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.

Background Brugada syndrome (BrS) primarily associates with loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (AC; or arrhythmogenic right ventricular cardiomyopathy, ARVC). Experimental models showed correlation between 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. Methods and Results We searched for PKP2 variants in genomic DNA of 200 patients with BrS diagnosis, no signs of AC, 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 site of cell contact. These deficits were restored by transfection of wild-type PKP2 (PKP2-WT), but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes (hIPSC-CMs) from a patient with PKP2 deficit showed drastically reduced INa. The deficit was restored by transfection of WT, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related INa deficiency to 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 co-existence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.

Rationale The early description of the intercalated disc defined three structures, all of them involved in cell-cell communication: desmosomes, gap junctions and adherens junctions. Current evidence demonstrates that molecules not involved in providing a physical continuum between cells, also populate the intercalated disc. Key among them is the voltage-gated sodium channel (VGSC) complex. An important component of this complex is the cytoskeletal adaptor protein ankyrin-G (AnkG). Objective To test the hypothesis that AnkG partners with desmosome and gap junction molecules, and exerts a functional effect on intercellular communication in the heart. Methods and Results We utilized a combination of microscopy, immunochemistry, patch clamp and optical mapping to assess the interactions between AnkG, plakophilin-2 (PKP2) and Connexin43 (Cx43). Co-immunoprecipitation studies from rat heart lysate demonstrated associations between the three molecules. Using siRNA technology we demonstrated that loss of AnkG expression caused significant changes in subcellular distribution and/or abundance of PKP2 and Cx43, as well as a decrease in intercellular adhesion strength and electrical coupling. Regulation of AnkG and of Nav1.5 by PKP2 was also demonstrated. Finally, optical mapping experiments in AnkG-silenced cells demonstrated a shift in the minimal frequency at which rate-dependence activation block was observed. Conclusions These experiments support the hypothesis that AnkG is a key functional component of the intercalated disc, at the intersection of three complexes often considered independent: the VGSC, gap junctions and the cardiac desmosome. Possible implications to the pathophysiology of inherited arrhythmias (such as arrhythmogenic right ventricular cardiomyopathy; ARVC) are discussed.