been associated with different inherited cardiac conditions including Arrythmogenic Cardiomyopathy (ACM or ARVC), Brugada syndrome (BrS), and idiopathic ventricular fibrillation to name the most relevant. However, the assessment of pathogenicity regarding the genetic variations associated with Pkp2 is still a challenging task: the gene has a positive Residual Variation Intolerance Score and the potential deleterious role of several of its variants has been disputed. Limitations in facilitating interpretation and annotations of these variants are seen in the lack of segregation and clinical data in the control population of reference. In this review, we will provide a summary of all the currently available genetic information related to the Pkp2 gene, including different phenotypes, ClinVar annotations and data from large control database. Our goal is to provide a literature review that could help clinicians and geneticists in interpreting the role of Pkp2 variants in the context of heritable sudden death syndromes. Limitations of current algorithms and data repositories will be discussed.

Background/UNASSIGNED:pathogenic variant in a decedent. Methods/UNASSIGNED:Forensic investigation and molecular autopsy were performed on an 18-year-old female who died suddenly and unexpectedly. Co-segregation family study of the first-degree relatives and functional characterization of the variant were conducted. Findings/UNASSIGNED:arose de novo, which eliminated the need for exhaustive genome testing and annual cardiac follow-up for the parents and four siblings. Interpretation/UNASSIGNED:Molecular testing enables accurate determination of natural causes of death and precision care of the surviving family members in a time and cost-saving manner. We advocate for molecular autopsy being included under the healthcare coverage in US.

Background: Mutations in plakophilin-2 (PKP2) are the most common cause of familial Arrhythmogenic Right Ventricular Cardiomyopathy, a disease characterized by ventricular arrhythmias, sudden death, and progressive fibrofatty cardiomyopathy. The relation between loss of PKP2 expression and structural cardiomyopathy remains under study, though paracrine activation of pro-fibrotic intracellular signaling cascades is a likely event. Previous studies have indicated that ATP release into the intracellular space, and activation of adenosine receptors, can regulate fibrosis in various tissues. However, the role of this mechanism in the heart, and in the specific case of a PKP2-initiated cardiomyopathy, remains unexplored. Objectives: To investigate the role of ATP/adenosine in the progression of a PKP2-associated cardiomyopathy. Methods: HL1 cells were used to study PKP2- and Connexin43 (Cx43)-dependent ATP release. A cardiac-specific, tamoxifen-activated PKP2 knock-out murine model (PKP2cKO) was used to define the effect of adenosine receptor blockade on the progression of a PKP2-dependent cardiomyopathy. Results: HL1 cells silenced for PKP2 showed increased ATP release compared to control. Knockout of Cx43 in the same cells blunted the effect. PKP2cKO transcriptomic data revealed overexpression of genes involved in adenosine-receptor cascades. Istradefylline (an adenosine 2A receptor blocker) tempered the progression of fibrosis and mechanical failure observed in PKP2cKO mice. In contrast, PSB115, a blocker of the 2B adenosine receptor, showed opposite effects. Conclusion: Paracrine adenosine 2A receptor activation contributes to the progression of fibrosis and impaired cardiac function in animals deficient in PKP2. Given the limitations of the animal model, translation to the case of patients with PKP2 deficiency needs to be done with caution.

The Brugada syndrome is an inherited channelopathy associated with increased risk of ventricular arrhythmias and sudden death, often occurring during sleep or resting conditions. Although this entity has been described more than 20 years ago, it remains one of the most debated among channelopathies, with several open questions on its genetic substrate, arrhythmia mechanisms, and clinical management. Studies on the genetics and physiopathology bases of the Brugada syndrome have opened novel investigative pathways and concepts that are now entering the field of cardiovascular genetics and are applied to other inherited arrhythmias. In this perspective, Brugada syndrome can be seen as an example on how basic science discoveries have influenced clinical management and led to novel therapeutic approaches.

Background: Arrhythmogenic cardiomyopathy (also known as "ARVC") is an inherited disease characterized by fibrous or fibrofatty infiltration of the heart muscle, commonly of right ventricular (RV) predominance, ventricular arrhythmias, and high propensity for sudden death. Sudden cardiac arrest frequently associates with exercise and most often occurs in early adulthood during the subclinical ("concealed") phase of the disease. Understanding electrical remodeling in the early stage of the disease is paramount to understand sudden death mechanisms. Methods: We generated a cardiomyocyte-specif-ic, tamoxifen-activated, PKP2 knockout murine line (alphaMHC-Cre-ERT2/PKP2 fl/fl) which allowed us to control the onset of PKP2 loss of expression, limit it to adult cardiomyocytes, and establish a time line for progression of molecular and functional events. Results: The first consequence of PKP2 loss was RV mechanical dysfunction (14 days post-tamoxifen injection, 14 dpi), followed by fibrosis of RV predominance and RV dilation (21 dpi), then biventricular dilated cardiomyopa-thy and left ventricular (LV) failure (28 dpi and beyond). End-stage failure and death occurred between 30 and 49 dpi. Isoproterenol (ISO)-induced ventricular arrhythmias were first detected prior to LV dysfunction (17/17 mice), and ISO-induced fatal ventricular fibrillation was observed only at 16 dpi, i.e., during the concealed stage (3/9). Differential tran-scriptome analysis at 21 dpi revealed reduced transcript levels for a gene network involved in intracellular calcium ([Ca²⁺]i) cycling, most critically genes encoding Ca²⁺ channel proteins (RyR2 and CaV1.2) and structural molecules that scaffold the dyad (ankyrin-B and triadin). Nanoscale imaging (3D super-resolution microscopy, SICM, and FIB-SEM) showed preservation of T-tubular structure, reduced size and increased separation of CaV1.2 clusters, and displacement of functional CaV1.2 channels from the T-tubular domain. Calcium imaging showed disruption of [Ca²⁺]i homeostasis, potentially causative of ventricular arrhythmias. Flecainide i.p. prevented ISO-induced arrhythmias in all animals. Retrospective analysis of clinical cases showed instances of sudden cardiac arrest without structural disease and suspect diagnosis of catechol-aminergic polymorphic ventricular tachycardia (CPVT) later revealed to foster PKP2 nonsense mutations. Conclusions: Our data provide the first evidence that PKP2 deficiency in adult ventricular myocytes is sufficient to cause an arrhythmo-genic cardiomyopathy of RV predominance. Adrenergic-induced arrhythmias and sudden death occur before the onset of overt structural disease and can mimic a CPVT phenotype. Our data also document a transcript-based [Ca²⁺]i dysfunction as a new key mechanism of arrhythmias in PKP2-deficient hearts and suggest flecainide as potential effective antiarrhyth-mic treatment

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 CaV1.2) 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.