Faculty Bibliography

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is one of the most common causes of sudden cardiac death in young people. Patients diagnosed with ARVC may experience increased likelihood of development of anxiety and depression, emphasizing the need for accurate diagnosis. To assist future genetic diagnosis and avoidance of misdiagnosis, we evaluated the reported monogenic disease-causing variants in ARVD/C Genetic Variants Database, Human Gene Mutation Database, and ClinVar. Within the aforementioned databases, 630 monogenic disease-causing variants from 18 genes were identified. In the genome Aggregation Database, 226 of these were identified; 68 of which were found at greater than expected prevalence. Furthermore, 37/226 genetic variants were identified amongst the 409 000 UK biobank participants, 23 were not associated with ARVC. Among the 14 remaining variants, 13 were previously found with greater than expected prevalence for a monogenic variant. Nevertheless, they were associated with serious cardiac phenotypes, suggesting that these 13 variants may be disease-modifiers of ARVC, rather than monogenic disease-causing. In summary, more than 10% of variants previously reported to cause ARVC were found unlikely to be associated with highly penetrant monogenic forms of ARVC. Notably, all variants in OBSCN and MYBPC3 were found, making these unlikely to be monogenic causes of ARVC. This article is protected by copyright. All rights reserved.

Background: Plakophilin-2 (PKP2) is classically defined as a protein of the desmosome, an intercellular adhesion structure that also acts as a signaling hub to maintain structural and electrical homeostasis. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy (ARVC). A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events that occur consequent to its mutation. Here we sought to captureearly molecular/cellular events that can act as nascent substrates for subsequent arrhythmic/cardiomyopathic phenotypes.
Method(s): We used multiple quantitative imaging modalities, as well as biochemical and high-resolution mass spectrometry methods to study the functional/structural properties of cells/tissues derived from cardiomyocytespecific, tamoxifen-activated, PKP2 knockout mice ("PKP2cKO"). Studies were carried out 14 days post-tamoxifen injection, a time point preceding an overt electrical or structural phenotype.Myocytes from right or left ventricular free wall were studied separately, to detect functional/structural asymmetries.
Result(s): Most properties of PKP2cKO left ventricular (LV) myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca2+transients, increased frequency of spontaneous Ca2+release events, increased [Ca2+] in the cytoplasm and sarcoplasmic reticulum compartments, and dynamic Ca2+accumulation in mitochondria. In addition, RyR2 in RV presented enhanced sensitivity to Ca2+and preferential phosphorylation in a domain known to modulate Ca2+gating. RNAseq at 14 days post-TAM showed no relevant difference in transcript abundance between RV and LV, neither in control nor in PKP2cKO cells, suggesting that in the earliest stage, [Ca2+]i dysfunction is not transcriptional. Rather, we found an RV-predominant increase in membrane permeability that can permit Ca2+entry into the cell. Cx43 ablation mitigated the increase in membrane permeability, the accumulation of cytoplasmic Ca2+and the early stages of RV dysfunction.
Conclusion(s): Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca2+ dysregulation) that precedes the cardiomyopathy and that is, at least in part, mediated by a Cx43-dependent membrane conduit. Given that asymmetric Ca2+ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca2+ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage

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 (ARVC). 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 (post-TAM) injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. RESULTS:homeostasis. Similarly, PKC inhibition normalized spark frequency at comparable SR load levels. CONCLUSIONS:handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Human variants in plakophilin-2 (PKP2) associate with most cases of familial arrhythmogenic cardiomyopathy (ACM). Recent studies show that PKP2 not only maintains intercellular coupling, but also regulates transcription of genes involved in Ca²⁺ cycling and cardiac rhythm. ACM penetrance is low and it remains uncertain, which genetic and environmental modifiers are crucial for developing the cardiomyopathy. In this study, heterozygous PKP2 knock-out mice (PKP2-Hz) were used to investigate the influence of exercise, pressure overload, and inflammation on a PKP2-related disease progression. In PKP2-Hz mice, protein levels of Ca²⁺-handling proteins were reduced compared to wildtype (WT). PKP2-Hz hearts exposed to voluntary exercise training showed right ventricular lateral connexin43 expression, right ventricular conduction slowing, and a higher susceptibility towards arrhythmias. Pressure overload increased levels of fibrosis in PKP2-Hz hearts, without affecting the susceptibility towards arrhythmias. Experimental autoimmune myocarditis caused more severe subepicardial fibrosis, cell death, and inflammatory infiltrates in PKP2-Hz hearts than in WT. To conclude, PKP2 haploinsufficiency in the murine heart modulates the cardiac response to environmental modifiers via different mechanisms. Exercise upon PKP2 deficiency induces a pro-arrhythmic cardiac remodeling, likely based on impaired Ca²⁺ cycling and electrical conduction, versus structural remodeling. Pathophysiological stimuli mainly exaggerate the fibrotic and inflammatory response.

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.

Background: Nav1.5 is targeted to distinct subcellular microdomains of cardiomyocytes by the microtubule network, with sodium current being largest in the intercalated disc (ID) region. The microtubule plus-end tracking proteins End Binding 1 (EB1) and CLIP-associating protein 2 (CLASP2) are mainly located at the ID and regulate microtubule recruitment and stability. The small molecule SB216763 (SB2) acts on Glycogen synthase kinase 3 beta (GSK3beta) and is known to enhance the EB1-CLASP2 interaction, thereby increasing microtubule stability.
Objective(s): To investigate the effect of targeting EB1-CLASP2 on Nav1.5 localisation and sodium current density (INa) in subcellular microdomains.
Method(s): Patch clamp and Stochastic Optical Reconstruction Microscopy (STORM) imaging experiments were performed on human iPSC-derived cardiomyocytes (hiPSC-CMs) and freshly isolated murine ventricular cardiomyocytes.
Result(s): EB1 overexpression in hiPSC-CMs increased membrane Nav1.5 cluster density and consequently increased whole-cell INa and action potential (AP) upstroke velocity, without affecting INa kinetics or other AP parameters. Increased whole-cell INa was observed in murine cardiomyocytes after 2-4 hours of SB2 treatment (5micro M), while INa kinetics remained unaffected. Macropatch experiments revealed that SB2 specifically increased INa at the ID, while INa at the lateral membrane was unchanged. In contrast, SB2 did not affect INa or Nav1.5 cluster size or density in cardiomyocytes from mice Clasp2-deficient mice.
Conclusion(s): Targeting the plus-end tracking proteins EB1 and CLASP2 resulted in increased whole-cell peak INa in hiPSC-CMs and isolated murine cardiomyocytes. On the subcellular level, INa was specifically increased at the ID after pharmacologically enhancing the CLASP2-EB1 interaction by SB2. Treatment with SB2 in cardiomyocytes lacking CLASP2 did not affect INa or Nav1.5 distribution, demonstrating the central role for CLASP2 in the SB2-mediated effects. Thus, the microtubule-EB1-CLASP2 complex constitutes a promising target for modulating INa in a microdomain-specific manner.
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Background and purpose: Dysfunction of the cardiac sodium channel Nav1.5 is associated with cardiac arrhythmias but therapeutic options to restore Nav1.5 are limited. Nav1.5 is transported to the cell membrane by the microtubule network and is differentially distributed around subcellular domains, with enrichment at the intercalated discs of cardiomyocytes. We have previously demonstrated that Nav1.5-based sodium current (INa) is modulated by the microtubule plus-end binding proteins CLIP-associating protein 2 (CLASP2) and End binding 1 (EB1), which are both enriched in the intercalated disc. Inhibition of Glycogen synthase kinase 3 beta (GSK3beta) by SB216763 (SB2) is known to enhance the interaction between CLASP2, EB1, and microtubules, thereby increasing microtubule recruitment and stability. We therefore hypothesise that GSK3beta inhibition by SB2 increases INa by enhancing Nav1.5 trafficking specifically to the intercalated disc.
Methods and Results: Cells were incubated with 5 M of the pharmacological GSK3beta inhibitor SB2 followed by whole-cell patch clamp measurements. In adult mouse cardiomyocytes, an increased whole-cell peak sodium current density (INa) was observed after subacute (2-4 hour) incubation with SB2, while INa kinetics remained unaffected. Macropatch experiments were performed on freshly isolated murine cardiomyocytes to investigate the effect of SB2 on INa in subcellular microdomains. These experiments revealed that SB2 specifically increased INa at the intercalated disc, while INa at the lateral membrane remained unaffected. To prove the central role of CLASP2 in these effects of SB2 on INa wholecell measurements were performed on cardiomyocytes from Clasp2-KO mice, showing that SB2 does not affect INa in the absence of CLASP2. Conclusion and perspectives: The GSK3beta inhibitor SB2 increased whole-cell peak INa in isolated murine cardiomyocytes. On the subcellular level, INa was specifically increased at the intercalated discs, while the current was unaffected at the lateral membrane of cardiomyocytes. Treatment with SB2 in cardiomyocytes lacking CLASP2 did not affect INa, showing that SB2 affects INa through CLASP2. Thus, the microtubule/EB1/CLASP2 complex constitutes a promising target for modulating INa in a microdomain-specific manner, which could be of particular relevance for diseases caused by a loss of Nav1.5 at the intercalated disc, such as arrhythmogenic right ventricular cardiomyopathy (ARVC)

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. The aim of this study was to investigate the role of ATP/adenosine in the progression of a PKP2-associated cardiomyopathy.
Methods and Results: HL1 cells were used to study PKP2- and Connexin43 (Cx43)-dependent ATP release. HL1 cells silenced for PKP2 showed increased ATP release compared to control. Knockout of Cx43 in the same cells blunted the effect. A cardiac-specific, tamoxifenactivated PKP2 knock-out murine model (PKP2-cKO) was used to define the effect of adenosine receptor blockade on the progression of a PKP2-dependent cardiomyopathy. Transcriptomic data of PKP2-cKO mice revealed overexpression of genes involved in adenosine-receptor cascades. Treatment with Istradefylline (an adenosine 2A receptorblocker) tempered the progression of fibrosis and mechanical failure observed in PKP2-cKO mice (see Fig. B,C). In contrast, PSB115, a blocker of the 2B adenosine receptor, showed opposite effects.
Conclusion(s): 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. (Figure Presented)

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.

Arrhythmogenic cardiomyopathy (ACM) is a familial heart disease, associated with ventricular arrhythmias, fibrofatty replacement of the myocardial mass and an increased risk of sudden cardiac death (SCD). Malignant ventricular arrhythmias and SCD largely occur in the pre-clinical phase of the disease, before overt structural changes occur. To prevent or interfere with ACM disease progression, more insight in mechanisms related to electrical instability are needed. Currently, numerous studies are focused on the link between cardiac arrhythmias and metabolic disease. In line with that, a potential role of mitochondrial dysfunction in ACM pathology is unclear and mitochondrial biology in the ACM heart remains understudied. In this review, we explore mitochondrial dysfunction in relation to arrhythmogenesis, and postulate a link to typical hallmarks of ACM. Mitochondrial dysfunction depletes adenosine triphosphate (ATP) production and increases levels of reactive oxygen species in the heart. Both metabolic changes affect cardiac ion channel gating, electrical conduction, intracellular calcium handling, and fibrosis formation; all well-known aspects of ACM pathophysiology. ATP-mediated structural remodeling, apoptosis, and mitochondria-related alterations have already been shown in models of PKP2 dysfunction. Yet, the limited amount of experimental evidence in ACM models makes it difficult to determine whether mitochondrial dysfunction indeed precedes and/or accompanies ACM pathogenesis. Nevertheless, current experimental ACM models can be very useful in unraveling ACM-related mitochondrial biology and in testing potential therapeutic interventions.