Purpose:Selective inhibition of atrial proarrhythmicity can be therapeutic for reducing the atrial fibrillation (AF) burden. Atrial-selective K+-channel blockade (mainly Kv1.5 and Kv4.3 channels conducting the sustained IKur and transient Ito outward currents) promises to suppress AF with a favorable benefit-to-harm ratio. The mechanisms underlying the efficacy of K+ channel blockade under arrhythmic conditions and its association with electrophysiological and contractile remodeling in AF remain to be investigated. Methods:Using our electromechanically coupled model MBS2023, we have simulated the effects of 4-aminopyridine (4-AP) and AVE0118 at different basic cycle lengths (2-0.25s). We have dissociated the primary and secondary responses to determine the drug's underlying mechanisms of action. We have analyzed the effects of K+-channel blockers under arrhythmogenic conditions induced by either forward excitation-contraction coupling (ECC) or mechano-calcium feedback. Results:At the basal rate, the voltage-mediated increase in IKr induced by 4-AP shortens the action potential duration (APD) under sinus rhythm (SR), whereas a surge in ICaL prolongs APD under AF. 4-AP can exacerbate the vulnerability to phase 2 early afterdepolarizations (EADs) by slowing repolarization and prolonging myofilament activation. K+-channel blockade can decimate the susceptibility of delayed afterdepolarizations (DADs) by eliminating the cytosolic Ca2+ overload. The slowing of repolarization induced by 4-AP can suppress the reopening of Na+ channels during phase 3 EADs. Conclusion:In both types of EAD, a shorter, Ca2+-desensitized sarcomere can reduce the propensity for AF in the model. In general, K+ channel blockade has anti-arrhythmic potential to suppress phase 3 EADs by slowing repolarization.

BACKGROUND:Predicting cardiac resynchronization therapy (CRT) response remains problematic. A fluoroscopy imaging-based method to reconstruct the 3-dimensional trajectory (3DTraj) of the coronary sinus (CS) lead pacing cathode throughout the cardiac cycle was used in this study. OBJECTIVE:The aim was to assess the correlation of left ventricular (LV) reverse remodeling with site-specific short-term change in 3DTraj geometry induced by biventricular pacing at the start of CRT. 3DTraj potential as an intraprocedural guide for CS pacing site choice was thus evaluated. METHODS:In CRT implants in 6 centers, pacing cathode 3DTraj was reconstructed just before (T-1) and immediately after (T0) biventricular pacing start at fixed atrio-ventricular and interventricular intervals. The TRAJ-index was calculated as the percentage variation of the ratio between the 2 main axes of 3DTraj between T-1 and T0. The TRAJ-index was compared with the volumetric response at echocardiography defined as a less than -15% decrease in LV end-systolic volume at follow-up. Receiver-operator analysis found the TRAJ-index optimal threshold value at -17%. RESULTS:Among 82 patients, 50 (61%) were responders at 8-month median follow-up (interquartile range 7‒10). TRAJ-index predicted positive response in 88% of cases and negative response in 66% of cases, with 77% overall accuracy value (area under curve = 0.86). The TRAJ-index prediction of response was less effective in large LV volumes and, not significantly, in major mitral regurgitations. CONCLUSION:TRAJ-index seems to aid the intra-operative choice of CS-pacing site at CRT implant, with no additional tools.

AIMS:Loss-of-function (LOF) mutations of the cardiac Na+ channel (SCN5A) are causatively associated with the Brugada Syndrome (BrS). However, the onset of Ventricular Fibrillation (VF) is a rare event, and critical factors favouring the pathological phenotype remain often elusive. This study explores how concomitant triggering conditions may impact on VF onset in a symptomatic proband carrying the S805L/SCN5A BrS mutation. METHODS AND RESULTS:Clinical, in-vitro, numerical, and structural analyses were performed. A 67-year-old male was resuscitated after cardiac arrest, and clinical analysis upon hospitalisation revealed severe hypokalaemia (2.5 mEq/L). The ECG showed a coved type-I BrS pattern and the SCN5A mutation (S805L) was identified. Patch-clamp studies carried out in a heterologous expression system (HEK293 cells) revealed that WT/S805L channels exhibit two different phenotypes (normal and LOF); the main parameter controlling this distribution is the cell membrane potential. A protected/normal behaviour was observed at -80 mV; conversely, LOF occurred at more negative potentials (-100/-120 mV). Further analyses in isolated outflow tract ventricular cardiomyocytes showed that hypokalaemia (and bradycardia) induced diastolic potential hyperpolarisation, thus favouring the Na+ current LOF. Computational and molecular modelling confirmed our findings and revealed the structural determinant of this alteration. CONCLUSION:WT/S805L Na+ channels exhibit either a LOF or a wild-type-like behaviour depending on the membrane potential. Since hypokalaemia and slow pacing rate induce cell hyperpolarisation and the associated LOF, they represent concurrent elements creating the scenario responsible for the VF and cardiac arrest. These results may represent an interpretative paradigm applicable to other BrS mutations.

Brugada Syndrome (BrS) is a genetic disease associated with ventricular arrhythmias and is one of the causes of sudden cardiac death. In particular, dysfunctional cardiac Na + channels (SCN5A) represent the only mechanism supporting the autosomal dominant inheritance. A proband without previous medical history was referred to the intensive care unit after resuscitation showing ECG Type 1 BrS pattern and a significant hypokalemia (2.5 mEq/L). Genetic analysis identified a SCN5A de-novo mutation (S805L) in heterozygosis. This study investigates the causative association between the S805L mutation and the BrS event. First, we expressed wild type (WT) and/or mutated (Hetero and Homo) channels in HEK293T. The application of I/V protocols (hp -120 mV) revealed reduced I Na density in Homo and Hetero (-65% and -26% vs WT at -20 mV), indicating S805L as a loss-of-function (LOF) mutation. However, Hetero I Na density was comparable to WT at more depolarized hp (-80 mV), suggesting alterations in the voltage dependent availability of the channel. Accordingly, steady-state inactivation curves of both Hetero and Homo I Na were right shifted, indicating a gain-of-function (GOF) behaviour (Hetero availability: +13% vs WT at -80 mV). To investigate the possible role of hypokalemia in uncovering the LOF behaviour of the mutation, we reproduced in vitro the hypokalemic condition of the patient testing the effects of external K + (K + out ) on diastolic membrane potential (E diast ) of paced guinea-pig cardiomyocytes. Cells were isolated from the Right Ventricular Outflow Tract, known as site of BrS onset. In comparison to a normokalemic condition, E diast significantly hyperpolarized at 2.5 mM K + out and the effect was greater at low pacing rates (1 Hz) (-16.5 mV vs E diast at 5 mM K + and 4 Hz). Thus, the combination of bradycardia and hypokalemia might represent a mechanism able to unmask the LOF effect of S805L mutation in Hetero conditions. Computational approach on the human ventricular action potential confirmed these findings. In conclusion, S805L-SCN5A is, at the same time, a LOF and GOF mutation causing reduced channel expression with increased channel availability. While the balance between GOF-LOF effects guaranties a “protected” phenotype, the hypokalemic-induced cell hyperpolarization might reduce the GOF, leading to the appearance of the BrS event.

The resolution of inflammation is modulated by specialized pro-resolving lipid mediators (SPMs), which can be modified in some cardiovascular diseases. Among these SPMs, RvD1 and LXA4 prevent atrial fibrillation (AF) remodeling and cardiac hypertrophy, respectively in animal models. However, little is known about their electrophysiological effects on cardiac voltage-gated (VG) ion channels. We used the patch-clamp technique in heterologous systems and cardiomyocytes to assess the acute effects of RvD1, and LXA4, on VG potassium currents. In silico simulations were used to predict the effect of current modulation on the atrial and ventricular action potentials (AP). RvD1 (5 nM) reduced IKs (channel KV7.1/KCNE1) in COS-7 cells and guinea-pig cardiomyocytes by 50.3 ± 7.3 % and 29.9 ± 5.4 % at + 40 mV, respectively, without modifying its voltage dependence. RvD1 was more potent than LXA4. In heterologous systems, RvD1 was also tested on IKur (channel KV1.5), Ito (channel KV4.3/KChIP2), IKr (channel KV11.1), and IK1 (channel Kir2.1) with the largest inhibitory effect on IKs and IKr. In in silico simulations RvD1 prolonged repolarization significantly in both atrial and ventricular myocytes. All these results provide a comprehensive evaluation of RvD1 and LXA4 on cardiac human potassium channels, at pathophysiologically relevant concentrations, being RvD1 more potent than LXA4. The predicted effects on the AP suggest that, along with their antiinflammatory action, RvD1 may reverse AF-induced electrical remodeling in the atria by their modulation of K+ currents. The same action might instead contribute to ventricular functional remodeling; however, direct evidence for this is missing.

AbstractHuman induced pluripotent stem cells–derived cardiomyocytes have revolutionized the field of regenerative medicine, offering unparalleled potential forin–vitromodeling of normal and pathological human cardiomyocytes. The ability to produce stem cardiac myocytes in abundance has opened new avenues for drug efficacy and safety testing, as well as the study of conditions such as atrial fibrillation, a familial cardiac disorder. The development of atrial fibrillation is influenced by ion channel mutations, genetic variants, and other risk factors. Stem cells derived cardiomyocytes hold promise in personalized medicine, as they share the genetic heritage of the donor. While mathematical models have focused on immature stem cardiomyocytes phenotypes, they have primarily relied on a system of stiff ordinary differential equations. Computational modeling of diseased tissue presents an opportunity to evaluate drugs in a patient-specific manner, thereby improving therapeutic targets and ablation techniques. Previous studies categorized cell phenotypes based on action potential morphology, yet classification criteria remains ambiguous.This work introduces the first atrial-specificin–silicomodel of stem cells ionic currents, leveraging experimental data provided by Altomare et al. It begins by summarizing the baseline electrophysiological model and mathematical descriptions of atrial–specific additional currents. Model parameter tuning was performed through automatic optimization techniques to ensure realistic action potential shape and expedite the parameter adjustment process. The resulting model was validated against rate dependence and atrial–specific ion current blocking data. In summary, the development of an atrial-specificin–silicomodel represents a significant step forward in understanding cardiac electrophysiology and the potential for personalized medicine in treating conditions like atrial fibrillation. This model offers new tools for drug evaluation, therapeutic improvement, and a deeper comprehension of cardiac phenotypes.Author summaryHuman induced pluripotent stem cells have revolutionized regenerative medicine since their discovery in 2006, leading to a Nobel Prize in 2012. This kind of pluripotent cells can give rise to different types of specific tissue cells, such as derived cardiomyocytes. Differentiated cardiac cells offer an unlimited supply for studying human heart cells in normal and disease conditions, aiding a patient–specific drug testing and helping to explore pathogenic mechanisms behind different cardiomyopathies, including atrial fibrillation. Atrial fibrillation is a common heart condition, and stem cells with the same genetic heritage as the donor, are ideal for patient-specific treatments.Recent advances have produced mathematical models for the ionic currents in cardiomyocytes derived from stem cells, focusing on immature forms and enabling virtual drug testing. However, previous models did not capture the atrial–specific characteristics. We decided to create and introduce by this study the first atrial–likein–silicomodel for these cells, using novel experimental data. Thus, we describe the baseline model and additional atrial–specific currents, we tune the model parameters using automatic optimization technique, and we validate the model’s accuracy in simulating atrial action potentials and ion current blockage. This research paves the way for better understanding and treating atrial fibrillation and other heart conditions.

This work introduces the first atrial-specific in-silico human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) model, based on a set of phenotype-specific IKur,IKCa and IK1 membrane currents. This model is built on novel in-vitro experimental data recently published by some of the co-authors to simulate the paced action potential of matured atrial-like hiPSC-CMs. The model consists of a system of stiff ordinary differential equations depending on several parameters, which have been tuned by automatic optimization techniques to closely match selected experimental biomarkers. The new model effectively simulates the electronic in-vitro hiPSC-CMs maturation process, transitioning from an unstable depolarized membrane diastolic potential to a stable hyperpolarized resting potential, and exhibits spontaneous firing activity in unpaced conditions. Moreover, our model accurately reflects the experimental rate dependence data at different cycle length and demonstrates the expected response to a specific current blocker. This atrial-specific in-silico model provides a novel computational tool for electrophysiological studies of cardiac stem cells and their applications to drug evaluation and atrial fibrillation treatment.

Background: Cardiac pacemaking remains an unsolved matter from many perspectives. Extensive experimental and computational studies have been performed to describe the sinoatrial physiology across different scales, from the molecular to clinical levels. Nevertheless, the mechanism by which a heartbeat is generated inside the sinoatrial node and propagated to the working myocardium is not fully understood at present. This work aims to provide quantitative information about this fascinating phenomenon, especially regarding the contributions of cellular heterogeneity and fibroblasts to sinoatrial node automaticity and atrial driving.Methods: We developed a bidimensional computational model of the human right atrial tissue, including the sinoatrial node. State-of-the-art knowledge of the anatomical and physiological aspects was adopted during the design of the baseline tissue model. The novelty of this study is the consideration of cellular heterogeneity and fibroblasts inside the sinoatrial node for investigating the manner by which they tune the robustness of stimulus formation and conduction under different conditions (baseline, ionic current blocks, autonomic modulation, and external high-frequency pacing).Results: The simulations show that both heterogeneity and fibroblasts significantly increase the safety factor for conduction by more than 10% in almost all the conditions tested and shorten the sinus node recovery time after overdrive suppression by up to 60%. In the human model, especially under challenging conditions, the fibroblasts help the heterogeneous myocytes to synchronise their rate (e.g. −82% in σCL under 25 nM of acetylcholine administration) and capture the atrium (with 25% L-type calcium current block). However, the anatomical and gap junctional coupling aspects remain the most important model parameters that allow effective atrial excitations.Conclusion: Despite the limitations to the proposed model, this work suggests a quantitative explanation to the astonishing overall heterogeneity shown by the sinoatrial node.

Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in CAV3 lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.

The dynamic clamp technique has emerged as a powerful tool in the field of cardiac electrophysiology, enabling researchers to investigate the intricate dynamics of ion currents in cardiac cells. Potassium channels play a critical role in the functioning of cardiac cells and the overall electrical stability of the heart. This chapter provides a comprehensive overview of the methods and applications of dynamic clamp in the study of key potassium currents in cardiac cells. A step-by-step guide is presented, detailing the experimental setup and protocols required for implementing the dynamic clamp technique in cardiac cell studies. Special attention is given to the design and construction of a dynamic clamp setup with Real Time eXperimental Interface, configurations, and the incorporation of mathematical models to mimic ion channel behavior. The chapter's core focuses on applying dynamic clamp to elucidate the properties of various potassium channels in cardiac cells. It discusses how dynamic clamp can be used to investigate channel kinetics, voltage-dependent properties, and the impact of different potassium channel subtypes on cardiac electrophysiology. The chapter will also include examples of specific dynamic clamp experiments that studied potassium currents or their applications in cardiac cells.