Action Potentials In Cells Research Articles

Graphene Field‐Effect Transistors toward Study of Cardiac Ischemia at Early Stage

AbstractIschemia and reperfusion states are studied in a network of cardiomyocytes as a part of real‐state conditions of heart injuries and inflammations, specifically myocardial infractions. Arrays of graphene field‐effect transistors (GFETs) fabricated in this work are used for extracellular recordings of ischemia states of cardiac cells during the external triggering of the ischemia infarction. The low‐concentrated ischemic buffer solution allows to create a cell‐stress condition resulting in the reperfusion process. The results show that the action potentials recorded with the graphene transistors, especially their shape, and duration of the active segment in measured extracellular action potentials, can be used to characterize the real state of the studied cardiac cell culture. The unique property of GFETs to detect such small changes in the action potential of cells in cardiac healthy and unhealthy states provides prospects for building the next generation of ultrasensitive biosensors, enabling the detection of acute ischemic states at an early stage.

Computational modelling of mouse atrio ventricular node action potential and automaticity.

The atrioventricular node (AVN) is a crucial component of the cardiac conduction system. Despite its pivotal role in regulating the transmission of electrical signals between atria and ventricles, a comprehensive understanding of the cellular electrophysiological mechanisms governing AVN function has remained elusive. This paper presents a detailed computational model of mouse AVN cell action potential (AP). Our model builds upon previous work and introduces several key refinements, including accurate representation of membrane currents and exchangers, calcium handling, cellular compartmentalization, dynamic update of intracellular ion concentrations, and calcium buffering. We recalibrated and validated the model against existing and unpublished experimental data. In control conditions, our model reproduces the AVN AP experimental features, (e.g. rate=175bpm, experimental range [121, 191]bpm). Notably, our study sheds light on the contribution of L-type calcium currents, through both Cav1.2 and Cav1.3 channels, in AVN cells. The model replicates several experimental observations, including the cessation of firing upon block of Cav1.3 or INa,r current. If block induces a reduction in beating rate of 11%. In summary, this work presents a comprehensive computational model of mouse AVN cell AP, offering a valuable tool for investigating pacemaking mechanisms and simulating the impact of ionic current blockades. By integrating calcium handling and refining formulation of ionic currents, our model advances understanding of this critical component of the cardiac conduction system, providing a platform for future developments in cardiac electrophysiology. KEY POINTS: This paper introduces a comprehensive computational model of mouse atrioventricular node (AVN) cell action potentials (APs). Our model is based on the electrophysiological data from isolated mouse AVN cells and exhibits an action potential and calcium transient that closely match the experimental records. By simulating the effects of blocking specific ionic currents, the model effectively predicts the roles of L-type Cav1.2 and Cav1.3 channels, T-type calcium channels, sodium currents (TTX-sensitive and TTX-resistant), and the funny current (If) in AVN pacemaking. The study also emphasizes the significance of other ionic currents, including IKr, Ito, IKur, in regulating AP characteristics and cycle length in AVN cells. The model faithfully reproduces the rate dependence of action potentials under pacing, opening the possibility of use in impulse propagation models. The population-of-models approach showed the robustness of this new AP model in simulating a wide spectrum of cellular pacemaking in AVN.

Instant rerouting of photosynthetic electron transport to O2 reduction after the plasma membrane excitation of Chara in the presence of methyl viologen

—Action potential (AP) of excitable plant cells is an important signaling event that can differentially alter physicochemical and physiological processes in various parts of the same cell. In giant cells of characean algae, the AP propagation has minor effect on photosynthetic electron transport in areas with high activity of plasmalemmal H+-pump but inhibits linear electron flow in regions featuring high passive H+/OH− conductance of the plasma membrane (PM). Uneven spatial distributions of local periplasmic and cytoplasmic pH facilitate the operation of distinct (CO2-dependent and O2-mediated) pathways of photoinduced electron flow, which presumably accounts for differential influence of AP on photosynthesis. The excitation of Chara australis cell in the presence of methyl viologen (MV), a redox mediator with the prooxidant action, provides a convenient model system to clarify the influence of voltage-dependent ion fluxes across PM on photosynthetic activity of chloroplasts. This study shows that permeation of MV to their target sites in chloroplasts is restricted by PM in resting cells, but MV easily passes through ionic channels opened during the PM depolarization. This gated permeation of MV gives rise to strong non-photochemical quenching, decrease in the effective quantum yield of linear electron flow, apparent O2 uptake, and, finally, the enhanced ROS production, as detected by the fluorescent probe dichlorofluorescein. Taken together, the results indicate that the AP generation in the presence of MV acts as trigger for instant redirection of photosynthetic linear electron flow from CO2-dependent route to the path of O2 reduction with the eventual formation of H2O2 as a dominant and most stable ROS form.

Epilepsy-associated Kv1.1 channel subunits regulate intrinsic cardiac pacemaking in mice.

The heartbeat originates from spontaneous action potentials in specialized pacemaker cells within the sinoatrial node (SAN) of the right atrium. Voltage-gated potassium channels in SAN myocytes mediate outward K+ currents that regulate cardiac pacemaking by controlling action potential repolarization, influencing the time between heartbeats. Gene expression studies have identified transcripts for many types of voltage-gated potassium channels in the SAN, but most remain of unknown functional significance. One such gene is Kcna1, which encodes epilepsy-associated voltage-gated Kv1.1 K+ channel α-subunits that are important for regulating action potential firing in neurons and cardiomyocytes. Here, we investigated the functional contribution of Kv1.1 to cardiac pacemaking at the whole heart, SAN, and SAN myocyte levels by performing Langendorff-perfused isolated heart preparations, multielectrode array recordings, patch clamp electrophysiology, and immunocytochemistry using Kcna1 knockout (KO) and wild-type (WT) mice. Our results showed that either genetic or pharmacological ablation of Kv1.1 significantly decreased the SAN firing rate, primarily by impairing SAN myocyte action potential repolarization. Voltage-clamp electrophysiology and immunocytochemistry revealed that Kv1.1 exerts its effects despite contributing only a small outward K+ current component, which we term IKv1.1, and despite apparently being present in low abundance at the protein level in SAN myocytes. These findings establish Kv1.1 as the first identified member of the Kv1 channel family to play a role in sinoatrial function, thereby rendering it a potential candidate and therapeutic targeting of sinus node dysfunction. Furthermore, our results demonstrate that small currents generated via low-abundance channels can still have significant impacts on cardiac pacemaking.

Voltage-Gated Ion Channels: Structure, Pharmacology and Photopharmacology.

Voltage-gated ion channels are transmembrane proteins responsible for the generation and propagation of action potentials in excitable cells. Over the last decade, advancements have enabled the elucidation of crystal structures of ion channels. This progress in structural understanding, particularly in identifying the binding sites of local anesthetics, opens avenues for the design of novel compounds capable of modulating ion conduction. However, many traditional drugs lack selectivity and come with adverse side effects. The emergence of photopharmacology has provided an orthogonal way of controlling the activity of compounds, enabling the regulation of ion conduction with light. In this review, we explore the central pore region of voltage-gated sodium and potassium channels, providing insights from both structural and pharmacological perspectives. We discuss the different binding modes of synthetic compounds that can physically occlude the pore and, therefore, block ion conduction. Moreover, we examine recent advances in the photopharmacology of voltage-gated ion channels, introducing molecular approaches aimed at controlling their activity by using photosensitive drugs.

Deep Learning Enhanced Label-Free Action Potential Detection Using Plasmonic-Based Electrochemical Impedance Microscopy.

Measuring neuronal electrical activity, such as action potential propagation in cells, requires the sensitive detection of the weak electrical signal with high spatial and temporal resolution. None of the existing tools can fulfill this need. Recently, plasmonic-based electrochemical impedance microscopy (P-EIM) was demonstrated for the label-free mapping of the ignition and propagation of action potentials in neuron cells with subcellular resolution. However, limited by the signal-to-noise ratio in the high-speed P-EIM video, action potential mapping was achieved by averaging 90 cycles of signals. Such extensive averaging is not desired and may not always be feasible due to factors such as neuronal desensitization. In this study, we utilized advanced signal processing techniques to detect action potentials in P-EIM extracted signals with fewer averaged cycles. Matched filtering successfully detected action potential signals with as few as averaging five cycles of signals. Long short-term memory (LSTM) recurrent neural network achieved the best performance and was able to detect single-cycle stimulated action potential successfully [satisfactory area under the receiver operating characteristic curve (AUC) equal to 0.855]. Therefore, we show that deep learning-based signal processing can dramatically improve the usability of P-EIM mapping of neuronal electrical signals.

Biphasic action potentials in an individual cellular neural network cell

Hardware circuit that can effectively simulate biological neurons is an important basis for neuromorphic computation. Cellular neural network (CNN) cell is the basic information processor of a CNN, which acts like a neuron in the brain and has good circuit realizability. An individual memristive CNN cell is constructed by using a memristor instead of a linear resistor for imitating the ion channel time-varying conductance, in which abundant biphasic chaotic and periodic spiking activities are uncovered. This provides a new way to simulate biological neurons at the level of analog circuits. This paper first deduces the mathematical model of the memristive CNN cell, analyzes the equilibrium stability and then explores its dynamical behaviors based on numerical simulation. The results display that the different spiking activities can be effectively regulated by the system parameters and excitation parameters. Furthermore, the analog circuit of the memristive CNN cell is designed and the PSpice-based circuit simulations are performed to verify the correctness of the numerical simulations.

Application of k-Nearest Neighbors Method for Drug Concentration and Cardiotoxicity Classification Using Extracellular Field Potentials and Reconstructed Action Potentials of Cardiac Cells

Micro-electrode array (MEA) systems are important for measuring extracellular field potentials (FP) of cardiac cells, which is a crucial step in cardiotoxicity assessment. However, without modification, the MEA system is only capable of recording FPs. This limits the number of parameters for cardiotoxicity assessment only to FP parameters, while the action potential (AP) parameters remain unused. To address this issue the MEA systems are often modified to use electro- or optoporation to record the local extracellular APs (LEAPs), which allows to reliably quantify the AP morphology. As an alternative to MEA modification and cell membrane stimulation the AP can be reconstructed mathematically.This study explores how using additional parameters from reconstructed action potentials (RAPs), derived from FPs, can improve the accuracy of k-NN machine learning models for drug concentration and potential cardiotoxicity classification. The k-NN classifier was trained using combinations of FP and RAP parameters. The k-NN models were evaluated using five-fold stratified cross-validation and cross-channel validation. Their performances were compared using error rate, macro precision, macro recall and macro F1 score accuracy metrics. The results indicated that ncorporating RAP parameters into the feature set increased the F1 score of k-NN model for DMSO concentration classification by up to 10.78% compared to the training set with only FP features.

NEDD4-2 and the CLC-2 channel regulate neuronal excitability in the pathogenesis of mesial temporal lobe epilepsy

An increasing number of studies have focused on the role of NEDD4-2 in regulating neuronal excitability and the mechanism of epilepsy. However, the exact mechanism has not yet been elucidated. Here, we explored the roles of NEDD4-2 and the CLC-2 channel in regulating neuronal excitability and mesial temporal lobe epilepsy (MTLE) pathogenesis. First, chronic MTLE models were induced by lithium-pilocarpine in developmental rats. Coimmunoprecipitation analysis revealed that the interaction between CLC-2 and NEDD4-2. Western blot analyses indicated that NEDD4-2 expression was downregulated, while phosphorylated (P-) NEDD4-2 and CLC-2 expression was upregulated in adult MTLE rats. Then, the primary hippocampal neuronal cells were isolated and cultured, and the NEDD4-2 was knocked down by shRNA vector, resulting in decreased protein levels of CLC-2. While CLC-2 absence caused increased NEDD4-2 in cells. Next, in an epileptic cell model induced by a Mg2+-free culture, whole-cell current-clamp recording demonstrated that NEDD4-2 deficiency inhibited the spontaneous action potentials of cells, and CLC-2 absence caused more significant decrease in the spontaneous action potentials of cells. In conclusion, we herein revealed that NEDD4-2 regulates the expression of CLC-2, which is involved in neuronal excitability, and participates in the pathogenesis of MTLE.

Somatic ion channels and action potentials in olfactory receptor cells and vomeronasal receptor cells.

Olfactory receptor cells are primary sensory neurons that catch odor molecules in the olfactory system, and vomeronasal receptor cells catch pheromones in the vomeronasal system. When odor or pheromone molecules bind to receptor proteins expressed on the membrane of the olfactory cilia or vomeronasal microvilli, receptor potentials are generated in their receptor cells. This initial excitation is transmitted to the soma via dendrites, and action potentials are generated in the soma and/or axon and transmitted to the central nervous system. Thus, olfactory and vomeronasal receptor cells play an important role in converting chemical signals into electrical signals. In this review, the electrophysiological characteristics of ion channels in the somatic membrane of olfactory receptor cells and vomeronasal receptor cells in various species are described and the differences between the action potential dynamics of olfactory receptor cells and vomeronasal receptor cells are compared.

Extracellular electrophysiology on clonal human β-cell spheroids.

Pancreatic islets are important in nutrient homeostasis and improved cellular models of clonal origin may very useful especially in view of relatively scarce primary material. Close 3D contact and coupling between β-cells are a hallmark of physiological function improving signal/noise ratios. Extracellular electrophysiology using micro-electrode arrays (MEA) is technically far more accessible than single cell patch clamp, enables dynamic monitoring of electrical activity in 3D organoids and recorded multicellular slow potentials (SP) provide unbiased insight in cell-cell coupling. We have therefore asked whether 3D spheroids enhance clonal β-cell function such as electrical activity and hormone secretion using human EndoC-βH1, EndoC-βH5 and rodent INS-1 832/13 cells. Spheroids were formed either by hanging drop or proprietary devices. Extracellular electrophysiology was conducted using multi-electrode arrays with appropriate signal extraction and hormone secretion measured by ELISA. EndoC-βH1 spheroids exhibited increased signals in terms of SP frequency and especially amplitude as compared to monolayers and even single cell action potentials (AP) were quantifiable. Enhanced electrical signature in spheroids was accompanied by an increase in the glucose stimulated insulin secretion index. EndoC-βH5 monolayers and spheroids gave electrophysiological profiles similar to EndoC-βH1, except for a higher electrical activity at 3 mM glucose, and exhibited moreover a biphasic profile. Again, physiological concentrations of GLP-1 increased AP frequency. Spheroids also exhibited a higher secretion index. INS-1 cells did not form stable spheroids, but overexpression of connexin 36, required for cell-cell coupling, increased glucose responsiveness, dampened basal activity and consequently augmented the stimulation index. In conclusion, spheroid formation enhances physiological function of the human clonal β-cell lines and these models may provide surrogates for primary islets in extracellular electrophysiology.

Elucidating molecular mechanisms of protoxin-II state-specific binding to the human NaV1.7 channel.

Human voltage-gated sodium (hNaV) channels are responsible for initiating and propagating action potentials in excitable cells, and mutations have been associated with numerous cardiac and neurological disorders. hNaV1.7 channels are expressed in peripheral neurons and are promising targets for pain therapy. The tarantula venom peptide protoxin-II (PTx2) has high selectivity for hNaV1.7 and is a valuable scaffold for designing novel therapeutics to treat pain. Here, we used computational modeling to study the molecular mechanisms of the state-dependent binding of PTx2 to hNaV1.7 voltage-sensing domains (VSDs). Using Rosetta structural modeling methods, we constructed atomistic models of the hNaV1.7 VSD II and IV in the activated and deactivated states with docked PTx2. We then performed microsecond-long all-atom molecular dynamics (MD) simulations of the systems in hydrated lipid bilayers. Our simulations revealed that PTx2 binds most favorably to the deactivated VSD II and activated VSD IV. These state-specific interactions are mediated primarily by PTx2's residues R22, K26, K27, K28, and W30 with VSD and the surrounding membrane lipids. Our work revealed important protein-protein and protein-lipid contacts that contribute to high-affinity state-dependent toxin interaction with the channel. The workflow presented will prove useful for designing novel peptides with improved selectivity and potency for more effective and safe treatment of pain.

Electrical signals of the plasma membrane and their effect on chlorophyll fluorescence in <i>chara</i> chloroplasts <i>in vivo</i>

Action potentials of plant cells are involved in regulation of many cell processes, such as photosynthesis and cytoplasmic streaming. Excitable cells of characean algae submerged in media with elevated K+ content are able to generate hyperpolarizing electrical responses. This active response of plasma membrane arises upon the passage of inward electric current comparable in extent to natural currents circulating in illuminated Chara internodes. It remains currently unknown whether the hyperpolarizing electrical signals in Chara affect the photosynthetic activity. Here we show that the negative shift of cell membrane potential, which causes the K+ influx into the cytoplasm, is accompanied by a delayed decrease in the effective chlorophyll fluorescence yield (F′) and maximal yield (Fm′) under low background light (12.5 µmol m-2 s-1). The transient changes in F′ and Fm′ were evident under illumination only, which indicates their close relation to photosynthetic energy conversion in chloroplasts. The passage of inward current caused an increase in pH at the cell surface (pHo), which reflects a high H+/OH- conductance of the plasmalemma and points to the decrease in cytoplasmic pH due to H+ entry into the cell. The shifts in pHo arising in response to the first hyperpolarizing pulse disappeared upon repeated stimulations, thus indicating the long-term inactivation of plasmalemmal H+/OH- conductance. Despite the suppression of plasmalemmal H+ fluxes, the hyperpolarizing responses and the analyzed chlorophyll fluorescence changes did not disappear. The results indicate the participation of K+ flows between the outer medium, cytoplasm, and stroma in chloroplast functional changes that are reflected by the dynamics of F′ and Fm′.

Inhibitory Perturbations of Fluvastatin on Afterhyperpolarization Current, Erg-mediated K+ Current, and Hyperpolarization-activated Cation Current in Both Pituitary GH3 Cells and Primary Embryonic Mouse Cortical Neurons.

Fluvastatin (FLV), the first synthetically derived 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, is a potent inhibitor of cholesterol biosynthesis. While its primary mechanism of action is to reduce cholesterol levels, there is some evidence suggesting that it may also have effects on K+ channels. However, the overall effects of fluvastatin on ionic currents are not yet well understood. The whole-cell clamp recordings were applied to evaluate the ionic currents and action potentials of cells. Here, we have demonstrated that FLV can effectively inhibit the amplitude of erg-mediated K+ current (IK(erg)) in pituitary tumor (GH3) cells, with an IC50 of approximately 3.2µM. In the presence of FLV, the midpoint in the activation curve of IK(erg) was distinctly shifted to a less negative potential by 10mV, with minimal modification of the gating charge. However, the magnitude of hyperpolarization-activated cation current (Ih) elicited by long-lasting membrane hyperpolarization was progressively decreased, with an IC50 value of 8.7µM, upon exposure to FLV. More interestingly, we also found that FLV (5µM) could regulate the action potential and afterhyperpolarization properties in primary embryonic mouse cortical neurons. Our study presents compelling evidence indicating that FLV has the potential to impact both the amplitude and gating of the ion channels IK(erg) and Ih. We also provide credible evidence suggesting that this drug has the potential to modify the properties of action potentials and the afterhyperpolarization current in electrically excitable cells. However, the assumption that these findings translate to similar in-vivo results remains unclear.

Cellular excitability and ns-pulsed electric fields: Potential involvement of lipid oxidation in the action potential activation

Recent studies showed that nanosecond pulsed electric fields (nsPEFs) can activate voltage-gated ion channels (VGICs) and trigger action potentials (APs) in excitable cells. Under physiological conditions, VGICs’ activation takes place on time scales of the order 10–100 µs. These time scales are considerably longer than the applied pulse duration, thus activation of VGICs by nsPEFs remains puzzling and there is no clear consensus on the mechanisms involved. Here we propose that changes in local electrical properties of the cell membrane due to lipid oxidation might be implicated in AP activation. We first use MD simulations of model lipid bilayers with increasing concentration of primary and secondary lipid oxidation products and demonstrate that oxidation not only increases the bilayer conductance, but also the bilayer capacitance. Equipped with MD-based characterization of electrical properties of oxidized bilayers, we then resort to AP modelling at the cell level with Hodgkin-Huxley-type models. We confirm that a local change in membrane properties, particularly the increase in membrane conductance, due to formation of oxidized membrane lesions can be high enough to trigger an AP, even when no external stimulus is applied. However, excessive accumulation of oxidized lesions (or other conductive defects) can lead to altered cell excitability.

Electrical Signals at the Plasma Membrane and Their Influence on Chlorophyll Fluorescence of Chara Chloroplasts in vivo.

Action potentials of plant cells are engaged in the regulation of many cell processes, including photosynthesis and cytoplasmic streaming. Excitable cells of characean algae submerged in a medium with an elevated K+ content are capable of generating hyperpolarizing electrical responses. These active responses of plasma membrane originate upon the passage of inward electric current comparable in strength to natural currents circulating in illuminated Chara internodes. So far, it remained unknown whether the hyperpolarizing electrical signals in Chara affect the photosynthetic activity. Here, we showed that the negative shift of cell membrane potential, which drives K+ influx into the cytoplasm, is accompanied by a delayed decrease in the actual yield of chlorophyll fluorescence F' and the maximal fluorescence yield Fm' under low background light (12.5 µmol m-2 s-1). The transient changes in F' and Fm' were evident only under illumination, which suggests their close relation to the photosynthetic energy conversion in chloroplasts. Passing the inward current caused an increase in pH at the cell surface (pHo), which reflected high H+/OH- conductance of the plasmalemma and indicated a decrease in cytoplasmic pH due to the H+ entry into the cell. The shifts in pHo arising in response to the first hyperpolarizing pulse disappeared upon repeated stimulation, thus indicating the long-term inactivation of plasmalemmal H+/OH- conductance. Suppression of plasmalemmal H+ fluxes did not abolish the hyperpolarizing responses and the analyzed changes in chlorophyll fluorescence. These results suggest that K+ fluxes between the extracellular medium, cytoplasm, and stroma are involved in the functional changes of chloroplasts reflected by transients of F' and Fm'.

A-019 Integrative Analysis of Arrythmogenic Right Ventricular Cardiomyopathy of Potencial Biomarkers Through Bioinformatic Analysis

Abstract Background Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disease usually associated with heterozygous or compound mutations in cardiac desmosomes genes. Pathologically, it is a disease characterized by fibrofatty replacement primarily in the right ventricle of normal cardiac tissue and clinically by sudden cardiac death in healthy young people. Diagnosis is often difficult due to the nonspecific nature of the disease and the broad spectrum of phenotypic variations, being commonly diagnostic post mostem. Non-coding RNAs are biomarkers that regulate gene expression and participate in various pathophysiological processes, becoming an important research field in cardiomyopathology for their potential as non-invasive clinical biomarkers and therapeutic. In this study, we aim to identify miRNAs that may serve as potentially effective targets in diagnosis and treatment as well as the exploration of regulatory miRNAs-mRNAs networks is beneficial for comprehensively understanding the molecular mechanism of ARVC development. Methods This study screened RNAs expression profiling studies from the Gene Expression Omnibus (GEO) database. The keyword “arrhythmogenic right ventricular cardiomyopathy” was used to search for suitable datasets that followed the criteria of being a comparable case-control human study, it was selected the GSE29819 and GSE164490 GEO series. The microarray GSE29819 set was analyzed using GEO2R, an interactive web tool disponibility in GEODataset site, resulting in the differential expression genes (DEGs) of this series, while high throughput sequencing GSE164490 set was preprocessing by miARma-Seq v 1.7.5 and for identifying DEGs was used R software v4.2.2 in R Studio (version: 2022.12.0 + 353) and Deseq2 package. The 2 sets used P < 0.05 and |log FC| > 1.5 as the cutoff criteria. An online prediction database, DIANA-microT CDS v 5.0 was used to obtain interactions with high confidence levels between miRNAs and target mRNAs. STRING online database v9 was used to predict protein-protein interactions (PPI) and functional enrichment analysis was conducted using the web-based utility Enrichr to uncover the roles of these molecules. Results The integrated analysis of the 2 datasets from GEO sequencing data from ARVC, after a criterium analysis, identify 04 common DEGs: hsa-miR-30d-5p, hsa-miR-200a-3p, hsa-miR-34a-5p and hsa-miR-125a-5p between them. Also, by constructing a PPI network, it was identified 04 genes among the overlapping DEGs, including PIK3R2, RARRES1, SCN2B, and SLCO5A1 (scores ≥0.7). The functional enrichment analyses demonstrated that the DEGs were enriched in some GO biological processes such as regulation of atrial cardiac muscle cell membrane and voltage-gated sodium channel activity involved in cardiac muscle cell action potential. Conclusion Our results suggest that these new network gene associations possibly play a potential role in metabolic pathways related to the pathogenesis of ARVC and are good candidates for further functional validation assays to confirm these findings.

Haemodynamic changes in visceral hybrid repairs of type III and type V thoracoabdominal aortic aneurysms

The visceral hybrid procedure combining retrograde visceral bypass grafting and completion endovascular stent grafting is a feasible alternative to conventional open surgical or wholly endovascular repairs of thoracoabdominal aneurysms (TAAA). However, the wide variability in visceral hybrid configurations means that a priori prediction of surgical outcome based on haemodynamic flow profiles such as velocity pattern and wall shear stress post repair remain challenging. We sought to appraise the clinical relevance of computational fluid dynamics (CFD) analyses in the setting of visceral hybrid TAAA repairs. Two patients, one with a type III and the other with a type V TAAA, underwent successful elective and emergency visceral hybrid repairs, respectively. Flow patterns and haemodynamic parameters were analysed using reconstructed pre- and post-operative CT scans. Both type III and type V TAAAs showed highly disturbed flow patterns with varying helicity values preoperatively within their respective aneurysms. Low time-averaged wall shear stress (TAWSS) and high endothelial cell action potential (ECAP) and relative residence time (RRT) associated with thrombogenic susceptibility was observed in the posterior aspect of both TAAAs preoperatively. Despite differing bypass configurations in the elective and emergency repairs, both treatment options appear to improve haemodynamic performance compared to preoperative study. However, we observed reduced TAWSS in the right iliac artery (portending a theoretical risk of future graft and possibly limb thrombosis), after the elective type III visceral hybrid repair, but not the emergency type V repair. We surmise that this difference may be attributed to the higher neo-bifurcation of the aortic stent graft in the type III as compared to the type V repair. Our results demonstrate that CFD can be used in complicated visceral hybrid repair to yield potentially actionable predictive insights with implications on surveillance and enhanced post-operative management, even in patients with complicated geometrical bypass configurations.

Abstract P2036: Induced Pluripotent Stem Cell- Derived Cardiomyocytes Provide New Insights Of The Pathogenic Consequences Of The Novel Frameshift Variant (c.5769delG) In MYH7 Gene

Background: the genetic basis of hypertrophic cardiomyopathy is pleiotropic; A novel disease causing frameshift variant (c.5769delG, MYH7 ) in the beta-myosin heavy chain gene has been recently described. However, the mechanisms involved have not been adequately defined. We have used iPSC-CMs to investigate the underlying mechanisms. Methods: We have generated patient-specific iPSCs from clinically and genetically defined HCM patients carrying the pathogenic variant (c.5769delG) (n=3) and compared them to iPSCs from healthy controls (n=3). Optical mapping of calcium transients and cell action potential were performed under different stimulation conditions. iPSC-CMs contractility was recorded via widefield microscopy. Gene and protein expression analyses were performed using RT-PCR, immunoblotting and immunocytochemistry. RNA sequencing data from patients’ myocardial tissues was analyzed to validate gene expression data from iPSC-CMs. Results: HCM iPSCs-CMs showed a significant decrease in calcium release duration (i.e., time to peak) with decrease in calcium transient amplitude, compared to controls. This was associated with an accelerated contraction and a decreased contraction amplitude. Nonetheless, action potential analysis showed no significant changes. Transcriptome analysis of iPSC-CMs and patients’ myocardial tissues showed a significant down regulation of SERCA/ATP2A2 , MYH6 and KCNIP2 . Conclusion: iPSC-CMs provided new insights of the pathogenic consequences of the novel variant in MYH7 gene on calcium handling and contractility. Key words: Hypertrophic Cardiomyopathy, Disease model, MYH7 , iPSC-CMs, Calcium Handling, Contractility.

Uncertainty assessment of proarrhythmia predictions derived from multi-level in silico models

In silico methods can be used for an early assessment of arrhythmogenic properties of drug candidates. However, their use for decision-making is conditioned by the possibility to estimate the predictions’ uncertainty. This work describes our efforts to develop uncertainty quantification methods for the predictions produced by multi-level proarrhythmia models. In silico models used in this field usually start with experimental or predicted IC50 values that describe drug-induced ion channel blockade. Using such inputs, an electrophysiological model computes how the ion channel inhibition, exerted by a drug in a certain concentration, translates to an altered shape and duration of the action potential in cardiac cells, which can be represented as arrhythmogenic risk biomarkers such as the APD90. Using this framework, we identify the main sources of aleatory and epistemic uncertainties and propose a method based on probabilistic simulations that replaces single-point estimates predicted using multiple input values, including the IC50s and the electrophysiological parameters, by distributions of values. Two selected variability types associated with these inputs are then propagated through the multi-level model to estimate their impact on the uncertainty levels in the output, expressed by means of intervals. The proposed approach yields single predictions of arrhythmogenic risk biomarkers together with value intervals, providing a more comprehensive and realistic description of drug effects on a human population. The methodology was tested by predicting arrhythmogenic biomarkers on a series of twelve well-characterised marketed drugs, belonging to different arrhythmogenic risk classes.