Abstract
Background: Patients with arrhythmogenic cardiomyopathy may suffer from lethal ventricular arrhythmias. Arrhythmogenic cardiomyopathy is predominantly triggered by mutations in plakophilin-2, a key component of cell-to-cell adhesion and calcium cycling regulation in cardiomyocytes. Calcium dysregulation due to plakophilin-2 mutations may lead to arrhythmias but the underlying pro-arrhythmic mechanisms remain unclear.Aim: To unravel the mechanisms by which calcium-handling abnormalities in plakophilin-2 loss-of-function may contribute to proarrhythmic events in arrhythmogenic cardiomyopathy.Methods: We adapted a computer model of mouse ventricular electrophysiology using recent experimental calcium-handling data from plakophilin-2 conditional knock-out (PKP2-cKO) mice. We simulated individual effects of beta-adrenergic stimulation, modifications in connexin43-mediated calcium entry, sodium-calcium exchanger (NCX) activity and ryanodine-receptor 2 (RyR2) calcium affinity on cellular electrophysiology and occurrence of arrhythmogenic events (delayed-afterdepolarizations). A population-of-models approach was used to investigate the generalizability of our findings. Finally, we assessed the potential translation of proposed mechanisms to humans, using a human ventricular cardiomyocyte computational model.Results: The model robustly reproduced the experimental calcium-handling changes in PKP2-cKO cardiomyocytes: an increased calcium transient amplitude (562 vs. 383 nM), increased diastolic calcium (120 vs. 91 nM), reduced L-type calcium current (15.0 vs. 21.4 pA/pF) and an increased free SR calcium (0.69 vs. 0.50 mM). Under beta-adrenergic stimulation, PKP2-cKO models from the population of models (n = 61) showed a higher susceptibility to delayed-afterdepolarizations compared to control (41 vs. 3.3%). Increased connexin43-mediated calcium entry further elevated the number of delayed-afterdepolarizations (78.7%, 2.5-fold increase in background calcium influx). Elevated diastolic cleft calcium appeared responsible for the increased RyR2-mediated calcium leak, promoting delayed-afterdepolarizations occurrence. A reduction in RyR2 calcium affinity prevented delayed-afterdepolarizations in PKP2-cKO models (24.6 vs. 41%). An additional increase in INCX strongly reduced delayed-afterdepolarizations occurrence, by lowering diastolic cleft calcium levels. The human model showed similar outcomes, suggesting a potential translational value of these findings.Conclusion: Beta-adrenergic stimulation and connexin43-mediated calcium entry upon loss of plakophilin-2 function contribute to generation of delayed-afterdepolarizations. RyR2 and NCX dysregulation play a key role in modulating these proarrhythmic events. This work provides insights into potential future antiarrhythmic strategies in arrhythmogenic cardiomyopathy due to plakophilin-2 loss-of-function.
Highlights
Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the cardiac muscle, predominantly in the right ventricle
Using a population-of-models approach (Britton et al, 2013), we show the synergistic effects of beta-adrenergic stimulation (BARS) and Cx43-mediated calcium entry in the generation of proarrhythmic delayed-afterdepolarizations (DADs), as well as the role of ryanodine receptors 2 (RyR2) and sodium-calcium exchanger (NCX) dysregulation in modulating these events
In combination with an increased Cx43 calcium influx, BARS promoted spontaneous diastolic sarcoplasmic reticulum (SR) calcium-release events, which translated into the occurrence of delayed afterdepolarization (DAD) (Figure 4A, top)
Summary
Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disease characterized by fibrofatty replacement of the cardiac muscle, predominantly in the right ventricle. The consequences of PKP2 deficiency in the cardiomyocyte remain poorly known, but recent studies have shown the crucial role of PKP2 in the translation of signals originating at the cell junction into intracellular signals controlling structural and electrical cardiomyocyte components, especially connexin (Oxford et al, 2007), voltage-gated sodium channel (Sato et al, 2009), and calcium cycling (Cerrone et al, 2017; Austin et al, 2019) This influence on calcium homeostasis suggests a key role for PKP2 loss-of-function in arrhythmogenicity, even in absence of structural disease (Cerrone et al, 2017). Calcium dysregulation due to plakophilin-2 mutations may lead to arrhythmias but the underlying pro-arrhythmic mechanisms remain unclear
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