Faculty Bibliography

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.

It is known that monogenic traits may predispose young and otherwise healthy individuals to die suddenly. Diseases such as Long QT Syndrome, Brugada Syndrome and Arrhythmogenic Right Ventricular Cardiomyopathy are well known causes of arrhythmic death in young individuals. For several years the concept of "genetic predisposition" to sudden cardiac death has been limited to these uncommon diseases. In the last few years clinical data have supported the view that risk of dying suddenly may cluster in families, supporting the hypothesis of a genetic component for sudden cardiac death. In this review I will try to provide an overview of current knowledge about genetics of sudden death. I will approach this topic by discussing first where we stand in the use of genetics for risk stratification and therapy selection in monogenic diseases and I will then move to discuss the contribution of genetics to patient profiling in acquired cardiovascular diseases.

Induced pluripotent stem cells (iPSC) offer a unique opportunity for developmental studies, disease modeling and regenerative medicine approaches in humans. The aim of our study was to create an in vitro 'patient-specific cell-based system' that could facilitate the screening of new therapeutic molecules for the treatment of catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited form of fatal arrhythmia. Here, we report the development of a cardiac model of CPVT through the generation of iPSC from a CPVT patient carrying a heterozygous mutation in the cardiac ryanodine receptor gene (RyR2) and their subsequent differentiation into cardiomyocytes (CMs). Whole-cell patch-clamp and intracellular electrical recordings of spontaneously beating cells revealed the presence of delayed afterdepolarizations (DADs) in CPVT-CMs, both in resting conditions and after beta-adrenergic stimulation, resembling the cardiac phenotype of the patients. Furthermore, treatment with KN-93 (2-[N-(2-hydroxyethyl)]-N-(4methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N- methylbenzylamine), an antiarrhythmic drug that inhibits Ca(2+)/calmodulin-dependent serine-threonine protein kinase II (CaMKII), drastically reduced the presence of DADs in CVPT-CMs, rescuing the arrhythmic phenotype induced by catecholaminergic stress. In addition, intracellular calcium transient measurements on 3D beating clusters by fast resolution optical mapping showed that CPVT clusters developed multiple calcium transients, whereas in the wild-type clusters, only single initiations were detected. Such instability is aggravated in the presence of isoproterenol and is attenuated by KN-93. As seen in our RyR2 knock-in CPVT mice, the antiarrhythmic effect of KN-93 is confirmed in these human iPSC-derived cardiac cells, supporting the role of this in vitro system for drug screening and optimization of clinical treatment strategies.

Rationale: The recessive form of catecholaminergic polymorphic ventricular tachycardia is caused by mutations in the cardiac calsequestrin-2 gene; this variant of catecholaminergic polymorphic ventricular tachycardia is less well characterized than the autosomal-dominant form caused by mutations in the ryanodine receptor-2 gene. Objective: We characterized the intracellular Ca(2+) homeostasis, electrophysiological properties, and ultrastructural features of the Ca(2+) release units in the homozygous calsequestrin 2-R33Q knock-in mouse model (R33Q) R33Q knock-in mouse model. Methods and Results: We studied isolated R33Q and wild-type ventricular myocytes and observed properties not previously identified in a catecholaminergic polymorphic ventricular tachycardia model. As compared with wild-type cells, R33Q myocytes (1) show spontaneous Ca(2+) waves unable to propagate as cell-wide waves; (2) show smaller Ca(2+)sparks with shortened coupling intervals, suggesting a reduced refractoriness of Ca(2+) release events; (3) have a reduction of the area of membrane contact, of the junctions between junctional sarcoplasmic reticulum and T tubules (couplons), and of junctional sarcoplasmic reticulum volume; (4) have a propensity to develop phase 2 to 4 afterdepolarizations that can elicit triggered beats; and (5) involve viral gene transfer with wild-type cardiac calsequestrin-2 that is able to normalize structural abnormalities and to restore cell-wide calcium wave propagation. Conclusions: Our data show that homozygous cardiac calsequestrin-2-R33Q myocytes develop spontaneous Ca(2+) release events with a broad range of intervals coupled to preceding beats, leading to the formation of early and delayed afterdepolarizations. They also display a major disruption of the Ca(2+) release unit architecture that leads to fragmentation of spontaneous Ca(2+) waves. We propose that these 2 substrates in R33Q myocytes synergize to provide a new arrhythmogenic mechanism for catecholaminergic polymorphic ventricular tachycardia.

Dysregulated intracellular Ca(2+) signaling is implicated in a variety of cardiac arrhythmias, including catecholaminergic polymorphic ventricular tachycardia. Spontaneous diastolic Ca(2+) release (DCR) can induce arrhythmogenic plasma membrane depolarizations, although the mechanism responsible for DCR synchronization among adjacent myocytes required for ectopic activity remains unclear. We investigated the synchronization mechanism(s) of DCR underlying untimely action potentials and diastolic contractions (DCs) in a catecholaminergic polymorphic ventricular tachycardia mouse model with a mutation in cardiac calsequestrin. We used a combination of different approaches including single ryanodine receptor channel recording, optical imaging (Ca(2+) and membrane potential), and contractile force measurements in ventricular myocytes and intact cardiac muscles. We demonstrate that DCR occurs in a temporally and spatially uniform manner in both myocytes and intact myocardial tissue isolated from cardiac calsequestrin mutation mice. Such synchronized DCR events give rise to triggered electrical activity that results in synchronous DCs in the myocardium. Importantly, we establish that synchronization of DCR is a result of a combination of abbreviated ryanodine receptor channel refractoriness and the preceding synchronous stimulated Ca(2+) release/reuptake dynamics. Our study reveals how aberrant DCR events can become synchronized in the intact myocardium, leading to triggered activity and the resultant DCs in the settings of a cardiac rhythm disorder.